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The Clamp loader of Escherichia coli DNA Polymerase III

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Title:
The Clamp loader of Escherichia coli DNA Polymerase III kinetics of the ATP-dependent steps in the sliding-clamp loading reaction
Creator:
Williams, Christopher R., 1974-
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English
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xv, 288 leaves : ill. (in ILLUS in FF) ; 29 cm.

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Subjects / Keywords:
Adenosine triphosphatases ( jstor )
Anisotropy ( jstor )
Binding sites ( jstor )
DNA ( jstor )
Fluorescence ( jstor )
Hydrolysis ( jstor )
Kinetics ( jstor )
Molecules ( jstor )
Nucleotides ( jstor )
Syringes ( jstor )
DNA Binding Proteins -- genetics ( mesh )
DNA Polymerase III -- genetics ( mesh )
DNA Replication -- genetics ( mesh )
Escherichia coli -- genetics ( mesh )
Kinetics -- methods ( mesh )
Nuclease Protection Assays -- methods ( mesh )
Research ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D)--University of Florida, 2003.
Bibliography:
Bibliography: leaves 274-287.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Christopher R. Williams.

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University of Florida
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80287708 ( OCLC )
ocm80287708
0030445780 ( ALEPH )

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THE CLAMP LOADER OF Escherichia coli DNA POLYMERASE III:
KINETICS OF THE ATP-DEPENDENT STEPS IN THE SLIDING-CLAMP
LOADING REACTION














By

CHRISTOPHER R. WILLIAMS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA




THE CLAMP LOADER OF Escherichia coli DNA POLYMERASE III:
KINETICS OF THE ATP-DEPENDENT STEPS IN THE SLIDING-CLAMP
LOADING REACTION
By
CHRISTOPHER R. WILLIAMS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003


Copyright 2003
by
Christopher R. Williams


This document is dedicated to my parents, family, and Heather Runyan.


ACKNOWLEDGMENTS
Foremost I would like to thank my advisor, Linda Bloom, Ph.D., for outstanding
guidance and providing an exceptional working environment in a state-of-the-art research
laboratory. I thank my committee at UF, Drs. Daniel L. Purich, Arthur S. Edison, J. Bert
Flanagen, and Alfred S. Lewin, for their assistance not only in guiding my project, but in
guiding me to become more wise and perceptive as a scientist. I must also acknowledge
my committee at Arizona State University, Drs. Neal Woodbury, Yuri L. Lyubchenko,
and Kenneth J. Hoober, for their assistance in the early stages of this dissertation project.
I acknowledge Manju Flingorani, Ph D., for helpful discussions at the Keystone
Symposia meetings and protein preparation, Mike ODonnell, Ph.D. for the generous
gifts of clamp loaders, Martin Webb, Ph.D., Myron F. Goodman, Ph.D., and Jeffery
Bertram for the gift of the phosphate binding protein plasmid, and help in
characterization and use of MDCC-PBP, and Petr Kuzmic, PhD., for contribution of the
custom version of DynaFit and assistance with kinetic modeling. I would like to thank
my following colleagues for their friendship and invaluable discussions regarding this
project and science in general: Brandon Ason, Ryan Shaw, John C. Lopez, Gabriel
Montano, Ph D., Gregory Uyeda, Jos dement, Ph D., Joyce Feller, Ph.D. Finally, I
would like to thank my parents for their unwavering support of my endevours, and for
purchasing my first microscope and chemistry sets years ago.
IV


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS' ., iv
LIST OF TABLES x
LIST OF FIGURES xi
ABSTRACT : xiv
CHAPTER
1 INTRODUCTION STATEMENT OF PROBLEM 1
The Processive Escherichia coli DNA Polymerase III Holoenzyme 1
DNA Polymerase III Holoenzyme 1
The P Sliding Clamp 2
The DnaX Clamp Loader Stoichiometry and Organization of Subunits 3
Clamp Loader Subunit Functions 4
The Mechanism of p Clamp Loading 5
Importance of the E. coli Model Replication System 7
The General Problem Under Study and Research Questions Addressed 8
Conformational Dynamics of the Clamp Loading Machine 10
Enhancement of the Clamp Loading Machine by its Clamp 13
The x and \\i Subunits are Required for Optimum Activity of the Clamp Loader 14
Application of the Analyses of the Clamp Loading Machine to Other Complex
Molecular Machines 17
Novel Hybrid Devices Based on the Clamp Loader and Sliding Clamp 19
Off-the-Wair Example of the Clamp and Clamp Loader in a Novel Device..20
Design of Research Project 21
2 LITERATURE REVIEW 24
DNA Replication in Escherichia coli 24
DNA Polymerase III Holoenzyme 25
The x Subunit is the Coordinator of Pol III Holoenzyme Function and
Processivity 29
Structure of the p Sliding Clamp Processivity Protein 31
The DnaX Clamp Loading Machine 35
The AAA+ Superfamily of Motor Proteins 40


X-ray Crystal Structure of the Clamp Loading Machine 45
Structure of the Nucleotide Binding Site and the Proposed Conformational
Change of the y Subunit 48
X-Ray Crystal Structure of the 8 Subunit Bound to a p Monomer and the 1
Mechanism for Opening the p Sliding Clamp 52
DNA structural requirements for p clamp loading by y complex 56
Mechanism of the P Clamp Loading Reaction Cycle by y Complex 60
Mutations of the p Clamp, and y Complex 8 and y Subunits: Effects on the
Clamp Loading Mechanism 65
The Clamp Loading Machine Within Polymerase m Holoenzyme 68
Clamps and Clamp Loaders of Bacteriophage, Eukaryotic, and
Archaeal Organisms 71
Bacteriophage T4 Clamp and Clamp loader 73
Eukaryotic PCNA Clamp and Replication Factor-C Clamp Loader 75
Archaeal PCNA Clamp and Replication Factor-C Clamp Loader 80
3 MATERIALS AND METHODS 84
Proteins, Reagents, and Oligonucleotide Substrates 84
DNA Polymerase III Proteins 84
Reagents 85
Oligonucleotide Substrates 85
Purification of Escherichia coli Phosphate Binding Protein 86
Labeling of Phosphate Binding Protein with MDCC 89
Characterization of MDCC-Labeled Phosphate Binding Protein 90
Removal of Free-Inorganic Phosphate (P¡) Contamination with the Pi-mop 91
Concentration and Efficiency of Labeling of MDCC-PBP 91
Characterization of the fluorescence-molar response of MDCC-PBP to P¡ 92
Active Site Titration of MDCC-PBP 93
Fluorescence Anisotropy Binding Assays 94
Calculation of Anisotropy 94
Steady-state Measurement of Clamp Loader RhX-DNA Binding Kinetics 95
Pre-Steady-State Measurement of Clamp Loader RhX-DNA Binding
Kinetics 99
Fluorescence-based MDCC-PBP ATP Hydrolysis (ATPase) Assay 101
Steady-State Kinetics of ATP hydrolysis 101
Pre-Steady-State Kinetics of ATP Hydrolysis 104
Computer Modeling of ATP Hydrolysis Kinetic Data 109
Correlated Pre-Steady-State MDCC-PBP ATPase Assays and Fluorescence
Anisotropy Binding Assays 110
Stopped-Flow Dead Time Determinations 112
Determination of the Dead Time for the Biologic SFM-4 Stopped-Flow 112
Determination of the Applied Photophysics SX. 18MV Stopped-Flow
Reaction Analyzer Sequential- and Single-mix Dead Times 114
vi


4 ATP-DEPENDENT CONFORMATIONAL CHANGE IN THE CLAMP LOADER! 18
Introduction 118
Steady-State Characterization of y Complex ATP Hydrolysis, DNA Binding and
Clamp Loading Activities 120
Enhancement of Steady-State ATP Hydrolysis Kinetics of y Complex by (3
clamp 120
Steady-State DNA Binding and Clamp Loading Activities of y Complex 122
Pre-Steady-State Kinetics of DNA-Dependent ATP Hydrolysis by y Complex 127
Pre-Steady-State MDCC-PBP ATPase Assays for y Complex in the Absence
and Presence of (1 Clamp 127
Pre-Steady-State MDCC-PBP ATPase Assays at Different Concentrations of y
Complex in the Presence of p 129
ATPyS-Chase of Pre-Steady-State ATP Hydrolysis Activity by y Complex... 131
Pre-Steady-State Kinetics of p Clamp Loading by y Complex Initiated at
Different Steps of the Reaction Cycle 135
Kinetics of Clamp Loading when y Complex is Equilibrated with ATP 135
Kinetics of Clamp Loading when y Complex is Equilibrated with ATP and P 137
Kinetics of Clamp Loading when y Complex is Added Directly to a Solution
of ATP, p, and DNA 141
Kinetics of ATP Hydrolysis During Clamp Loading when y Complex is
Equilibrated with ATP 142
Kinetics of ATP Hydrolysis when y Complex is not Equilibrated with ATP 144
Kinetics of Formation of the Two Populations of y Complex 146
Computer Modeling of ATP Hydrolysis Reaction Kinetics 149
Discussion 153
5 CHARACTERIZATION OF THE MINIMAL CLAMP LOADER COMPLEX
AND COMPARISON TO GAMMA COMPLEX 163
Introduction 163
7388 is the Minimal Clamp Loader Complex Which Can Bind DNA 165
Analysis of DNA Binding Activity of the Individual Subunits or
Sub-complexes of the Clamp Loader 165
Analysis of the DNA Binding Activity of 7388 Minimal Complex in the
Absence and Presence of P 167
Comparison of p Clamp Binding Affinity of the Minimal Complex and 7 Complexl69
Equilibrium ppyrene Binding Activity of the Minimal Complex and 7 Complexl69
Apparent Dissociation Constant for ATP Binding the Minimal Complex or 7
Complex 171
Kinetics of ATP Hydrolysis by the Minimal Complex Measured Using the MDCC-
PBP ATPase Assay 173
Steady-State ATP Hydrolysis Kinetics of 7388 Minimal Complex in the
Absence and Presence of P Clamp 173
Pre-Steady-State Kinetics of ATP Hydrolysis by 7388 Minimal Complex in
the Absence and Presence of P Clamp
vii
176


Kinetics of ATP Hydrolysis when the Minimal Complex is not equilibrated
with ATP 180
ATPyS-Chase of Pre-Steady-State ATP Hydrolysis Activity by the 7388
Minimal Complex !... 183
ATPyS-Chase of Steady-State ATP Hydrolysis Activity by the 7388 Minimal
Complex or y Complex 186
Clamp Loading Activity of the Minimal Complex is More Sensitive to ADP
than y Complex 188
Pre-Steady-State Kinetics of Clamp Loading by the Minimal Complex Initiated at
Different Steps of the Reaction Cycle 190
Kinetics of Clamp Loading when the Minimal Complex is Equilibrated with
ATP and P 191
Kinetics of Clamp Loading when the Minimal Complex is Equilibrated with
ATP 194
Kinetics of Clamp Loading When the Minimal Complex is Mixed Directly
with a Solution of ATP, p, and DNA 195
Direct Real Time Correlation of the Minimal Complex DNA Binding and ATP
Hydrolysis Kinetics in the Presence and Absence of p Clamp 196
Discussion 200
Understanding y Complex Kinetics by Characterization of and Comparison
with 7388 Minimal Complex 200
7388 is the Minimal Complex with DNA Binding Ability, and Binds ATP and
P with Affinity Similar to 7 Complex 202
Pre-Steady-State ATP Hydrolysis and DNA Binding Kinetics: Analyses of the
Active and Inactive Clamp Loader States 202
The % and V Subunits, Missing from the Minimal Complex, May Facilitate the
Conformational Dynamics of 7 Complex 210
P Clamp Enhances the Switch from Inactive to Active Clamp Loader
Populations 211
Experiments when the Minimal Complex was not Equilibrated with ATP
Reveal Slower Conformational Change Kinetics than 7 Complex 213
The Nature of Nucleotide Binding to the Minimal Complex and 7 Complex .214
6 CONCLUSIONS AND RECOMMENDATIONS 221
Introduction 221
Steady-State Kinetics of the Clamp Loader in the Absence or Presence of p 224
Kinetics of ATP-Dependent Conformational Changes within the Clamp Loader .. .226
A Possible Mechanism for p Clamp Enhancement of Clamp Loader Activity 233
The Missing % and y Subunits are Responsible for the Kinetic Differences
Between the Minimal Complex and 7 Complex 236
The x and vp Subunits are E. coli Clamp Loader AAA+ Adaptor Proteins 237
Programmatic Recommendations 239
Pre-Steady-State Kinetics of p Clamp Binding 239
Fluorescence Lifetime Measurement of MDCC-PBP Titrated with Inorganic
Phosphate 241
viii


Further Investigation of ATP Binding and Nucleotide Exchange by the Clamp
Loader 242
Analysis of Clamp Loader Conformational Dynamics by Circular Dichroism
Spectroscopy 244
Identification of the Putative Clamp Loader DNA Binding Surface 245
The i and vp AAA+ adaptor hypothesis 246
APPENDIX
COMPUTER MODELING OF EXPERIMENTAL KINETIC DATA 248
DynaFit Script For Fitting Shown in Figure 4-9 248
DynaFit Script For Fitting in Shown in Figure 4-10C 249
DynaFit Script For Fitting in Shown in Figure 5-6 251
Simulation Mechanisms for Equilibration Steps: KinTekSim Program 252
DynaFit Output Indices 255
Fitting for y Complex Data in Figure 4-9 255
Fitting for y Complex Data in Figure 4-10C 258
Fitting for Minimal Complex Data in Figure 5-6 260
Fitting for Minimal Complex Data For Estimation of Conformational Rate
Constants From a Single Data set 262
Alternate Dynafit Model with a Branch Step at After Hydrolysis of Second ATP265
Alternate Dynafit Model Applied to y Complex in Figure 4-9 265
Alternate Dynafit Model Applied to y Complex in Figure 4-10C 267
Alternate Dynafit Model Applied to the Minimal Complex in Figure 5-7 269
Alternate Dynafit Model Applied to y Complex in a DNA Binding Assay in the
Absence of p Clamp 271
LIST OF REFERENCES 274
BIOGRAPHICAL SKETCH 287
ix


LIST OF TABLES
Table page
2-1. Clamps and clamp loaders through evolution 72
3-1. Assay and protein buffers 85
3-2. TG-plus media contents 87
4-1. Steady-state ATP hydrolysis kinetics of y complex in the absence and
presence of ¡5 121
5-1. Steady-state ATP hydrolysis kinetic parameters for the minimal complex and y
complex in the absence and presence of fl 174
5-2. Steady-state ATPyS-chase assay results: comparison of the minimal complex and y
complex in the presence or absence of [5 187


LIST OF FIGURES
Figure page
1 -1. Schematic of the p clamp loading reaction for processive DNA synthesis 6
2-1. The crystal structure of p sliding damp 32
2-2 The crystal structure of a AAA+ motor protein 43
2-3. The crystal structure of the 7388 clamp loader 46
2-4. The crystal structure of the S-Pi complex 53
2-5. A schematic cartoon of the basic steps in the clamp loading reaction and initiation
complex formation 62
2-6. Architecture of the polymerase III holoenzyme at the replication fork organized by
the DnaX clamp loading machine 69
3-1. P; titration analysis of MDCC-PBP 93
3-2. A plot of observed reaction decay rates as a function of NBS concentration 113
3-3. Fluorescent decay amplitudes plotted as a function of experimentally observed
decay rate constants to determine dead time of the SFM-4 stopped-flow 114
3-4. Sequential-mix reaction fluorescent decay amplitudes plotted as a function of
experimentally observed decay rate constants to determine dead time of the
SX. 18MV stopped-flow 116
3-5. Single-mix reaction fluorescent decay amplitudes plotted as a function of
experimentally observed decay rate constants to determine dead time of the
SX.18MV stopped-flow 117
4-1. Steady-state DNA binding and clamp loading activities of y complex 123
4-2. Steady-state kinetics of clamp loading as a function of ATP concentration 125
4-3. Kinetics of ATP hydrolysis by y complex in the presence and absence of P 128
4-4. Quantification of the number of ATP molecules hydrolyzed by y complex in the
first turnover of p clamp loading 130
xi


4-5. Pre-steady-state kinetics of ATP hydrolysis by y complex in the presence and
absence of the [! clamp in assays with and without an ATPyS chase
132
4-6. Kinetics of clamp loading measured in reactions that were initiated at different
stages of the loading cycle 138
4-7. Kinetics of ATP hydrolysis during the clamp loading reaction when y complex is
equilibrated with ATP 143
4-8. Kinetics of ATP hydrolysis in reactions initiated by the addition of y complex to
ATP and DNA 145
4-9. Kinetics of ATP hydrolysis when y complex is incubated with ATP for a defined
period of time prior to addition of DNA 147
4-10. Kinetic modeling of ATP hydrolysis reactions 150
5-1. Change in steady-state anisotropy for RhX-ss DNA in the presence of individual
subunits, sub-complexes, and y complex with or without ATP 166
5-2. Steady-state binding of the minimal complex or y complex with RhX-pt DNA with
and without (1 168
5-3. Steady-state anisotropy binding activity of y complex or the minimal complex with
ppyrene jn tjje presence or absence of ATP 170
5-4. Minimal complex or y complex binding ppyrene as a function of ATP concentration 172
5-5. Kinetics of ATP hydrolysis by the minimal complex or y complex in the presence
and absence of p 178
5-6. Kinetics of ATP hydrolysis of the minimal complex or y complex directly mixed
with pt DNA and ATP 181
5-7. Pre-steady-state kinetics of ATP hydrolysis by the minimal complex when chased
with non-hydrolyzable ATPyS 184
5-8. Effect of increasing ADP concentration on the steady-state p clamp loading
reaction for the minimal complex and y complex 189
5-9. Pre-steady-state kinetics of the clamp loading reaction initiated at different steps 192
5-10. Direct correlation of the kinetics of DNA binding and ATP hydrolysis by the
yj88 minimal complex in the absence and presence of p clamp 198
5-11. Kinetic modeling of ATP hydrolysis reactions 203
6-1. Kinetic modeling of ATP hydrolysis reactions 229
xii


A-l. KinTekSim simulation ofy complex, no equilibration with ATP 253
A-2. KinTekSim simulation of the minimal complex equilibrated with ATP 254
A-3. KinTekSim simulation of the minimal complex, no equilibration with ATP 255
A-4. DynaFit Fitting of the minimal complex (Figure 5-6), no equilibration time with
, ATP 262
A-5. DynaFit fitting of th minimal complex (Figure 5-7B, black trace), Single
experimental data set, 1000 ms equilibration with ATP 265
A-6. Figure 4-9 data for y complex fit to alternate model with a branch step after
hydrolysis of two ATPs 267
A-7. Figure 4-10C data for y complex fit to alternate model with a branch step after
hydrolysis of two ATPs 269
A-8. Figure 5-7, black trace data for the minimal complex fit to alternate model with a
branch step after hydrolysis of two ATPs 271
A-9. Example of the alternate model applied to DNA binding (anisotropy) data for a
reaction of y complex and Rhx-pt DNA 273
xiii


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE CLAMP LOADER OF Escherichia coli DNA POLYMERASE III:
KINETICS OF THE ATP-DEPENDENT STEPS IN THE SLIDING-CLAMP
LOADING REACTION
By
Christopher R. Williams
December 2003
Chair: Linda B. Bloom
Major Department: Biochemistry and Molecular Biology
DNA polymerase III holoenzyme, the principal enzyme responsible for E. coli
chromosomal replication, synthesizes stretches of DNA thousands of nucleotides long at
a rate approaching 750 nucleotides s'1 without dissociation. A ring-shaped DNA sliding-
clamp P topologically links the polymerase to DNA. A clamp loader, y complex,
assembles p on DNA in an ATP-dependent reaction. This dissertation project was
undertaken for investigation of the mechanism of p clamp loading by the clamp loader for
processive replication. ATP binding and hydrolysis activities of the clamp loader
promote conformational changes modulating its binding affinity for the clamp and DNA.
Using fluorescence-based steady-state and real-time stopped-flow methods, the kinetics
of these dynamic conformational changes were measured. Pre-steady-state ATP
hydrolysis assays performed in the absence or presence of P resulted in biphasic or
monophasic kinetics respectively. Biphasic kinetics suggested that the clamp loader,
equilibrated with ATP, exists in a mixture of two dominant species. Addition of p
xiv


converted this mixture into a single activated population, effectively increasing its
concentration. These experiments, in addition to adenosine 5-0-(3-thiotriphosphate)
(ATPyS)-chase assays showed that activated y complex hydrolyzed one ATP per y-
subunit at a rate faster than ATP dissociation. The hypothesis that y complex exists in
multiple species with ATP, was confirmed by measuring pre-steady-state clamp loading
kinetics in DNA-binding assays. When y complex was equilibrated with ATP, a mixture
of two species formed which when mixed with DNA and p exhibited biphasic DNA
binding kinetics. When equilibrated with ATP and (1 rapid monophasic DNA binding
kinetics resulted. Direct mixing of y complex with DNA p and ATP displayed slow
monophasic kinetics, limited by the conformational change rate. The rate of the
conformational changes separating the two dominant species was determined by
investigating their evolution in equilibration time with ATP. Computer modeling of
these experimental data revealed a conformational change rate of ~4.5 s'1. Comparison of
the kinetics of a minimal clamp loader complex missing x and y subunits, revealed that
these subunits facilitate the conformational changes in y complex required for modulation
of p and DNA binding affinities during the clamp loading reaction.
XV


CHAPTER 1
INTRODUCTION STATEMENT OF PROBLEM
The Processive Escherichia coli DNA Polymerase III Holoenzyme
DNA polymerase IH holoenzyme (pol III holoenzyme) is the principal enzyme
involved in replication of the Escherichia coli chromosome (Komberg and Baker, 1992).
The pol III holoenzyme copies the parent chromosome with surprising speed and
processivity, moving along the DNA at a maximum velocity reaching approximately 750
nucleotides per second, without release from the template at distances of over thousands
of nucleotides. Polymerase III holoenzyme must possess these functional characteristics
to complete rapid replication of the chromosome for cell division. Rapid and processive
DNA synthesis is required for all forms of DNA metabolism and genome maintenance
including DNA repair and recombination, and is evolutionary conserved in all branches
of life, underscoring the significance of advancing the detailed understanding of the
proteins providing these functional mechanisms.
DNA Polymerase III Holoenzyme
Pol III holoenzyme is a multi-protein complex consisting of ten distinct subunits (a,
e, 0, (I, T, y, 8,8, x and \|). A core of the holoenzyme consisting of the a-5 to 3
polymerase, £-3 to 5 exonuclease, and 0 subunits is paired-up by the x % protein-dimer
(McHenry, 1982; McHenry and Crow, 1979). The T2-dimerized pol III core, termed pol
III, was originally purified from cells and isolated from pol III holoenzyme. The
replication activities of pol III core and pol III were determined to be distributive in
kinetic and product size determination assays, whereas holoenzyme could synthesize
1


2
stretches ofDNA with high processivity (Fay et al., 1981; LaDuca et al., 1983). Clearly,
these were incomplete forms of pol III holoenzyme, and were missing important factors
necessary for complete and rapid replication of the chromosome.
Another form of pol III, termed pol III*, contained all of the subunits of pol III
holoenzyme except one, the ¡3 subunit (Fay et al., 1982). Addition of this p subunit was
all that was required to convert the distributive pol III* enzyme into a highly processive
enzyme with DNA synthesis activity matching pol III holoenzyme. The p subunit is
initially associated with the replication fork DNA at sites called preinitiation complexes
where polymerase III holoenzyme assembles for DNA synthesis. Several early
biochemical studies revealed that a complex consisting of the r,y,8,8, x and y subunits
within pol HI holoenzyme comprised a complex responsible for ATP-dependent
placement of the p subunit at primed sites on DNA for formation of the preinitiation
complexes, and were required for conferring processivity in replication (Maki et al.,
1988; Maki and Komberg, 1988).
The p Sliding Clamp
Solution of the X-ray crystal structure of the p subunit revealed that it was a ring-
shaped dimer of crescent-shaped protomers (Kong et al., 1992). p was termed a DNA
sliding-clamp due to its ability to topologically link pol III holoenzyme to template
DNA in such a way that holoenzyme remains tightly associated with DNA, yet has
significant freedom of movement on the DNA. The P clamp has an inner pore lined with
a-helices that act akin to skates, traversing the clefts of the major and minor grooves in
the DNA backbone as the clamp slides along. This inner pore has a diameter large
enough to encircle duplex DNA as well as hybrid RNA-DNA duplex structure found at


3
the sites of preinitiation complex formation. A combination of hydrogen bonding,
hydrophobic and ionic interactions at the dimer interfaces strengthen and hold together
the p clamp subunits. P binds directly to the core of pol III holoenzyme through the a
subunit (Stukenberg et al., 1991). The P clamp has a dissociation constant for
dimerization in the range of ~ 6.0 x 10'" molar, and is stable enough to remain on
circular DNA for ~ 100 minutes (Yao et al., 1996). The extraordinary stability of the
circular p dimer in solution and on the circular chromosome stresses the need for some
mechanism to open p for its loading onto and disassembly from DNA.
The DnaX Clamp Loader Stoichiometry and Organization of Subunits
The other processivity proteins within pol 111 holoenzyme (x,y,8,8,x and i|f) form a
complex that performs the duty of opening and loading p onto DNA for formation of
preinitiation complexes on primed DNA at the leading and lagging strands at the
replication fork (Kelman and O'Donnell, 1995). These proteins form the DnaX complex
clamp loader, with the subunit stoichiometry [(DnaX)3,8|8iXiVi]. in which each
subunit executes a unique function (Pritchard et al., 2000). The dnaX gene forms both x
and y subunits by a translational ffameshift that forms a stop codon defining the y subunit
C-terminus, approximately two-thirds the length of the complete mRNA (Flower and
McHenry, 1990; Tsuchihashi and Komberg, 1990). Therefore, y and t subunits are
identical in the first two-thirds of their sequence and structure. As a consequence, the N-
terminal domain of x acts akin to the y subunit, and the unique C-terminal domain of x
has a specialized function in coordinating the holoenzyme at the replication fork
(Dallmann et al., 2000). Due to the requirement of the x subunit for dimerization of the
pol III core, the DnaX complex associated with pol III holoenzyme most likely has a


4
stoichiometry of T2T1 (Pritchard et al., 2000). Each of the other subunits (8,8,x and ?) is
present in a single copy in this clamp loader (Onrust et al., 1995). For in vitro
reconstitution of the DnaX clamp loader, the % and f subunits bind the y subunit through
y-y interaction (Glover and McHenry, 2000), and greatly increase the affinity for the 88
subunits for the complex (Olson et al., 1995). The structure of the E. coli clamp loader
recently also revealed existence of a trimer of y (DnaX) subunits (Jeruzalmi et al.,
2001a).
Clamp Loader Subunit Functions
The x subunit interacts with single-stranded DNA binding protein (SSB) at the
replication fork (Glover and McHenry, 1998). This x-SSB interaction is thought to be
involved in a primase-to-polymerase switch prior to formation of preinitiation complexes
(Yuzhakov et al., 1999). The \p subunit has no known function other than forming a
structural bridging contact for x to the clamp loader through v|/ interaction with the y
subunit (Glover and McHenry, 2000; Xiao et al., 1993b). The function of the x-SSB
interaction is well characterized, however there is no known function for the x and f
subunits directly in loading the clamp in DNA.
It is the 8 subunit that binds to, and alone has the ability to open, the p clamp
(Jeruzalmi et al., 2001b; Leu et al., 2000). The surface of 8 subunit which binds to p is
concealed by the 8 subunit when the clamp loader is in an inactive state (Jeruzalmi et
al., 2001a). The x and y subunits transduce the energy from ATP binding and hydrolysis
into mechanical work within this clamp loading machine to load the clamp on DNA
(Bertram et al., 2000; Onrust et al., 1991).


5
All of the subunits of the clamp loader, except x and f are members of a diverse
superfamily of AAA+ (ATPases Associated with a variety of cellular Activities)
molecular motors (Neuwald et al., 1999). This AAA+ superfamily contains conserved
sequences and structures for nucleotide binding and hydrolysis that drive conformational
changes in these motor proteins. In the DnaX clamp loader ATP binding and hydrolysis
by the x (i.e., through the N-terminal y-region) and y subunits promote conformational
dynamics that modulate the binding affinity for the (3 clamp as well as DNA for the
loading reaction. The 5 and 8 have been identified as AAA+ superfamily members
based on sequence and structural homology of 8, and structural homology in the case of
8, however, neither has the ability to bind or hydrolyze ATP, although they are believed
to actively participate in the conformational dynamics of the clamp loader for the loading
reaction (Jeruzalmi et al., 2001a; Podobnik et al., 2003).
The Mechanism of p Clamp Loading
Figure 1-1 depicts a simplified schematic of the clamp loading mechanism (under
study in this dissertation) for processive DNA synthesis by polymerase III. Initially, ATP
binds to the clamp loaders x and y subunits. Binding of up to three ATP molecules
causes conformational changes in the x and y subunits, and therefore changes the overall
structure of the clamp loader. Although the exact nature of these ATP-dependent
conformational changes is not yet known, there are two major consequences following
the conformational changes. The P clamp interaction surface of the 8 subunit becomes
exposed, and a DNA binding surface on the clamp loader either forms or becomes
exposed Interaction of the 8 subunit with p has been biochemically and structurally
characterized, showing that 8 induces a conformational change in the p subunit that


6
A. B.
Primed-Template DNA conformational
changes
Figure 1-1. Schematic of the p clamp loading reaction for processive DNA synthesis. A)
p clamp, and the DnaX clamp loader are present with a primed-tempiate DNA
substrate. B) Binding of ATP increases the affinity of the clamp loader for
both p and DNA by conformational changes producing exposure of the 8
subunit-p interaction surface, and formation of a putative DNA binding
surface. Interaction of 8 with p induces conformational changes in the clamp
that open it at a single interface. C) Primed-tempiate DNA triggers ATP
hydrolysis and rapid dissociation of the clamp loader from the loaded clamp.
D) DNA Polymerase then binds the same surface of p that was previously
occupied by the clamp loader. E) Incorporation of deoxyribonucleotide-
triphosphates (dNTPs) by polymerase then proceeds without polymerase
dissociation from the template.
triggers opening at a single dimer interface (Jeruzalmi et al., 2001b; Turner et al., 1999).
The nature of the putative DNA binding surface on the ATP-bound clamp loader remains
unknown. However, it is clear that ATP binding is required for the clamp loader to bind
DNA, and further that the clamp loader preferentially places p at the 3-end of the primer
on a replication-proficient template (Ason et al., 2003). Binding primed-tempiate DNA
triggers the clamp loader to hydrolyze ATP. ATP hydrolysis causes release of the clamp


7
loader from (3, which then tightly closes on DNA when the 8 subunit-interaction is
removed. The discharged clamp loader is then in some inactive state at this point in the
cycle, and most likely must undergo some as yet undefined conformational changes in
order to continue loading clamps. Immediate removal of the clamp loader from the
loaded clamp is essential since the clamp loader and the polymerase a subunit share the
same binding surface on the clamp. Once bound to its clamp, polymerase can
processively replicate thousands of nucleotides without further dissociation from the
template.
Importance of the E. coli Model Replication System
In E. coli, the clamp loader and (I clamp are utilized in DNA replication from
initiation at the origin to DNA partitioning of parent and daughter chromosomes upon
termination (Katayama, 2001; Levine and Marians, 1998). Aside from its interaction
with pol III core, p clamp has been found to interact with all five E. coli DNA
polymerases, as well as several other partner proteins in DNA metabolism such as the
MutS, UvrB, and DNA ligase (O'Donnell and Lopez de Saro, 2001; Tang et al., 1999).
The p clamp and DnaX clamp loading machine subunits are functionally conserved
across evolution to all branches of life suggesting a fundamentally similar mechanism for
processivity in all DNA metabolism (Ellison and Stillman, 2001). Increasing structural
analyses also reveal that the composition of these clamps and clamp loading machines
from such organisms as diverse as a bacteriophage and a human are remarkably similar,
and that all clamp loaders utilize AAA+ motor proteins (Davey et al., 2002). Using E.
coli as a model replication system has been outstandingly valuable for the determination
of how complex multi-protein machines interact and work together in a well-regulated
efficient manner for DNA replication. It is important to determine how the individual


8
protein subunits of these complex biological machines carry out their own functions and
communicate with each other for completion of their tasks. Additionally, understanding
of these mechanisms will allow for a more complete comprehension of the important
DNA metabolic tasks in which they are involved.
The General Problem Under Study and Research Questions Addressed
The general problem under study in this project is the many remaining unknown
and questionable aspects of the mechanisms by the clamp loader for p clamp loading and
the results and interpretations of other investigations. Conformational changes in the
clamp loader are a requirement for modulation of clamp loading. However, the kinetics
of clamp loader conformational changes are unknown. Different nucleotide-dependent
clamp loader conformational species have been identified in proteolytic digestion
experiments, but how their abundance and dynamics control the loading reaction cycle is
not understood mechanistically. How do the nucleotide-dependent conformational
dynamics within the clamp loader subunits drive this machine for clamp loading?
Chapter 4 of this dissertation details an investigation that addresses this question. The
studies describe the existence of distinct conformational species of the clamp loader and
the kinetics of their activity in the clamp loading mechanism. Chapter 4 further addresses
the questions concerning how p and ATP affect the kinetics of clamp loader
conformational changes. An additional key question asked in chapter 4 is that of the
nature of nucleotide binding to the clamp loader, and whether there is interdependency
between ATP binding and the conformational dynamics. Computer modeling was
applied to the experimental data for analysis of these important questions.
Sliding clamps of all organisms studied to date are known to enhance the activity of
their respective clamp loader. How the p clamp affects the y complex clamp loader for


9
promotion of its own loading onto DNA was tested in this research project. Although it
is understood that P increases the ATP hydrolysis energetics of the clamp loader, it
remains unknown what the kinetic mechanism of this enhancement may be. By
comparison of y complex with the 7388 minimal clamp loader in chapter 5, along with
experiments presented in chapter 4, this dissertation addresses how p clamp affects the
specificity and stability of nucleotide binding by the clamp loader, and therefore the
kinetics of the conformational dynamics during the clamp loading reaction for its
enhancement.
In comparison to the AAA+ clamp loading machines of other organisms outlined in
chapter 2, the E. coli clamp loader contains two additional subunits (x and y). It is
known that x and y assist in the assembly of other subunits in the clamp loader, and the x
subunit has a role in primase-to-polymerase switching at the replication fork, but do they
serve any direct function in the clamp loading mechanism by y complex? Chapter 5 of
this work outlines a detailed characterization of the 7388 minimal clamp loader. This
minimal complex is missing the x and y subunits, and is used to assess their roles in the
clamp loading mechanism by direct experimental comparison to 7 complex. Are the x
and y subunits AAA+ adaptor proteins of 7 complex? Do they provide an example of
a novel adaptor function through their effect on the conformational dynamics of the
clamp loader? Although not originally hypothesized, the findings presented here along
with findings presented elsewhere suggest that they are.
Finally, what can the details learned here about the E. coli clamp loading machine
tell us about other molecular machines driving and regulating complex and diverse
cellular tasks in other organisms? This machine is essentially a molecular switch that


10
catalyzes a fundamental reaction in all forms of life. Can the knowledge gained through
study of this clamp loading machine be used for development of novel biological or
hybrid molecular machines?
Conformational Dynamics of the Clamp Loading Machine
Solution of the X-ray crystal structures of a minimal 7388 clamp loader complex
and a C-terminal truncated y subunit has recently given information revealing some of the
structure-function relationships of the DnaX clamp loading machine (Jeruzalmi et al.,
2001a; Podobnik et al., 2003). The subcomplex of (73818*1) forms a minimal complex
with clamp loading activity similar to the complete DnaX clamp loader (Onrust et al.,
1991). Each of the y3,8, and 8 subunits is a AAA+ superfamily member, and is
composed of three structural domains that form C-shaped molecules. The N-terminal
domains-I and II encompass the conserved nucleotide binding site (in y subunits only),
and the third C-terminal domain-111 forms an oligomerization region for this
heteropentameric complex. Domain-11 generally forms a mobile hinge between
domains-I and III. ATP binding and hydrolysis by the y-AAA+ motor subunits directly
trigger conformational changes in these subunits. Although the 8 and 8 subunits are
AAA+ proteins, they do not have the ability to bind and hydrolyze ATP, but most likely
do undergo conformational changes caused by y subunit conformational movements. The
structure of the 7388 clamp loader complex showed that the complex was arranged as a
heteropentameric ring through the C-terminal oligomerization domain, and that there was
extensive contact between each of the five subunits extending down in asymmetric
orientation from the oligomerization domain. The nucleotide binding sites were found in
the interfaces between the 8*-yi, 71-72, and 72-73 subunits. At least one and possibly two
of these interfacial nucleotide binding sites is thought to be constitutively open whereas


11
the third is deeply buried in an interface. This lead to a model describing a sequential
series of individual y-subunit conformational changes whereupon ATP binding to an
open interface caused a conformational change opening the adjacent interface and so on
(Jeruzalmi et a!., 2001a), where the status of ATP binding to the nucleotide binding sites
is communicated between the subunits of the clamp loader modulating its affinity for p
clamp and DNA.
There are several limitations to the mechanistic inferences made in light of these
structures. The clamp loader structure was determined in the absence of nucleotide.
Therefore, the observed subunit interactions most likely do not represent the functional
complex, as it would appear in the cell, and the overall conformation was thought to be
largely due to a crystal-packing artifact. The truncated-y subunit structure was
determined in the presence of non-hydrolyzable adenosine 5-0-(3-thiotriphosphate)
(ATPyS), and crystallized as a tetramer (not as it would appear in a functional complex).
The structure contained electron density consistent with ATPyS molecules bound to two
protomers, ADP bound to a third, and no nucleotide in the fourth. ATPyS is sufficient for
both p clamp and DNA binding by the clamp loader but does not confer clamp loading
activity in biochemical assays (Bloom et al., 1996). Therefore, the ATPyS-bound y
subunit structure revealed important features of the conformational change in this motor
subunit. However, the y subunit was truncated and a substantial portion is missing from
its C-terminus. Only the conserved AAA+ nucleotide binding domains-I and II were
present. This means that the different conformations observed in each of these structures
may be considerably different than the actual structure in solution, or within the cell. The
experiments presented in this dissertation do not address structure specifically, however


12
these in vitro studies do directly address the kinetics of the conformational dynamics of
the clamp loader in solution conditions during real-time clamp loading reactions.
There are significant nucleotide-dependent conformational changes in the clamp
loader that must be made during the clamp loading reaction for intersubunit
communication modulating p clamp and DNA binding. Currently, only proteolytic
protection assays (Hingorani and O'Donnell, 1998) have provided biochemical proof of
the nucleotide-dependent conformational changes, however there is no kinetic detail
known regarding the nature of these conformational changes during the clamp loading
reaction. It has been previously hypothesized that the rate-limiting step of the clamp
loading mechanism occurs in the clamp loader, away from DNA (Bloom et al., 1996),
and could be ADP-release or additional conformational changes that reset the clamp
loader for resuming the clamp loading cycle. The results of this dissertation address the
kinetics of the nucleotide-dependent conformational dynamics of the clamp loader and
add detail to what is known about the clamp loading mechanism, initially asking, how do
ATP-dependent conformational dynamics drive and regulate this molecular machine for
clamp loading?
The kinetics of nucleotide binding and hydrolysis by the clamp loader, DNA
binding, and p clamp loading have been determined using several biochemical analyses
in this project. Together, these explorations have lead to a hypothesis based on
conformational dynamics for the internal workings of the clamp loading machine. Here,
it is proposed that the clamp loader exists, dominantly, in two distinct conformational
states that are either activated or inactive for clamp and DNA binding when in
equilibrium with ATP. This project has addressed the kinetics of the ATP-dependent


13
conformational changes separating the two clamp loader species in the clamp loading
mechanism, and additionally shows that there is complexity in the conformational
dynamics between these two major states. This added complexity probably arises due to
the proposed asymmetry in the three nucleotide binding sites, and the possibility of
having differential affinities for ATP within these sites causing a complex mixture of
conformational intermediates within the clamp loader. Elucidation of the nature of the
additional mixture of conformational states will require further study, but overall this
project reveals, kinetically, higher populations of two major states modulating the
function of the clamp loader in the p clamp loading mechanism.
Enhancement of the Clamp Loading Machine by its Clamp
It has been known for some time that the P clamp enhances, but does not trigger,
the ATP hydrolysis activity of the clamp loader in the presence of DNA (Onrust et al.,
1991). This feature of a sliding clamp enhancing the DNA-dependent ATP hydrolysis
activity of its clamp loader is a general characteristic in bacteriophage (Pietroni et al.,
2001), eukaryotic (Yoder and Burgers, 1991), and archaeal organisms (Oyama et al.,
2001); thus it is a fundamental attribute in all clamp loading mechanisms. However, the
mechanism of how a clamp enhances the activity of its clamp loader has remained a
mystery. Part of the originally proposed research project was to determine how the E.
coli p clamp affected the kinetics of ATP hydrolysis by the clamp loader. This
dissertation addresses the mechanism of P clamp enhancement of clamp loader DNA-
dependent ATP hydrolysis, and reveals several important findings interrelated with
nucleotide binding and the conformational dynamics of the clamp loader. It comes as no
surprise that the mechanism of p clamp enhancement of ATP hydrolysis activity is
related to a conversion of the equilibrium between the dynamic conformational states of


14
the clamp loader. It is hypothesized that the p clamp converts the conformational
equilibrium of the clamp loader with ATP into a completely active population (i.e., a
complex of ATP-bound clamp loader and P). An extension of this hypothesis is that the p
clamp affects the affinity and specificity of the clamp loader for ATP, effectively
trapping the nucleotide within the clamp loader promoting formation of the active p-
bound complex poised for loading onto DNA. Selective binding and trapping the active
clamp loader conformation may also stabilize a putative DNA binding surface on the
clamp loader as well. An increase in the apparent concentration of this active complex
poised for clamp loading would provide a direct mechanism for the increase in ATP
hydrolysis turnover rate observed in kinetic assays. Due to the considerable evolutionary
conservation of clamp loader function across all branches of life (Davey et al., 2002), the
hypothesized mechanism of clamp enhancement of clamp loader activity presented in this
dissertation could also apply to clamp loading for processive DNA synthesis in all other
organisms as well.
The x and y Subunits are Required for Optimum Activity of the Clamp Loader
The x and \p proteins do not bind DNA, do not bind or hydrolyze ATP, and are
dispensable for clamp loading activity. At the replication fork in vivo, the x subunit is
involved in an important interaction with SSB, and a primase-to-polymerase hand-off
through association with the clamp loader, but not directly involved in preinitiation
complex formation by the clamp loader. A subcomplex of the clamp loader (7388), of
which the crystal structure was determined, does not contain the % and \p subunits, yet
maintains nearly the same ATP hydrolysis and clamp loading activity as the clamp loader
with x and y (Onrust et al., 1991). Although the structure of the x-y dipeptide is known,
it is still a mystery where it binds to the clamp loader. For these reasons the 7388 clamp


15
loader has been called the minimal clamp loader complex, and in some cases, it has been
called the clamp loader itself.
This dissertation encompasses a detailed biochemical characterization of the (7368)
minimal clamp loader, and comparison of its activities to the 7 complex clamp loader.
This work shows that there are in fact several mechanistic distinctions between the
minimal clamp loader and 7 complex clamp loader. These differences have generally
revealed that the minimal complex is slightly hindered in its nucleotide binding and
hydrolysis properties and hence clamp loading.activity. Therefore the % and y subunits
are required for optimum clamp loading activity, conceivably by strengthening the
intersubunit communication within the clamp loading machine. Previous studies
revealed that the % and y subunits did in fact stabilize the clamp loader by increasing the
affinity of x and 7 subunits for 88 (Olson et al., 1995). One of the most obvious
deficiencies in minimal complex function is the loss of the ability to stabilize ATP
binding in an active complex with p clamp. This is hypothesized to be the result of
possibly slower conformational changes in the minimal complex, and perhaps looser
conformational dynamics in the minimal complex leading to more conformational
complexity (i.e., more inactive intermediate conformational states) between the major
two states hypothesized for the clamp loader. This research shows that the population of
inactive conformations of the minimal complex is several-fold greater than the population
of active conformation in equilibrium with ATP. p clamp does sustain the ability to
enhance the ATP hydrolysis activity of the minimal complex. However, the results of
kinetic analysis of ATP hydrolysis activity reveal that this enhancement is less than that
of 7 complex, and that p must do more work to convert the large population of inactive


16
conformational states of the minimal complex into the trapped active conformation. To
date, there is no other steady-state and time-resolved biochemical analysis comparing y
complex to the minimal complex in such a comprehensive manner as that presented in
this dissertation, and the results have allowed a much more clear appreciation for the
clamp loading mechanism catalyzed by the DnaX clamp loader.
The clamp loader is a AAA+ protein containing machine. The x and y subunits are
structurally unrelated and much smaller than the AAA+ subunits of the clamp loader.
Since the x and >(/ subunits seem to be unique to the E. coli clamp loader, and most likely
other gram-negative bacteria (Xiao et al., 1993a; Xiao et al., 1993b), they provide some
unique functions in these prokaryotic clamp loaders compared to those of other
organisms. They present a form of substrate specificity to the clamp loader for the SSB-
coated lagging strand at the replication fork for assistance in preinitiation complex
formation for Okazaki fragment synthesis. These characteristics of the x and vp subunits
associate them with a new and growing list of AAA+ adaptor proteins. AAA+ adaptor
proteins provide a simple and effective way for modulation of AAA+ machine function,
giving the machine better control over substrate specificity and rapid redirection of its
specific activity (Dougan et al., 2002). Along with their function at the replication fork,
the x and vp subunits are proposed here to provide increased structural stability and
facilitated conformational changes for intersubunit communication within the AAA+
clamp loading machine. It is thus hypothesized that they are in fact AAA+ adaptor
proteins and reveal a novel adaptor function for this newly defined subset of proteins.


17
Application of the Analyses of the Clamp Loading Machine to Other Complex
Molecular Machines
Like the clamp loader, there are many other enzyme complexes that perform
complicated biological tasks such as assembly and disassembly of new protein-protein
complexes, protein-cofactor complexes, and protein-DNA complexes. Orchestration of
protein-protein and protein-nucleic acid interactions is likely to utilize converging
mechanisms to drive the diverse functions of these multi-protein enzyme complexes. The
intersubunit conformational communication observed structurally and functionally by the
clamp loaders drive several essential jobs within the large replication complexes in all
forms of life. The clamp loader catalyzes assembly of a new protein-nucleic acid
complex without making or breaking any covalent bonds of either component. The
energy for this assembly is derived from within the clamp loader by ATP-dependent
conformational changes that essentially switch the clamp loader between on and off
states. Understanding of subunit recognition and conformational communication driving
similar switching mechanisms in molecular machines is a complicated task in science,
but explorations such as that presented in this dissertation for the y complex clamp loader
could help accelerate other investigations. Not only for ATP- and GTP-utilizing energase
enzymes, but also for transmembrane transport machinery, cellular cargo-transport
motors, chaperone and protein modulating complexes, proteases, etc.
The investigations outlined in this dissertation are a highly focused and detailed in
vitro examination of the mechanism of a molecular machines mechanism of switching
between active and inactive states for assembly of a protein-DNA complex at the expense
of energy from ATP binding and hydrolysis. The details learned here about protein
conformational changes and communication of these changes within the machine could


18
be applied to other solution-based structure-function studies for a broad range of proteins.
A key result of this and prior work shows that the information for conformational
changes in these proteins is stored in the structure of the proteins themselves, akin to the
storage of protein folding information encoded within secondary and tertiary peptide
structure (Fersht and Shakhnovich, 1998). For example, the P clamp is not simply an
unintelligent ring-shaped protein dimer, but contains structural information for release of
a spring-like trigger for opening of one of its sturdy interfaces (Ellison and Stillman,
2001). In the clamp loading machines, nucleotide-dependent intersubunit communication
induces the extraction of intramolecular-stored information for subunit conformational
changes that, in turn, can induce changes in partner proteins.
Molecular motors and switching mechanisms are common in many energase
enzymes other than the AAA+ proteins that make up the clamp loader under study here.
For example, several molecular motors move along the cellular architecture of
microtubule and actin cytoskeletal networks (Mehta et al., 1999; Vale, 2003). These
motors power transport of organelles and other cargo throughout the cell, drive
chromosomal segregation in cellular division during mitosis, give cells the capability of
motility, and power the contraction of muscle fibers that give animals the extraordinary
strength needed for a diverse range of movement, and let their hearts beat for entire
lifetimes (Vale and Milligan, 2000). The machines powering these processes are not
much unlike the clamp loading machine detailed in this dissertation. They each generally
contain several domains including nucleotide binding sites, protein-protein and protein-
nucleic acid binding regions, hinge-like domains, multisubunit oligomerization domains,
and regulatory subunit or cofactor interfaces (Vale and Milligan, 2000). The


19
conformational movements made by several of these nucleotide dependent energases
drive their functions, give them processive activity, and allow them to generate molecular
power that is matched on a relative scale only by mans largest macromolecular machines
(Baker and Bell, 1998; Ellison and Stillman, 2001). Several molecular switches also act
through conformational dynamics allowing protein-protein and protein-nucleic acid
interactions in signal transduction cascades (Franco et al., 2003; Phillips et al., 2003),
chromatin structural modification (Hakimi et ah, 2002), transcriptional and translational
regulation (Mazumder et ah, 2003), and ion channels driving action potentials for
neuronal signaling. The ras-GTPase activating protein provides an example of protein-
protein contact directly related to the subunit interfaces within the clamp loader. A
conserved arginine-finger residue is positioned between two proteins for catalysis of
nucleotide hydrolysis, inducing conformational changes that switch the activity status of
the protein (Ahmadian et ah, 1997). Nearly all cellular processes are driven or regulated
in some way by phosphorylation / dephosphorylation mechanisms, but the activities of
energase molecular motors and switches (Purich, 2001) also appear to have great
importance in maintaining cellular function in all organisms. Therefore the significance
of investigating the inner workings of a molecular machine such as the E. coli clamp
loader extends far beyond the understanding of the mechanisms of DNA metabolism.
Novel Hybrid Devices Based on the Clamp Loader and Sliding Clamp
Understanding of the mechanism of the clamp loader molecular machine;
structurally and functionally how protein subunits come together and communicate with
each other based on nucleotide binding status can be exploited for development of novel
molecular hybrid devices. In even simple terms, the clamp loader is a molecular switch,
activated by ATP binding, and inactivated by DNA-dependent hydrolysis of ATP. The


20
inactive and active states of this molecular switch are driven by conformational changes,
and further affected by binding a partner protein (the clamp). Essentially, the reaction
mechanism under study in this dissertation is a molecular switch that entails placement or
removal of (protein) rings on (nucleic acid) rods or hoops. This activity produces a
topological connection for another machine (polymerase) to the rod or hoop structure,
essentially increasing its concentration at the preferred catalytic site (template DNA).
The clamp loader switching mechanism could be utilized in other systems that
require regulated activation/deactivation or in systems such as memory or
communications devices that require a simple (binary) on/off signal. By modification of
the protein properties of the clamp loader subunits it is feasible that this energase
machine/switch could be custom-tailored for guided assembly of proteins (or other
materials) other than the clamp, onto specific substrates in order to control the putative-
complex concentration, and activate or deactivate it specifically through NTP binding and
hydrolysis.
Off-the-Wall Example of the Clamp and Clamp Loader in a Novel Device
There is a need for understanding and improvement of industrial catalysis systems
(Coontz et al., 2003). Briefly, for homogenous catalysts, where the molecular catalyst
and reactant are dispersed in the same phase, there exists a seemingly simple problem of
separation of the reactants and catalysts from products. The development of
heterogeneous catalysts have provided a solution by direct separation of catalyst and
reactant in different phases, but in most cases do not provide the surface area for cost-
efficient devices. One could envision use of a clamp loading machine to place (and
remove) a ring-shaped clamp, either with the ability to bind a specific catalyst, or
containing the catalyst itself, onto the reactant. This process would allow a substantial


21
increase the heterogeneous catalytic surface area on an insoluble nano-sized rod- or hoop
shaped reactant. Perhaps the products would easily be separated in solution or gas
phases. These devices would be smart devices where the catalytic components contain
the information needed for their assembly and disassembly for disposal or recycling. For
example, a spring-loaded clamp that can be placed and removed at will by a specialized
machine with easily regulated activity. Improvement of industrial catalysts is only a
single (imaginative) example of how the functional knowledge about sliding clamps and
clamp loading machines could be used for design of a novel hybrid device. With
imagination and lots of money, these and other, perhaps more significant biologically
functional devices could be developed.
Design of Research Project
This research project was developed on two fluorescence-based experimental
methodologies for investigation of the kinetics of DNA dependent ATP hydrolysis and p
clamp loading activities of the y complex clamp loader and the y388 minimal clamp
loader complex. The two experimental methods utilized were an anisotropy binding
assay, and an E. coli phosphate binding protein-based MDCC-PBP ATP hydrolysis assay.
The anisotropy binding assay was employed for investigation of the dynamics of the
interactions between the clamp loader and fluorescent-labeled DNA or fluorescent-
labeled p clamp. Studies of the DNA dependent ATP hydrolysis kinetics of the clamp
loaders were performed with the MDCC-PBP ATPase assay. Both methodologies
allowed for steady-state and time-resolved measurements of clamp loading kinetics.
For the anisotropy binding assay, depolarization of the emission from a fluorescent
probe reports on the rotational dynamics of the fluorescent-labeled species (Lakowicz,


22
1999). By monitoring changes in anisotropy, one can measure binding dynamics and
observe intermediates that arise in real time during a given reaction. This enables
elucidation of the kinetics of discrete steps involving interactions with the labeled species
in a reaction mechanism. To study binding kinetics of the clamp loader with (3 clamp as
well as the binding kinetics of the clamp loader with DNA in the presence and absence of
P clamp on steady-state, and pre-steady-state time scales, the fluorescence depolarization
of X-rhodamine-labeled DNA (RhX-DNA) or pyrene-labeled p clamp (p^1') was
measured.
ATP hydrolysis kinetics were measured with the MDCC-PBP ATPase assay. This
assay utilizes a site-specific mutant of E. coli phosphate binding protein (phoS gene
product) that allows covalent attachment of an environmentally sensitive fluorescent
probe (N-[2-( 1 -maleimidyl)ethyl]-7-(diethy lamno)coumarin-3-carboxamide, MDCC)
near the phosphate binding cleft (Brue et al., 1994). Binding of inorganic phosphate (P¡)
product (i.e. from ATP hydrolysis by the clamp loader) to the labeled-phosphate binding
protein is both rapid and tight, and produces a substantial increase in fluorescence
intensity readily measurable in a fluorimeter or stopped-flow real-time detection system.
Computer modeling was performed using two different programs for simulation
and fitting of experimental data for investigation of the clamp loader conformational
change mechanism. The KinTekSim program (Barshop et al., 1983; Dang and Frieden,
1997) was used to test model reaction mechanisms by simulation and visual comparison
to the experimental data. A fitting program (DynaFit) was used to fit the experimental
time course of the reaction with a least-squares regression methodology (Kuzmic, 1996).


23
Computer modeling of the reaction kinetics provided a way to validate, and predict
several of the mechanistic features determined in this project.


CHAPTER 2
LITERATURE REVIEW
DNA Replication in Escherichia coli
A complex assembly of DNA replication proteins forms at each replication fork for
synthesis of a nascent Escherichia coli chromosome. At the leading edge of the
replication fork is a topoisomerase, an enzyme that relieves torsional stress created by
duplex DNA unwinding. Opening up the replication fork and priming the leading and
lagging strand templates is a machine called the primosome. The primosome is made up
of two proteins, DNA helicase, which unwinds parental DNA, and primase, which
synthesizes RNA primers, the sites of initiation of replication of the parent templates.
Stabilizing the single strand template DNA that is exposed upon unwinding by the
helicase is single-stranded DNA binding protein. Associated with the primosome is a
complex of enzymes that synthesize and proofread the nascent DNA. This machine is
DNA polymerase III holoenzyme, a dimer of the synthesizing and proofreading units that
coordinate simultaneous high fidelity elongation of the leading strand, and Okazaki
fragments on the lagging strand of nascent DNA. The dimeric DNA polymerase III is
physically linked to the parental template leading and lagging strands by ring-shaped
sliding clamp processivity proteins, such that it cannot easily dissociate from the
template. A clamp loading machine, located within dimerized DNA polymerase III
utilizes the energy of ATP binding and hydrolysis to load the circular sliding clamp on
the leading strand template and on each Okazaki fragment of the lagging strand template.
DNA polymerase I and DNA ligase follow at the tail end of the replication fork, where
24


25
DNA polymerase I is poised to excise the RNA primers and replace them with DNA, and
DNA ligase seals the remaining nicks produced between the Okazaki fragments on
lagging strand DNA.
As a whole, this assembly of replication machines has been termed the replisome.
The mass of the replisome approaches 1 million Daltons, and together, two replisomes
move along the parental chromosome in opposite directions simultaneously replicating
the nascent daughter chromosome from a single point of initiation and meeting at the
point of termination. This process of replicating of the entire chromosome lasts about 1
hour for a pair of replisomes; however, under some conditions dichotomous replication of
the chromosome can occur reducing the time of chromosomal replication to nearly 20
minutes. Up to six replisomes, at six separate replication forks, can be simultaneously
synthesizing three daughter chromosomes from the single original parental chromosome
during this process.
DNA Polymerase III Holoenzyme
At the heart of the replisome is the DNA polymerase III holoenzyme (pol III
holoenzyme). DNA polymerase III was identified as the essential polymerase for E. coli
chromosomal replication, distinct from the two previously discovered activities of DNA
polymerase I, and II by analysis of mutants temperature sensitive for DNA synthesis and
for cell viability (Gefter et al., 1971; Hirota et al., 1972).
Pol III holoenzyme replicates DNA in a semidiscontinuous manner. Pol III
holoenzyme is capable of megabase processivity for continuous synthesis of the leading
strand, and responsible for controlled 1 2 kilobase Okazaki fragment synthesis of the
lagging strand. Pol III holoenzyme performs these activities simultaneously at speeds
approaching 1 kilobase per second, and has an extraordinary fidelity of a single


26
nucleotide misincorporation in 1 x 109 nucleotides polymerized (Komberg and Baker,
1992).
Through purification and biochemical characterization of DNA polymerase III and
accompanying proteins found to be involved in synthesis elongation, the individual
subunits of this machine began to be identified. Pol III holoenzyme consists of 10
distinct subunits: a, e, 0, t, y, 8, S, x, V, and |3. Five of these subunits are present in two
copies bringing the total number of pol III holoenzyme subunits to 20.
The core of pol III holoenzyme (pol III core) was later purified and resolved into
individual subunits: a, e, 0 (McHenry and Crow, 1979). The a subunit encoded by the
dnaE gene (M, = 140,000 Daltons), was found to be the subunit responsible for catalysis
of 5-3 DNA synthesis activity (Maki et al., 1985; Spanos et al., 1981). The e subunit
encoded by the dnaQ gene (Mr = 25,000 Daltons) is the 3-5 exonuclease or
proofreading subunit (Scheuermann and Echols, 1984). The 0 subunit encoded by the
holE gene (Studwell-Vaughan and O'Donnell, 1993) (Mr = 10,000 Daltons) is tightly
bound to a and e in pol III core, but a 0-specific function has not been identified. The a
subunit was further characterized after overexpression and purification of the dnaE gene
product (Maki et al., 1985). Gap-filling replication assays and complementation assays
were performed to show that the a subunit was responsible for the 5-3 polymerase
activity of pol III holoenzyme, and that elevated levels of a in vivo did not increase the
amount of pol III holoenzyme in the cell
Within pol III core, the a and e subunits are tightly bound and complement each
others function with the overall effect of increasing the fidelity of chromosomal
replication. Using purified a and e subunits, (Maki and Komberg, 1987) showed that a


27
complex formed of a-e had increased 5-3 polymerase activity over a subunit alone, and
highly increased 3-5 e-exonuclease activity. The affinity for DNA of the a subunit in
pol III core resulted in an increase in apparent affinity of £ for the 3 -hydroxyl terminus,
thus stimulating the exonuclease activity. In the cell, this proofreading activity during
synthesis was found to represent a 5-fold stimulation compared to exonuclease activity
uncoupled from synthesis which suggested that the fidelity of DNA replication may be
controlled by the relative abundance of the a and e subunits.
Polymerase III core is not processive in DNA replication whereas polymerase III
holoenzyme is highly processive. Using a kinetic assay, along with product size
determination assays it was originally shown that pol III holoenzyme was processive over
thousands of nucleotides, and pol III core had distributive activity, capable of
synthesizing 10-30 nucleotide stretches only (Fay et al., 1981).
It was found that pol III holoenzyme could be isolated from cells in two distinct
forms other than pol III core, each form catalyzing synthesis of a characteristic length of
product DNA (Fay et al., 1982; LaDuca et al., 1983). Polymerase IIP was the first of
these smaller forms of pol III to be purified. At the time pol III was purified and
characterized, the x subunit was discovered and predicted to dimerize the pol III core
assemblies forming pol IIP (McHenry, 1982). The processivity of pol IIP was increased
6-fold over pol III core, and pol IIP exhibited greater ability in synthesizing long single-
stranded templates coated with spermidine, a polybasic amine that stabilizes the helical
structure of DNA. The Polymerase III* form was also purified, and contained all of the
same subunits found in pol III holoenzyme except the p subunit-dimer. The activity of
pol III* showed an increase in processivity of at least 20-fold over pol III core (LaDuca et


28
al.; 1983). When pol III* was reconstituted with the (1 dimer, the characteristic
processivity of the holoenzyme was restored, suggesting (3 as the major factor in
conferring processivity on pol III holoenzyme.
Pol III holoenzyme must place the p dimer on primers formed by primase in order
to form preinitiation complexes. The formation of these preinitiation complexes requires
ATP binding and hydrolysis by pol III holoenzyme, and is absolutely required for
initiation of processive synthesis (Burgers and Komberg, 1982). The p subunit binds
directly to the a subunit within pol III holoenzyme (Stukenberg et al., 1991), and pol III
holoenzyme is dimerized by the x subunit by direct interaction also with a (Studwell-
Vaughan and O'Donnell, 1991). Through these interactions, pol III holoenzyme
simultaneously and processively synthesizes DNA. Therefore, preinitiation complexes
must be formed at least once on the leading strand for its continuous synthesis, and many
times on the lagging strand for Okazaki fragment synthesis.
Fragmented synthesis of the lagging strand by pol III holoenzyme is in conflict
with the level of processivity gained through binding the p processivity subunit. The
polymerase must detach from each completed Okazaki fragment and rapidly cycle to the
next preinitiation complex on the lagging strand (O'Donnell, 1987). During
chromosomal replication, anywhere from 2,000 to 4,000 Okazaki fragments are
synthesized at a rate of approximately 1 per second (Komberg and Baker, 1992). Such
polymerase cycling activity requires precise coordination of protein-protein and protein-
DNA interactions at the replication fork. The coordinated efforts of several subunits,
including processivity proteins within pol III holoenzyme are responsible for this rapid
and ordered activity, and consequentially give the holoenzyme structural asymmetry,


29
while the DNA synthesis activity of the polymerase cores remain symmetric between the
leading and lagging strands.
The T Subunit is the Coordinator of Pol HI Holoenzyme Function and Processivity
The dnaX gene codes for both the i and y subunits of pol III holoenzyme. The full-
length dnaX gene product is the x subunit (Mr = 71,000 Daltons). The y subunit (Mr =
47,500 Daltons) is created by a -1 translational frameshift adjacent to a hairpin-loop
structure in the mRNA that causes a stop codon (UGA) to appear approximately two-
thirds the way through the dnaX gene (Flower and McHenry, 1990; Tsuchihashi and
Komberg, 1990). Therefore, the y polypeptide is identical in sequence with the first two-
thirds of t. This translational frameshift occurs with about 50% yield of x and y
polypeptides. Although they are extensively identical, the larger x polypeptide has
several important functions specific to its C-terminus, which greatly distinguishes it from
y (Gao and McHenry, 2001).
A dimer of x subunits (x) acts as the glue of pol III holoenzyme, holding it
together and providing a structural scaffold for the asymmetric function of holoenzyme
on the leading and lagging strands. A tight interaction between X2 and the a subunit of
core occurs through the C-terminus of x bridging two molecules of pol III core (Kim et
al., 1996b; Studwell-Vaughan and O'Donnell, 1991). The C-terminus of x also binds to
the DnaB helicase, physically linking the replication machine of pol III holoenzyme with
the primosome machine composed of DnaB and primase. The physical and
communications link between pol III holoenzyme and the primosome enhances the DNA
unwinding activity of DnaB helicase and connects pol III holoenzyme to the RNA
priming activity involved in Okazaki fragment length determination and polymerase
cycling on the lagging strand (Dallmann et al., 2000; Kim et al., 1996a). A single DnaB


30
helicase couples with both the leading and lagging strands, and through interaction with
the T-bridged polymerase may help to keep both strands closely associated with the
replication fork.
The r subunit protects the p processivity protein from removal off of the leading
strand indicating a direct role of x in the high processivity of the leading strand (Kim et
al., 1996c). This activity is most likely communicated through the close proximity of x-a,
and p-a binding sites on the C-terminus of a. Within this proximity, x could potentially
contact p providing direct protection from removal, or the x-a interaction may cause a
rearrangement of the P-core complex preventing removal of p. On the lagging strand, x
still protects the p processivity protein, but must be able to switch between protective and
non-protective states to allow polymerase cycling. Two distinct triggers for polymerase
cycling on the lagging strand have been identified (Li and Marians, 2000). The dynamic
action of primase binding the replisome through DnaB helicase and synthesizing a primer
is thought to trigger polymerase cycling, and collision of the lagging strand polymerase
with the 5-end of the previously synthesized Okazaki fragment also is thought to trigger
cycling. With respect to the second case, a processivity switch requiring the x subunit
was identified (Leu et al., 2003; Wu et al., 1992). This x processivity switch is off
during processive synthesis, conferring protection of the P-core interaction with high
processivity for completed synthesis of the Okazaki fragment. The processivity switch is
turned on only upon incorporation of the final dNTP of the Okazaki fragment when the
5-end of the previously synthesized primer is reached. The resulting nick in DNA
activates the processivity switch, and through actions of the C-terminus of x, core is
released from p and allowed to cycle to the next preinitiation complex.


31
Pol III holoenzyme contains the processivity proteins necessary to form
preinitiation complexes at newly primed sites on template DNA. The x subunit is also
part of this complex of processivity proteins that coordinates the protein-protein and
protein-DNA interactions required for preinitiation complex formation. Unlike the
functions of the x C-terminus, x activity in preinitiation complex formation is located in
the N-terminal region of the protein identical to the y subunit. A DnaX-complex forms
within pol m holoenzyme which functions in loading p onto primed template DNA. For
preinitiation complex formation, this clamp loader is discussed in detail below. The
DnaX complex within holoenzyme contains two x subunits (i.e., same two that dimerize
core) in a complex with y, and the 8,8, & ty subunits (stoichiometry: X2y58xv)
(Pritchard et al., 2000). The DnaX complex binds and hydrolyzes ATP forming
preinitiation complexes on leading and lagging strand primers in what may be an ordered
activity. In their report, (Glover and McHenry, 2001) showed that formation of the
leading strand preinitiation complex required ATP binding but not hydrolysis by DnaX
complex, and that ATP hydrolysis was required for formation of the lagging strand
preinitiation complex. Further, a non-hydrolyzable analogue of ATP (ATPyS) caused
removal of the polymerase presumably bound to a lagging-strand template, extending the
knowledge of pol III holoenzyme as an intrinsically asymmetric dimer with
distinguishable leading and lagging strand polymerases.
Structure of the (5 Sliding Clamp Processivity Protein
When the X-ray crystal structure of the fi processivity protein was solved, it was
immediately clear how this homodimer conferred processivity to polymerase III


32
holoenzyme (Kong et al., 1992). The P sliding clamp is composed of two identical
crescent-shaped monomers arranged in head-to-tail fashion (Figure 2-1).
Figure 2-1. The crystal structure of p sliding clamp. Two views of the clamp composed
of crescent-shaped protomers (red / yellow) are shown. A) Front view of p
showing continuous P-sheet structure surrounding an inner core of a-helices.
Asterisks denote the dimer interfaces. Green lines drawn through the left
protomer represent imaginary boundaries between structural subdomains.
From the top of the left (red) protomer, the subdomains are numbered counter
clockwise: 1 (N-terminal), 2 (middle), and 3 (C-terminal). B) Side view of the
p dimer showing the asymmetry of the faces. On the left face is the extended-
loop region to which y complex and pol III a subunit compete for binding.
Images were composed using DeepView / Swiss-Pdb Viewer Ver. 3.7,
http//www.expasy.org/spdbv/, with structural coordinates downloaded from
the Protein Data Bank (Berman, Westbrook et al. 2000),
http/Avww.rcsb.org/pbd/. (PDB code: 2POL)
The p clamp topologically links the polymerase to template DNA in a non-specific
manner providing a tight interaction with DNA while allowing essentially unlimited two-
dimensional diffusion along the template. Several structural characteristics of p clamp
confer its extraordinary ability to provide processivity to DNA polymerase. Each P


33
monomer is formed from three structurally similar domains containing p sheets on the
outside and a helices on the inside. As a homodimer, the six domains form an inner pore
lined with 12 a helices that are surrounded by an essentially continuous p sheet structure.
The axis of the a helices lining the inner pore are almost exactly perpendicular to the
local direction of the phosphate backbone of DNA facilitating rapid movement along the
duplex. For example, the a helices traverse the major and minor grooves so as not to fall
into them. The height of the p clamp is ~ 80 . The width of p clamp (~ 34 ) would
cover about one full turn of duplex B-form DNA, or ~ 10 base pairs. It was shown
biochemically that an 11-base pair primer was required by p to form a productive
preinitiation complex synthesized by pol III (Yao et al., 2000). The diameter of the inner
pore is ~ 35 , enough space to encircle either duplex B-form DNA (~ 20 ) or a RNA -
DNA hybrid duplex (i.e. a RNA-primed DNA template) (~ 21 ). The extra space within
the inner pore, between the clamp and DNA, is expected to be filled by one or two layers
of water molecules.
The two faces of the p clamp are asymmetric (figure 2-1B). One face has extended
loop C-terminal structures, and the other face is essentially flat giving the clamp an
overall shallow cone-shaped thickness. The extended loops on the extended C-terminal
face contain the binding sites for both the DnaX clamp loader and the a subunit of pol III
core. The presence of overlapping binding sites for both the clamp loader and
polymerase determine the proper orientation of the p clamp when loaded onto primed-
template DNA and result in competition for the binding sites. DNA mediates this
competition for overlapping binding sites. In the absence of DNA, p clamp binds the


34
clamp loader, but when p is loaded onto DNA, the DNA causes the clamp loader to lose
affinity for the clamp, and polymerase binds (Naktinis et al., 1996).
Calculation of the electrostatic field generated by the p clamp revealed that the
protein has an overall negative electrostatic potential. However, the surface lining the
inner pore has a focused positive electrostatic potential, precisely where the negatively
charged DNA molecule would be. The calculated electrostatic features suggest that the
inner pore of the clamp has inherent affinity for DNA. This electrostatic interaction is
most likely lubricated by water molecules allowing a great degree of freedom for linear
diffusion while still maintaining some affinity for DNA.
The dimer interfaces appear as a continuation of the p sheet structure from the outer
surface of each monomer across a molecular boundary, and contribute at least four
hydrogen bonds at each interface. In addition, two distinct sets of interactions between
neighboring a -helical side chains stabilize the p dimer interface. A small hydrophobic
core is formed in the center of the interface by the packing of Phe and lie side chains with
lie and Leu side chains between monomers. Six potential intermolecular ion pairs
between Glu, Arg, and Lys side chains surround the small hydrophobic core. The P
dimer consequentially is very stable with a monomer-dimer dissociation constant of <60
pM, and a half-life on closed-circular DNA of ~ 100 minutes (Yao et al., 1996).
Considering that the cellular concentration of p is -250-500 nM, no monomeric p would
be present in vivo (Komberg and Baker, 1992). The number of specific and potentially
strong interactions at the dimer interfaces underscores the requirement for a clamp
loading machine to load this clamp on DNA.


35
The DnaX Clamp Loading Machine
Three major parts make up the pol III holoenzyme within the E. coli replisome.
The replicative polymerase III core, the DnaX complex that contains the r subunit -the
cement of pol III holoenzyme, and the (3 sliding clamp processivity protein. The DnaX
complex has an additional function in polymerase processivity by loading the p clamp
onto primed-template DNA for preinitiation complex formation. Originally identified as
the y-8 complex or y complex (Maki and Komberg, 1988; O'Donnell, 1987), this DnaX
complex was shown to load p onto DNA in an ATP-dependent manner (Onrust et al.,
1991).
The x and y subunits are expressed at roughly equal levels in cells due to a
translational frameshift in the DnaX gene (Flower and McHenry, 1990). Therefore it was
expected that a mixed x / y containing DnaX complex would reside in the pol III
holoenzyme. In fact, several DnaX complexes expressed from an artificial operon were
isolated from cells including two forms of mixed x / y complex, and a y-only complex
(Pritchard et al., 2000). No x-only complex was isolated from cells in that study. One of
the two isolated forms of mixed x / y complex had the stoichiometry of x 2 y 1 of the DnaX
gene products, and this form was predicted to function within the pol III holoenzyme
based on the requirement of the x subunit dimer for holoenzyme function. The separate
y3 only complex, and / or the mixed x 1 / 72 complex were predicted to function separately
from holoenzyme, for example, in DNA repair activity with DNA polymerase II (Bonner
et al., 1992).
Identification of all five of the subunits of y complex (Maki and Komberg, 1988),
and the genes encoding them (Dong et al., 1993; Xiao et al., 1993a), lead to in vitro
reconstitution and use of this y-only complex as the model P sliding clamp loader (Onrust


36
et al., 1995). Recently, sequence and structural analysis have identified several subunits
of y complex as AAA+ superfamily members and y complex as a AAA+ molecular
machine (Davey et al., 2002). These studies have provided significant insight into the
mechanism of opening the stable ring-shaped dimer structure of (1 clamp and loading it
onto DNA at the speed required by polymerase III catalyzed Okazaki fragment synthesis.
The y complex contains five subunits with the stoichiometry: 73,81,8i, Xi> and \|/i.
The x and t)/ subunits bind a y subunit through y (Glover and McHenry, 2000), and
greatly increase the affinity for the 88 subunits to the complex (Olson et al., 1995). The
Y subunit, alone in solution, was found to exist in a monomer-tetramer equilibrium,
however when formed in a complex with 88 became a trimer of Y3 (Pritchard et ah,
2000). The structural asymmetry derived by single copies of the 8,8, x, and vp subunits
of the DnaX clamp loader within holoenzyme imparts structural asymmetry in the
holoenzyme, and implicates their function with the lagging strand polymerase.
The 8 and 8 subunits, products of the holA and hoiB genes, respectively, were
purified and characterized for their interactions and function in y complex (Dong et ah,
1993). These subunits have similar mass, (8: Mr = 38,700 Daltons, and 8: Mr = 36,900
Daltons), and high affinity for each other and were usually co-purified as a consequence.
The 8 subunit binds (1 through a P interaction element (PIE), while 8 binds tightly to
the y subunit. During the clamp loading cycle, an inactive form of y complex, the 8
subunit is thought to occlude 8 from binding p and a conformational change upon binding
of ATP to y complex causes exposure of the piE of the 8 subunit (Naktinis et ah, 1995).
Genetic knockouts of the holA or holB genes are not viable revealing that the function of
8 in clamp loading absolutely requires the 8 subunit (Song et ah, 2001). Song, Pham et


37
al. (2001) also showed that 8 and 8 are required not only for ATP-dependent
preinitiation complex formation, but are essential for DNA synthesis elongation as well.
They participate in the DnaX clamp loader complex function in coupling to DnaB
helicase at the replication fork to pol III holoenzyme for leading and lagging strand
synthesis.
The 8 subunit alone was found to have the ability to unload P from DNA without
any requirement for ATP binding or hydrolysis (Leu et al., 2000). In this study, the
approximate quantity of each subunit of y complex was determined from whole cell
lysates by protein absorbance measurements and western blot analyses. Surprisingly they
discovered a ~4-fold excess of the 8 subunit present separate from any within y complex.
In Okazaki fragment synthesis, about 2000 to 4000 p clamps are used for complete
replication of the lagging strand. However, with only about 300 molecules of p present
in the cell, there is a need to recycle the clamps left behind on the DNA as the lagging
strand polymerase cycles between Okazaki fragments. To keep up with the pace of
Okazaki fragment synthesis p must actively be unloaded at a rate of at least 0.01 s'1. It
was found that the quantity of y complex within the cell was limited to -140 molecules
by the 8 subunit. Since pol III holoenzyme and any free y complex are present in low
quantities and perform several DNA metabolic functions such as replication and repair, it
was envisioned that the excess 8 subunit aided in removal of p clamps abandoned by pol
III at the end of each Okazaki fragment. In their investigation (Leu, Hingorani et al.,
2000) also showed that in vitro removal of p from circular DNA substrates by purified 8
happened at a rate of 0.011 s'1. This rate was comparable to measured rates of P
unloading by y complex (0.015 s'1) and pol III holoenzyme (0.007 s'1) in similar assays.


38
As all of the 5 subunit is expected to be stoichiometrically associated with y complex,
the 5 subunit would only regulate the activity of 5 in clamp loading, leaving free-8
subunit to recycle leftover |3 clamps, keeping a sufficient level of free sliding clamp
available for rapid Okazaki fragment synthesis.
Although not necessary for in vitro clamp loading activity, y complex contains the
xand \|/ subunits. The x (Mr -16,600 Daltons) and vp (Mr -15,000 Daltons) subunits are
the products of the holC and holD genes, respectively, and have important functions
adapting the lagging strand polymerase for Okazaki fragment synthesis (Onrust et al.,
1995; Xiao et al., 1993a). As stated above, the y subunit binds to y, bridging the
interaction of the x subunit to the complex. The x-y dipeptide forms a rod-like structure
(Kelman et al., 1998), however it is not known where on the y subunit x-y binds in the
clamp loader complex (Jeruzalmi et al., 2001a). A sub-complex of y~xy, or x-y alone
had no independent function in promoting DNA synthesis activity by pol III (Xiao et al.,
1993b).
The x subunit through the y structural bridge to the y subunit in the DnaX complex
strengthens DNA polymerase III holoenzyme interactions with the single-stranded DNA
binding protein (SSB)-coated lagging strand template facilitating preinitiation complex
formation and processive synthesis elongation. Isolated pol III core or pol in, which are
without the x-y subunits, are inhibited in synthesizing DNA on SSB-coated template
DNA (Glover and McHenry, 1998). Removal of x-y from pol III holoenzyme results in
reduced efficiency of clamp loading activity and an increase in salt sensitivity in the
physiological range (40-150 mM) (Kelman et al., 1998). Investigation of a common
point mutation in SSB (SSB-113) revealed that the direct interaction with the x subunit is


39
located at the C-terminus of SSB, and that this interaction is most likely hydrophobic in
nature. Through this direct x-SSB contact on template DNA, the % subunit was thought to
provide resistance to elevated ionic strength for proper clamp loading activity, as well as
possibly keeping the lagging strand polymerase localized to the SSB-coated template for
greater efficiency in synthesis elongation.
The % subunit has been found to be required in a primase to polymerase handoff for
Okazaki fragment synthesis. A three-point switch was proposed where the x subunit,
through direct interaction with SSB, causes primase to dissociate from a newly formed
primer allowing initiation complex formation and synthesis of the Okazaki fragment to
proceed (Yuzhakov et al., 1999). Primase (DnaG) interacts distributively with DnaB
helicase in formation of the primosome for controlled RNA primer synthesis (Tougu and
Marians, 1996). After synthesizing a primer, primase remains attached to the nascent
primer, and is thought to protect it from exogenous exonuclease activity. Primase is also
directly attached to SSB, and therefore blocks initiation complex formation. Through the
X-SSB interaction, but not a direct x-primase interaction, primase is displaced, perhaps
through a conformational change in SSB. The x-DnaB helicase interaction provides a
contact point to hold pol III holoenzyme to the replication fork while lagging strand
polymerase cycles (Kim et al., 1996a). Contact of pol III holoenzyme to the newly
primed template, and bringing the x subunit through the DnaX clamp loader complex into
the vicinity of the SSB-coated lagging strand to displace primase through SSB would
allow preinitiation complex formation for polymerase cycling for rapid lagging strand
Okazaki fragment synthesis.


40
The Dna X (x or y) subunits of the clamp loader bind ATP (Tsuchihashi and
Komberg, 1989). In the reconstituted 7 complex clamp loader, interaction with the 58
subunits causes a solution-monomer-tetramer equilibrium of 7 subunit to form a trimer
giving the clamp loader a stoichiometry of 73,81,8i, xi, and q/i (Pritchard et al., 2000).
The 7 subunits in isolation (i.e. in monomer-tetramer equilibrium) do not hydrolyze ATP
at an appreciable rate with respect to activity needed for replication of the lagging strand.
However, when reconstituted into a complex with the 8,5,x and t|t subunits, or just the 8,
and 8 subunits, the rate of ATP hydrolysis is much higher, and further enhanced in the
presence of p to a rate that would be rapid enough for preinitiation complex formation for
Okazaki fragment synthesis (i.e. ~2 s'1) (Onrust et al., 1991). This means that the clamp
loader can bind and hydrolyze a maximum of three molecules of ATP to power the clamp
loading reaction. Since the 7 / x subunits of the clamp loader are the only subunits which
bind and hydrolyze ATP to power the clamp loading reaction they are known as the
motor subunits of the clamp loader.
The AAA+ Superfamily of Motor Proteins
Through sequence analysis and ultimately structural analysis the 7,8, and 8
subunits of the clamp loader have been identified as members of the AAA+ superfamily
of ATPases (Davey et al., 2002; Neuwald et al., 1999). As the name of this superfamily
implies: ATPases Associated with a variety of cellular Activities, proteins of this class
are involved in many cellular processes. Whole genome analyses have indicated that the
AAA+ class is ancient and has undergone substantial functional divergence prior to the
emergence of the major divisions of life, thus this class is large and diverse (Neuwald et
al., 1999). Proteins of the class generally assist in protein transitions, such as remodeling,


41
assembly, and disassembly. AAA+ proteins assist not only in protein-protein transitions,
but also in protein-nucleic acid transitions.
AAA+ proteins including GTPase proteins, which are mechanochemical proteins
that transduce the energy of nucleotide hydrolysis into useful work, are becoming
recognized as a quickly growing group of energase enzymes (Purich, 2001). An
energase can simply be defined as an enzyme that couples the change in energy of a
covalent bonding state directly to changes in non-covalent substrate- and product-like
states. For AAA+ machines such as the y complex clamp loader, the chemical energy of
ATP hydrolysis activity is transduced into mechanical changes in the clamp loader
structure. As a AAA+ machine, y complex clamp loader could be described as a
molecular matchmaker. The y complex modifies a protein (i.e., p clamp) such that it
becomes enabled for interaction with DNA in such a way that it would not normally
interact.
The AAA+ superfamily includes chaperone-like ATPases such as regulatory
components of proteases, transcriptional regulators, vesicle synthesis and fusion proteins,
and dynein motor proteins to name a selected few. Aside from AAA* proteins of y
complex, other important replication proteins are members of the AAA+ class. For
example, bacterial DnaA, DnaC, and RuvB proteins, and the subunits of the eukaryotic
origin recognition complex, Mcm2-7 helicase, and replication factor-C subunits of the
eukaryotic clamp loader (Erzberger et al., 2002; Neuwald et al., 1999).
Increasing investigation of AAA+ proteins reveal that adaptor proteins may
modulate their functions. As a consequence, a growing list of these structurally unrelated
adaptor proteins are being identified for many of the known AAA* proteins (Dougan et


42
al.; 2002). Generally smaller than their AAA+ partner, AAA+ adaptor proteins provide a
simple and effective way to modulate the function of the AAA4- machine. Adaptor
proteins have been found to give the AAA+ protein better control over substrate
specificity, allowing quick response to changing local conditions and redirection of
AAA+ activity. The y complex has two proteins, % and \p, that are not related in sequence
or structure to the other AAA+ class subunits of the clamp loader. Questions concerning
the function of x and vp in the clamp loading reaction by y complex are of major
importance in this dissertation, and it is attractive to propose that they are in fact adaptor
proteins in complex with the AAA+ clamp loading machine. As described above, the x
subunit, through v|/, has important interactions with SSB at the replication fork for
preinitiation complex formation. As will be discussed in the results of this dissertation, x
and vp are also important for activity beyond interaction with SSB at the replication fork,
and are important for optimal activity of the clamp loading machine itself. This
dissertation explores the structural and conformational stability provided to y complex by
the x and vp proteins, and therefore may reveal a novel example of how adaptor proteins
modulate the activity of their partner AAA+ machines. The X-ray crystal structures of a
minimal y3SS clamp loader and a x-y complex have been solved, but it still remains a
mystery as to where the x-'|' dipeptide binds to the y subunit of the clamp loader. The
limited examples of AAA+ -adaptor protein complexes have shown that the adaptor
proteins generally bind to the least conserved N-terminal domain of their AAA+ partner.
This fact may provide direction for further structural analysis of the complete y complex
including the x and tp proteins.


43
Figure 2-2 The crystal structure of a AAA+ motor protein. The y1'243 crystal structure
(Podobnik, Weitze et al. 2003) is shown here as a representative AAA+ motor
protein. This structure was truncated at the C-terminus, and only the AAA+
domains-I (red) and -II (yellow) are shown. A molecule of ATPyS bound to
the NTP-binding site is shown in space-filling representation. The conserved
structural and functional features of AAA+ motors are indicated: Sensor-2
(blue), P-loop (green), and Sensor-1 (pink). Also marked are the conserved
Arg (y-215) of sensor-2 and Thr (y-155) and (SRC) Arginine (y-169)-fmger
of sensor-1. A Cys-coordinated Zn~ atom is also present in Domain-I.
Images were composed using DeepView/ Swiss-Pdb Viewer Ver. 3.7,
http//www.expasy.org/spdbv/, with structural coordinates downloaded from
the Protein Data Bank (Berman, Westbrook et al, 2000),
http//www.rcsb.org/pbd/. (PDB code: 1NJF)
AAA+ proteins have several common structural features that define their function
(Figure 2-2). All AAA+ proteins have an approximately 220 amino acid conserved
structural ATPase core, although sequence is not always conserved (Jeruzalmi et al.,
2001b; Neuwald et al., 1999). The AAA+ conserved structure has two domains (I and


44
II), and a third oligomerization domain (III) is common in multimeric complexes of
AAA+ protein subunits. AAA+ proteins generally contain the phosphate-loop (P-loop)-
type NTP-binding site having the Walker-A and Walker-B motifs that are common in
ATPase and GTPase proteins. The Walker-A motif (GVxxxGKT) is involved in the
phosphate binding of ATP (or GTP), and the Walker-B motif (DExx) is involved in metal
(Mg44) binding and catalysis (Walker et al., 1982).
The P-loop a,p-foId provides a platform upon which other AAA+ structural motifs
are mounted. Above the P-loop, in domain-II is the sensor-2 motif containing a highly
conserved Arg residue (Arg-215 in the y subunit). This Arg residue is predicted to
interact with the p- and y-phosphate of bound ATP (Podobnik et al., 2003). Domain II
containing the sensor-2 motif is typically a hinge-like domain around which movements
in domains-I and III are made.
In domain-I, flanking the P-loop from the underside is the sensor-1 motif. The
sensor-1 motif contains the highly conserved Ser-Arg-Cys (SRC) sequence that is
predicted to interact with the y-phosphate of the bound ATP through hydrogen bonding.
The SRC sequence includes an arginine-finger. This arginine-finger is thought to
stimulate catalysis of ATP hydrolysis by analogy to the similar, conserved switch II
region of GTPase activating proteins (Ahmadian et al., 1997). In an oligomeric AAA+
complex such as y complex, the SRC-sensor-1 motif of one AAA+ subunit interacts with
the nucleotide-binding site of a neighboring AAA+ subunit, and is implicated in
interfacial stimulation of ATP hydrolysis (Jeruzalmi et al., 2001a; Podobnik et al., 2003).
The X-ray crystal structure of the y complex AAA+ clamp-loading machine is described
below. It has five AAA+ proteins that make up three parts of the clamp loading machine:


45
three y-ATPase-motor subunits, the S subunit, harboring the p interaction element (PIE),
and the 8 stator, upon which movement of the other subunits is supported.
X-ray Crystal Structure of the Clamp Loading Machine
The X-ray crystal structure of a reconstituted minimal clamp loader complex
(7388) was solved at a resolution of 3.0 using multiwavelength anomalous diffraction
methodology (Jeruzalmi et al., 2001a). The subunits crystallized as a heteropentameric
circle having the stoichiometry (8V73-81) (Figure 2-3).
While the amino acid composition of the 8 and 8 subunits was complete, all three y
subunits were truncated by 57 amino acids from protease-labile C-terminii. All subunits
showed a three domain C- shape previously observed in the structure of the 8 subunit
alone (Guenther et al., 1997), giving the complex an overall form analogous to five
fingers extending down from the palm of a hand. Both of the AAA+ domains I and II
were observed for all five subunits. This was a surprising observation for the 8 subunit,
as it shared only 7-8 % sequence identity with other AAA+ proteins including those
within the clamp loader. All of the ATPase motor y subunits crystallized in nucleotide-
free form, and the 8 and 8 subunits were already known not to bind ATP. The X-ray
crystal structure of a truncated form of the y subunit (y1 243) including the AAA+ domains
I and II only was later solved in the presence of nucleotide (Podobnik et al., 2003), and
will be used for the discussion of the nucleotide binding sites and consequences of
nucleotide binding the y motor subunit.
The C-terminal domain III of all five subunits of the clamp loader forms a tight
circular collar at the top of the complex (Figure 2-3B). The subunits are arranged
where the 8 and 8 subunits surround the three y subunits.


Figure 2-3. The crystal structure of the 7388 clamp loader. The clamp loader
forms a circular heteropentameric structure, each subunit having three
domains (I II and III). A) Fronf-view: The point of view is directly
into the open interface between the 8 (white) and 8 (blue) subunits.
The three 7 subunits (different shades of red) are present behind 8 and
8. Structural domains are indicated: Domain-Ill (the C-terminal
collar), Domain-II (hinge), and Domain-I (N-terminal
functional). B) The clamp loader has been tilted forward 90 for a
slab-view of the top showing the tight pentameric collar of the C-
terminal domain-III only. Subunit nomenclature is indicated. C) The
clamp loader has been tilted back 90 from the view in (A), and
domains-Il and III have been dimmed for the bottom view. The N-
terminal domain-I of each subunit is colored for emphasis on their
asymmetrical disposition. The NTP binding sites of the 7 subunits are
occupied with sulfate molecules from crystallization (space-filling
representation), and the Zn" atoms (yellow) bound to 8, 71,72, and 73
subunits are shown. The (1-interaction element ((HE), and the
conserved hydrophobic-wedge residues of the (HE (8-Leu-73 and -Phe-
74) are shown (green). Images were composed using DeepView /
Swiss-Pdb Viewer Ver. 3.7, http//www.expasy.org/spdbv/, with
structural coordinates downloaded from the Protein Data Bank
(Berman, Westbrook et al, 2000), http/Avww.rcsb.org/pbd/. (PDB
code: 1JR3)


47
Hydrophobic interactions within a helical packing arrangement strengthen subunit
interactions in this oligomerization domain. Only the C-terminal domain of the 8 subunit
appeared loose within this oligomerization domain, and was proposed to have
consequences for movement of the 8 subunit within the complex.
The N-terminal domains of the five subunits containing the AAA+ structural
domains-I and II are highly asymmetric with respect to each other extending down from
the C-terminal collar (Figure2-3C). Each N-terminal domain is increasingly splayed out


48
with respect to the circular C-terminal collar forming an open o-shape. Splaying-out of
N-terminal domains in this structure resulted in considerable exposure of the 8 subunit
and thus the pIE. Based on biochemical studies, a nucleotide-ffee structure would be
predicted to be more closed where the 5 subunit-piE would be obstructed by the 8
subunit. The authors speculate that the observed structure of the clamp loader may be an
artifact of the crystal packing forces, but overall would reflect that of an open or
activated complex.
The nucleotide binding sites of the y subunits are buried within the subunit
interfaces as is common with other oligomeric AAA+ ATPases. The AAA+ domains are
arranged such that domains-I and II of one subunit cradle domain-1 of the neighboring
subunit providing a mechanism for conformational communication based on nucleotide
binding status. Three interfacial nucleotide binding sites are between the 8-yi, Y1-Y2, and
Y2-Y3 subunits. The N-terminal AAA+ domains of the y subunits were highly asymmetric
in the complex, resulting in asymmetry in these interfacial nucleotide binding sites. The
S-Yi interface and Y2-Y3 interface were open in this nucleotide-free structure, while the yi-
Y2 interface had the most extensive contact and therefore appeared inaccessible to
nucleotide.
Structure of the Nucleotide Binding Site and the Proposed Conformational Change
of they Subunit
The X-ray crystal structure of C-terminal truncated y1-243 was solved in the presence
of ATPyS to a resolution of 2.2 using multiwavelength anomalous dispersion
methodology (Podobnik et al., 2003). The resulting structure had four y subunits
arranged in the asymmetric crystal unit. One of these y subunits had complete electron
density corresponding to ATPyS in the nucleotide binding site, and another was missing


49
electron density corresponding to the y-phosphate, and therefore was taken as the
structure of ADP-bound y. Two other y subunits in the asymmetric unit had no
nucleotide bound. Slow hydrolysis of ATPyS through the crystallization procedure was
thought to result in producing the ADP-bound y. This structure allowed identification of
several as yet unknown features of nucleotide binding and the conformational changes
induced due to nucleotide binding. The results have important implications for the
mechanism of communication between the AAA+ domains within the clamp loader and
therefore the overall clamp loading mechanism.
The structure showed that the nucleotide was bound in the expected position in the
cleft between domains-I and II formed by the Walker-A P-loop (see figure 2-2). The
adenine ring was located in a non-polar environment, and formed hydrogen bonds from
the N-6 position with the main chain carbonyl groups of Val-19 and Val-49. This feature
provides a mechanism for distinguishing ATP from GTP by the y subunit. Both the 2-
and 3-hydroxyl oxygens of the ribose moiety were hydrogen-bonded to the carbonyl
oxygen of Ala-7 of the N-terminal helix extending from domain I. Three residues of the
conserved Walker-A P-loop motif coordinate the (1- and y- phosphates of the nucleotide.
The Lys-51 residue forms a salt-bridge, and Thr-52 and Ser-53 form hydrogen bonds to
the phosphate groups from the sensor-1 loop.
Comparison of the nucleotide-bound y1-243 structure with the nucleotide-empty
y388 structure reveals that the y subunit domain-11 a-helix containing the sensor-2 motif
blocked the region corresponding to the position of the ribose ring. This observation was
extended to all of the empty nucleotide-binding sites in the y388 structure. Thus it was
predicted that nucleotide binding to a given y subunit alters the position of domain-I with


50
respect to domain-III by conformational change. The reorientation of domain-I would
open up the nucleotide binding site from a collapsed state, and clearly affect the positions
of the sensor-1 and sensor-2 motifs within the complex. Also, rotation of the sensor-2
region containing the conserved Arg-215 residue occurs upon nucleotide binding and
provides space for the phosphate groups of the nucleotide. The sensor-1 motif does not
move with respect to the P-loop, and therefore follows the general movement with the N-
terminal domain-1 upon nucleotide binding.
Conformational changes induced by nucleotide binding to the P-loop region are
directly linked to movement of the catalytic arginine-finger of sensor-1, thereby
coupling (i.e. communicating) changes in nucleotide binding status to the neighboring
subunits of the complex. In the 7388 structure, the sensor-1 motif containing the
arginine-finger, was either too far away from the neighboring nucleotide binding site that
it is thought to stimulate in the case of the S-yi interface, or in steric-clash with the
neighboring nucleotide binding site as in the case of the 71-72 interface. Therefore,
conformational changes in individual subunits must be made to correctly position the
sensor-1 arginine-finger with the neighboring nucleotide for catalysis. These
conformational changes that occur upon ATP binding not only bring the subunits into
proper orientation for ATP hydrolysis, but more importantly cause exposure of the 8
subunit from occlusion by the S stator so that it can bind p clamp.
Crude modeling of the p clamp bound to the clamp loader structure was
performed using information from the 8-P structural interaction (described in detail
below). This simplistic docking of dimeric p with the clamp loader revealed that the
orientation of the 8-p interaction would cause the clamp to make contact with all subunits


51
in the clamp loader (Jeruzalmi et al., 2001a). One of the key points addressed in this
dissertation is the question of how p affects the clamp loader in the clamp loading
mechanism. The multipoint contact suggested by the modeling of p bound to the clamp
loader provides a structural basis for several conclusions concerning individual subunit
conformational changes and how p may trap an ATP-bound activated clamp loader.
This contact may potentially explain how the clamp loader stabilizes the open p ring
while a single dimer interface remains open (see below) until a primer template junction
is located.
The exact nature of how the clamp loader binds to DNA is not yet fully understood.
A cysteine-coordinated zinc module is present in the 8 and all three y subunits of the
complex (figure 2-3) and may provide a interaction zone for duplex DNA, however, the
function of the zinc modules in the clamp loader complex has not been investigated.
Calculation of the surface electrostatic potential of the clamp loader structure invites
some interesting speculation on the nature of where DNA binds and stimulates the
activity of the clamp loader. Two extensive regions of positive electrostatic potential
were observed on the structure. Both were seen on the N-terminal asymmetric bottom
side of the clamp loader that is predicted to interact with the p clamp. One of these
regions was on the 8 subunit, adjacent to the PIE presumably where DNA would thread
through the p clamp when bound to 8. The other region was observed as a partially
continuous belt that tracks along the outer surface of the N-terminal domains of the y
subunits. Each of these regions of positive electrostatic potential predict that when DNA
is encircled within the p clamp under the clamp loader, single-stranded DNA could be
positioned to interact with the outer regions of the ATP binding domains causing


52
additional rearrangement of sensor-2 and sensor-1 motifs stimulating hydrolysis of ATP.
The requirements for formation of the DNA binding site of y complex are further
explored in the results of this dissertation.
The X-ray crystal structure of the 8 subunit bound to a (3 monomer was also solved,
and provides a possible mechanism of clamp opening by the 8 subunit as well as how the
P clamp may interact with y complex through binding the 8 subunit-piE.
X-Ray Crystal Structure of the 8 Subunit Bound to a p Monomer and the
Mechanism for Opening the p Sliding Clamp
The X-ray crystal structures of two complexes between the 8 subunit and a mutant
monomeric form of P (Pi) were solved by multiwavelength anomalous diffraction
(Jeruzalmi et al., 2001b). Attempts to crystallize the 8 subunit with wild-type p2 were
unsuccessful, and therefore a mutant p that remained monomeric due to mutations within
the dimer interface was used (Stewart et al., 2001). The 8: pi structures either contained
full-length 8 subunit, composed of all three domains observed in the y complex structure
including the AAA+ region, or truncated 8 subunit (amino acids 1 140) 8M4 containing
the N-terminal domain I only. Both complexes of S:Pi and SM40:Pi crystallized in 1:1
complexes at resolutions of 2.9 and 2.5 respectively (Figure 2-4).
Examination of both structures revealed a mechanism by which P clamp is opened
upon the 8 subunit inducing or trapping a conformational change in p such that a stable
dimeric interface cannot be formed. Some possibilities for ring opening were that 8
forcibly breaks the dimer interface, but due to the fact that ATP is not required by 8 to
open p, this was not very probable. Another possibility was that the P clamp could
oscillate between conformations and the 8 subunit could have high affinity for a
conformation inconsistent with ring closure.


53
Figure 2-4. The crystal structure of the S-pi complex. The fall structure of the p subunit
(blue) is shown bound to Pi monomer. Domains (I II and III) of 5 are
indicated. The subdomains of pi are colored: N-terminal subdomain-1
(white), middle subdomain-2 (pink), and C-terminal subdomain-3 (red). The
conserved hydrophobic-wedge, pIE (green) Leu-73 and Phe-74 of 8 subunit
are marked. The a-helix of the p subunit involved in the spring-loaded
conformational change is colored yellow. Images were composed using
DeepView / Swiss-Pdb Viewer Ver. 3.7, http//www.expasy.org/spdbv/, with
structural coordinates downloaded from the Protein Data Bank (Berman,
Westbrook et al.), http//www.rcsb.org/pbd/. (PDB code: 1JQJ)
This also was not a very probable mechanism due simply to the high stability of the p
dimer alone on circular DNA. A third possibility was that the 8 subunit could induce and
stabilize a conformation in p that is inconsistent with ring closure. The results from
analysis of these structures are consistent with the third possibility, and it was proposed
that 8 subunit binding induces a conformational change in the p clamp that results in


54
straightening of a crescent-shaped (3 monomer such that a single dimer interface is
opened to allow DNA passage into the clamp.
The piE is on the N-terminal domain of the 8 subunit, and consistent with earlier
biochemical studies, makes contact with the C-terminal-extended-loop face of the p
subunit (Naktinis et al., 1995). Only one 8 subunit bound Pi in the full length 8: pi
structure, and it was observed that domains II and III of the 5 subunit impeded binding of
a second molecule of 8 to Pi. In the 51I40:Pi structure showed that the pIE forms a
hydrophobic wedge that binds to a hydrophobic pocket on the Pi subunit between
subdomains 2 and 3 (of the p subunit) (Figure 2-4). Subdomains 2 and 3 are the middle
and C-terminal structural domains of the P protomer (Kong et al., 1992). The Met-71,
Leu-73, and Phe-74 residues form the hydrophobic wedge on the 8 subunit. The
hydrophobic pocket on p, into which this wedge is inserted, is formed by Leu-177, Pro-
242, Val-247, Val-361, and Met-362 of p. The piE hydrophobic wedge of the 8 subunit
is highly conserved among all bacterial 8 subunit sequences examined. Likewise, the
residues forming the hydrophobic pocket on p are also highly conserved across evolution,
and therefore found on sliding clamps of bacteriophage, eukaryotes, and archaea (see
below). Conservation of this interaction between the clamp loader and sliding clamp
suggests a common mechanism for clamp opening in all known organisms.
These structures have revealed that interaction of 8 with p requires conformational
changes in p that weaken the dimer interface, and importantly, 8 does not interact directly
with the dimer interface. The structure of the P dimer interface consists of a hydrophobic
core surrounded by ionic interactions (Kong et al., 1992). An a-helix participating in
these interactions at the dimer interface is distorted in the wild-type dimeric p clamp such


55
that additional hydrophobic residues contained on this distorted helix do not extend into
the interface. The hydrophobic pocket binding site where the 8-subunit-piE binds is near
a 5 -residue loop adjacent to the distorted a-helix in p. The binding of 8 is thought to
impose strain on the distorted helix that causes its relaxation into a more ideal a-helical
conformation. This results in extension of the hydrophobic residues from the previously
distorted portion of the a-helix directly into the dimer interface. Removal of dimer
interface stability was proposed to trigger release of spring-like tension in the crescent
shaped p monomer such that it becomes straightened. Thus the clamp is not simply an
inert ring, but contains structural information for its own opening. The crescent shape of
the p subunit to which 8 bound was calculated to straighten by 12 at the unopened
interface and by 5 at the opened interface by comparison with the structure of dimeric p.
Comparison of the 8 subunit in the 8:Pi structure with the structure of the 8 subunit
from the 7388 structure revealed that the N-terminal domain of 8 is in a considerably
different position when bound to p. An a helix (a4), containing the piE, in domain-I
translates by ~5.5 and rotates -45 with respect to the rest of domain-I, and the piE or
hydrophobic wedge is extended further away from the a4-helix for contact with p. The
reverse conformational change in 8, from the p-bound form to the p-free form of results
in exposure of specific hydrophobic residues that are important for interaction with the 8
stator of the clamp loader.
Although the two structures were solved with mutants of p that do not form dimers,
the above conclusions regarding release of spring-like tension in opening an interface
were consistent with molecular dynamics simulations performed on the dimeric P clamp.
In these simulations, a p protomer was simply released from dimeric constraints


56
determined from the original p2 clamp structure. The p protomer followed a trajectory in
the simulation that resulted in straightening of its crescent-shape such that it
superimposed well with the structure determined from the 8-bound P monomer
DNA structural requirements for p clamp loading by y complex
The p clamp loading mechanism and preinitiation complex formation for
processive synthesis by pol III holoenzyme has been studied on several different DNA
substrates including synthetic poly-dT-primed poly-dA-templates, as well as circular
phage genomes in different replicative forms (i.e. single- or multi-primed, nicked-duplex
etc.). It was shown that short synthetic primed-template DNA (pt DNA) substrates (i.e.
an 80-105 nucleotide template with a 30 nucleotide primer) whose sequence was based
on the M13 phage where sufficient for proficient elongation by pol III core in the
presence of p clamp and reconstituted y complex, and therefore for the study of the clamp
loading mechanism (Bloom et al., 1996).
Using these short synthetic pt DNA substrates in experiments along side circular
phage genome substrates containing supercoiled or relaxed-structures, the DNA structural
requirements for p clamp loading where investigated (Ason et al., 2000; Ason et al.,
2003; Yao et al., 2000). For preinitiation complex formation at the replication fork it was
known that RNA primers were required. The exact structural nature of these primers
were unknown except that a 3-hydroxyl group was annealed at the single-stranded DNA
(ss DNA) / double-stranded RNA/DNA (ds RNA/DNA) junction where the preinitiation
complex was formed. In experiments with supercoiled DNA and closed relaxed circular
DNA substrates it was shown that reconstituted y complex did not require a 3-end to
load p, as it was able to load the clamp onto supercoiled DNA, but not closed relaxed


57
circular DNA (Yao et al., 2000). It was hypothesized that supercoiled DNA may produce
unwound regions due to superhelical tension, and at these ds RNA/DNA / ss DNA
junctions y complex could load p forming the preinitiation complex.
Using circular single-stranded M13 phage DNA primed with synthetic DNA
primers differing in specific structural features such as length and unannealed 15-
nucleotide "flaps at 5- or 3-ends, or both 5- and 3-ends, it was shown that y complex
can load p onto a wide range of these substrates (Yao et al., 2000). The minimal primer
length requirement was found to be 10 base pairs, consistent with the size of the inner
pore width of p, and with the known fact that in vivo, primers formed by primase, are ~
10-12 nucleotides in length (Kitani et al., 1985; Kong et al., 1992). Yao, Leu et al. also
showed that y complex could load p onto primers with 5- and/or 3- 15-nucleotide-long
unannealed flaps, supporting their conclusion that y complex only requires a ds DNA / ss
DNA junction to load the clamp.
By placing protein- or DNA secondary-structural blocks on or within the primer in
replication assays, the polarity and primer spatial requirement for clamp loading were
determined. Using physical (i.e. protein) blocks tightly bound at the annealed primer 5-
or 3-ends it was shown that y complex exhibits polarity in loading the p clamp
specifically at the 3-end of the primer. This result was expected due to the fact that
polymerase synthesis elongation extends from the primer 3-end in the 5 to 3 direction.
Placement of DNA secondary structural elements at different distances upstream from the
3-end of the primer, allowed determination of the spatial requirement of the primer for
p-bound y complex and p-bound pol III core. It was shown that with y complex, 14-16
base pairs of primer were needed to load the clamp, and 20-22 base pairs were needed for


58
P to bind pol III core. This result indicated that y complex interacts directly with 4 to 6
base pairs of the primer, and that the clamp is pushed back an additional ~6 base pairs
when pol III core binds p at the ds DNA / ss DNA junction (Yao et al., 2000).
Utilizing fluorescence-based steady-state and pre-steady-state kinetic anisotropy
assays with a X-Rhodamine-labeled short synthetic pt DNA in solution, a DNA-triggered
switch in y complex was discovered (Ason et al., 2000). This DNA-triggered switch
caused a change in affinity of y complex for DNA, and was found to be pt DNA- and
ATP hydrolysis-dependent. Initially, in steady-state assays, it was observed that y
complex had higher affinity for ss DNA than pt DNA. Even more surprising, was that
the 5-single-stranded template overhang of the pt DNA was the same sequence and
length as the ss DNA substrate examined in these experiments. However, pre-steady-
state real-time assays revealed DNA binding kinetics suggesting that y complex bound
transiently with high affinity to the pt DNA substrate. Use of non-hydrolyzable ATPyS
in both steady-state and pre-steady-state assays with pt DNA substrates removed this
dynamic switch in y complex affinity for DNA resulting in a high affinity state only. It
was therefore proposed that ATP hydrolysis by y complex was required for cycling
between high and low affinity states, and that the low affinity state may have been an
ADP-bound y complex. With ss DNA substrates y complex with p maintained high
affinity for the DNA, hydrolyzed ATP, but did not load p (Bloom et al., 1996; Turner et
al, 1999).
The dynamic interaction with pt DNA and the DNA triggered switch in y complex
accomplishes two major goals in the clamp loading reaction. The primer-template, ds
DNA / ss DNA junction, provides the proper site for clamp loading and preinitiation


59
complex formation, and modulates the DNA binding affinity of the clamp loader, so as
not to allow y complex competition with pol III core bound to (5 at previously formed
initiation complexes. The nature of the low affinity state of y complex remains a
mystery, and this dissertation addresses several possibilities [and questions] including the
dynamics of conformational changes defining different states of y complex with respect
to the clamp loading reaction.
A recent investigation addressed the possibility that the pt DNA-triggered
modulation of y complex provides a dynamic mechanism for recognition of appropriate
sites for p clamp specific for proficient DNA synthesis. This was accomplished by
investigation of the DNA structural features required to trigger y complex into the low
affinity state and release p (Ason et al., 2003). In fluorescence-based steady-state and
pre-steady-state anisotropy assays in solution, y complex binding and clamp loading were
studied with elongation-proficient DNA substrates (i.e. synthetic template DNA primed
on its 3-end or center), or elongation-deficient DNA substrates (i.e. synthetic template
DNA primed such that a blunt duplex was formed at the 5-end of the template, or
unprimed-ss DNA). In steady-state titrations of y complex to the elongation-proficient or
deficient DNA substrates, results similar to the previous investigation defining the DNA-
triggered switch were observed. The y complex showed high affinity for elongation-
deficient DNA substrates, but did not appear to bind well to the elongation-proficient
DNA substrates. Pre-steady-state assays revealed biphasic up-down-kinetics (i.e., a
rapid rise to a defined peak followed by a decay phase into steady-state DNA binding
activity), representing transient high affinity of y complex for the elongation-proficient
DNA substrates as well as characteristic changes in anisotropy for the clamp loading


60
reaction when p was present. For elongation-deficient DNA substrates, pre-steady-state
binding kinetics were monophasic suggesting a simple high affinity bimolecular
equilibrium, whether p was present or not. Additionally, a correlated DNA-binding and
ATP hydrolysis assay with y complex and elongation-proficient pt DNA clearly showed
that a binary complex consisting of y complex with pt DNA transiently formed just prior
to DNA-triggered ATP hydrolysis and release of y complex without any further binding.
Overall, the results show that y complex uses a dynamic mechanism driven by ATP
binding and hydrolysis for targeting p clamp only to DNA that can serve as a template for
synthesis elongation by pol III core, and prevent further interaction with polymerase-
bound p.
Mechanism of the p Clamp Loading Reaction Cycle by y Complex
Formation of a processive pol III holoenzyme requires the formation of
preinitiation complexes on the leading and lagging strands at the replication fork. The
ring-shaped sliding clamp must be opened and loaded onto the circular E. coli
chromosome in order to form a topological link between pol III and DNA. The clamp
loading machine performs this task through dynamic protein-protein and protein-DNA
interactions. Some 26 years ago, Sue Wickner presented a model for the mechanism of
DNA elongation catalyzed by DNA polymerase III that still generally holds true today
(Wickner, 1976). Wickners model described a mechanism where primed-template DNA
was activated by the ATP-dependent transfer of DNA elongation factor I (the sliding
clamp) by DNA elongation factor III (the clamp loader), to which DNA polymerase III
then bound in an ATP-independent manner, followed by DNA elongation. Combined
biochemical analysis and structural details have in recent times given us a detailed, yet


61
still incomplete, view of the clamp loading mechanism required for processive synthesis
by pol III holoenzyme.
The y complex (73,8,8xy)> or a minimum complex of five subunits (73,8,8) is the
energase complex that transduces the energy from ATP binding and hydrolysis into
mechanical work to load [1 onto DNA (Figure 2-5).
Within this machine, the three 7 subunits serve as the motor subunits, 8 has the 13-
interaction element (|3IE), and 8 is the stator upon which movement of the other
subunits is thought to be supported. In general terms, ATP binding and hydrolysis by the
7 subunits promote conformational changes that modulate the dynamic protein-protein
and protein-DNA interactions involved in clamp loading. To work at the speed and
efficiency required at the replication fork within the cell, there must be precise
communication between all subunits of the clamp loading machine to power and regulate
its activity.
Initially, ATP binding to the 7 subunits, but not hydrolysis, promotes changes in the
clamp loader that modulate the binding affinity for the clamp and DNA, and therefore
powers most of the steps in the clamp loading reaction (Bertram et al., 1998; Hingorani
and O'Donnell, 1998). Asymmetry of the interfacial nucleotide binding sites in the clamp
loader structure provided a model for activation upon ATP binding for p clamp loading.
The 8-7i and 72-73 interfacial nucleotide binding sites were open in the structure and
could provide space for the first one or two molecules of ATP to bind. These
conformational changes could also result in the opening of the 71-72 ATP binding site,
and further splaying-out of the 7 subunits from the backbone of the 8 stator, and
ultimately exposure of the 8 subunit piE (Figure 2-5B). This model would bring the


62
maximum number of ATP molecules needed for exposure of the 8 subunit piE to three in
open y complex.
Figure 2-5. A schematic cartoon of the basic steps in the clamp loading reaction and
initiation complex formation. The cartoon of the clamp loader in closed and
open states is based on the y388 crystal structure, and the y complex
subunits are colored as in figure 2-3. A) ATP binding to the y subunits. B)
ATP-dependent conformational changes occur within the clamp loader
exposing the 8 subunit pIF. (green). C) p clamp binding to the clamp loader
through the piE induces a conformational change in p, opening the clamp at a
single interface. D) The P-bound clamp loader complex binds pt DNA and
positions the clamp at the ds DNA / ss DNA junction at the 3-OH of the
primer. E) pt DNA triggers ATP hydrolysis, the release of inorganic
phosphate (Pi), and dissociation of ADP-bound clamp loader from loaded p
clamp. F) Polymerase III core (a, e, and 0 subunits) can then bind P and
commence processive elongation of the template.
Originally it was shown that ATP binding caused a conformational change in y complex
that resulted in characteristic proteolytic cleavage of the exposed 8 subunit, and that this
change did not require p (Flingorani and O'Donnell, 1998; Naktinis et al., 1995). The


63
interaction of the exposed 8 subunit pfE with P then induces or stabilizes conformational
changes in p that result in ring opening at a single dimer interface (Figure 2-5C). The
kinetics of ATP binding and the dynamics of the conformational changes within the
clamp loader are a major component of this dissertation and will be discussed further in
chapters 4 and 5.
ATP binding to y complex is also required for interaction with DNA in the clamp
loading reaction (figure 2-5D) (Bloom et al., 1996; Stukenberg and O'Donnell, 1995).
Studies described in this dissertation show that none of the individual subunits of the
clamp loader have significant DNA binding affinity, and it is hypothesized here and that
the interaction of all clamp loader subunits form the DNA binding site. With a clamp
bound and opened, the clamp loader then binds DNA at a ds DNA / ss DNA junction,
specifically on elongation proficient primed-template DNA (Ason et al., 2003; Yao et al.,
2000). The DNA binding kinetics and positioning of p at the 3-hydroxyl of the primer
happen in less than 100 ms with an approximate bimolecular binding constant on the
order of 2.0 4.0 x 108 M-1 s'1 just prior to the hydrolysis of one molecule of ATP for
each y subunit in the clamp loader (Ason et al., 2000).
Primed-template DNA triggers ATP hydrolysis and dissociation of the clamp
loader (Figure 2-5E). Earlier investigations of ATP hydrolysis by the clamp loader
showed that mutations of the conserved lysine in the y subunit Walker-A motif of
domain-I, to alanine or arginine (K51A or K51R), resulted in complete abrogation of
ATP hydrolysis activity by the clamp loader (Xiao et al., 1995). Our laboratory has also
shown that these mutations of the conserved lysine in y complex abolished both p and
DNA binding in solution (unpublished), therefore it is likely that the lysine-mutants


64
cannot bind ATP. Use of non-hydrolysable ATPyS allowed y complex to bind p and
DNA, consistent with the notion that nucleotide binding powers most of the steps in the
clamp loading reaction. In these reactions, the complex of p-bound y complex remained
in a dynamic steady-state interaction with DNA for periods of time extending to 90
minutes (Bertram et al., 1998; Bloom et al., 1996). These investigations showed that
although ATP binding can bring P-bound y complex to DNA; ATP hydrolysis was
absolutely necessary for release of y complex, and completion of the clamp loading
reaction. The pre-steady-state ATP hydrolysis assays showed that the ATPs were
hydrolyzed at a minimal rate of 20-34 s'1 (Bertram et al., 2000). Closure of the p clamp
on DNA was found not to require any further energy (Turner et al., 1999), therefore ATP
hydrolysis by the clamp loader is not needed to close p onto DNA. The y complex crystal
structure predicted a mechanism wherein ATP hydrolysis drives final conformational
changes in which p is pushed off the 8 subunit piE as 8 returns to its occluded interaction
with the rigid 8 subunit. This is consistent with an earlier investigation that showed ATP
hydrolysis was required for release of y complex from p on DNA, leaving the clamp
behind (Bloom et al., 1996). Dissociation of y complex from the loaded clamp is
essential, and would provide space for the interaction of P with polymerase III, which is
known to bind the same surface of p that y complex had occupied (Figure 2-5F). Pre
steady-state analysis of DNA binding activity and ATP hydrolysis have shown that a
single turnover of the clamp loading reaction takes approximately 300 ms before
entering a steady-state that continues at a rate of-2-2.5 s1 (Ason et al., 2000).
Ultimately ATP hydrolysis would then allow the clamp loading machine to reset
itself for continued clamp loading, most likely through nucleotide exchange and


65
conformational changes. The y complex released from the clamp loaded on DNA is
presumably in some inactive closed state that would have to release ADP and become
able to bind ATP again. ADP release could cause conformational changes that may
transiently bring y complex through some state in which there is no nucleotide bound.
ATP binding would then cause the conformational changes that reactivate y complex for
another clamp loading reaction. In previous pre-steady-state ATP hydrolysis assays,
there was a pause in hydrolysis activity observed during the last 200-250 ms of the first
turnover of ATP during transition into the steady-state (Ason et al., 2000). Given the
way these assays were initiated (i.e., with preincubation of y complex with ATP and p
before mixing with pt DNA), during this transition, the rate-limiting step of the reaction
cycle was believed to occur. The exact nature of the rate-limiting step of the clamp
loading reaction is not yet known. Possibilities include ADP-release, ATP binding,
conformational dynamics within y complex, and p clamp binding. The steady-state and
pre-steady-state kinetics of DNA binding and ATP hydrolysis by the clamp loader are
further studied in this dissertation, and reveal some novel kinetic features that predict that
conformational changes within y complex are most likely the rate-limiting step in the
reaction and regulate the clamp loading mechanism.
Mutations of the p Clamp, and y Complex 8 and y Subunits: Effects on the Clamp
Loading Mechanism
Mutations in the p clamp as well as in subunits of y complex have been studied to
provide greater detail for the interactions modulating this clamp loading mechanism.
The steady-state and pre-steady-state kinetics of DNA binding and ATP hydrolysis
activities were investigated using a P clamp with mutations at two positions within the
dimer interface. The leucine to alanine (L273A, LI08A) mutations were thought to


66
weaken the dimer interfaces, although this mutant p was still able to form dimers in
solution (Bertram et al., 1998). The p interface mutants were found to bind y complex
and DNA similar to wild-type p, but they remained bound to DNA in a ternary complex.
The DNA binding assay results appeared similar to assays performed in the presence of
non-hydrolyzable ATPyS where the P-bound clamp loader appeared stuck in a dynamic
steady-state interaction with DNA, without loading p. In correlated pre-steady-state
DNA binding and ATPase assays, the P interface mutants bound to DNA and hydrolyzed
ATP with nearly identical kinetics as wild-type-p. However, the clamp loader steady-
state ATPase activity with the P mutants was significantly decreased, coincident with
extremely slow dissociation of y complex from mutant p on DNA (Ason et al., 2000).
The results confirmed the idea that the clamp loader must cycle off of p that is loaded
onto DNA, and undergo a rate-limiting step that is separated from the DNA-bound state
in order to continue loading clamps as previously suggested by Bloom et al. 1996. The
results of these investigations also showed that although p does not require any energy
from ATP hydrolysis to close on DNA, ATP hydrolysis is tightly coupled to release of
the clamp on DNA. It seemed interesting that mutations within the p dimer interface so
greatly affected the steady-state DNA binding and ATP hydrolysis activities of the clamp
loader. Now, with better knowledge of the interaction between the clamp loader-8
subunit and p clamp it is more clear how p interface mutations would cause these effects
in the clamp loader. Perhaps the conformational change of p cannot be properly
promoted by the 8 subunit-piE interaction, disallowing loading onto DNA. This
possibility could result in a decrease in the interaction of y complex with DNA, and
reduced DNA stimulated ATP hydrolysis activity.


67
To gain further insight into the AAA+ machine activity of 7 complex, mutations in
the sensor-1 (SRC) motif were made, and steady-state ATP binding, hydrolysis, and
clamp loading activities were examined (Johnson and O'Donnell, 2003). In one of these
mutations, arginine-158 of the 8 stator subunit (8-R158A) SRC motif was changed to
alanine. This mutation removed the arginine-finger from the S-7i nucleotide binding
interface. The results showed that the reconstituted S-RI58A-y complex still bound
approximately three molecules of ATP, but had reduced ATP hydrolysis activity and
diminished ability to load p in DNA synthesis assays. These results implied that the
arginine-finger contributed from the S subunit to the S-y i nucleotide binding site was
in fact catalytic in nature and therefore, this stator subunit was coupled with the motor
function of the clamp loader, as well as performing as the motor support subunit. Recent
results from our laboratory have shown that this reconstituted 8-R158A-y complex
mutant has little ability to bind p, as well as significantly reduced DNA binding and
clamp loading abilities (Snyder, A.K., personal communication). This is consistent with
the reduced DNA synthesis activity observed in the original study.
The arginine-finger was removed from the y subunits to create a second clamp
loader mutant (Johnson and ODonnell, 2003). The y-RI69A mutation removed the
arginine-finger from the 71-72 and 72-73 nucleotide binding interfaces, thus only
affected two nucleotide binding sites in the clamp loader. This reconstituted 73-Rl 69A-
complex still maintained the ability to bind three molecules of ATP and p, but had no
ATP hydrolysis activity, clamp loading activity, and was unable to stimulate DNA
synthesis. Our laboratory has since shown that this y3-R169A-complex can bind p at a
level near that of wild-type 7 complex in an ATP-dependent manner in solution, but


68
showed no DNA binding activity in steady-state and pre-steady-state solution-based
assays (Snyder, A.K., personal communication). Johnson and ODonnell discussed the
S- and y-subunit mutants in terms of an ordered ATP binding and hydrolysis mechanism
where ATP molecules are hydrolyzed in the reverse order than they were bound,
requiring proper positioning of the arginine-fingers for catalysis. Our laboratorys
results indicate that there is more than just a disruption in catalysis of ATP hydrolysis in
these mutants. Taken together, all of the mutation analysis results exemplify the need for
precise subunit conformational communication within the clamp loading machine for p
binding and also for formation of the putative DNA binding surface on the clamp loader.
The Clamp Loading Machine Within Polymerase HI Holoenzyme
Most of the biochemical analyses and all of the structural analyses of the clamp
loader and clamp loading mechanism have been performed with reconstituted y complex.
How do these results apply to the clamp loader within pol III holoenzyme? The clamp
loader within holoenzyme contains both the t and y subunits encoded by the dnaX gene.
In the t subunit, the region of the DnaX protein structure extending from the C-terminus
of the y subunit region into the C-terminal domain of x contains many proline residues
and is thought to be unstructured and therefore a highly flexible region (O'Donnell et al.,
2001). This flexible region divides the DnaX (t) protein into the y-AAA+ motor region,
and the x-C-terminal replisome interaction region. Within holoenzyme, there are at least
two t subunits, and a single y subunit (Figure 2-6).
The structural arrangement of the DnaX subunits of the clamp loader within
holoenzyme is likely to be [5i, T2>yi,8i,Xi,Vi]or [8i,yi, X2,8i,xi,v|/i]. The xand \\i
subunits only bind to the y subunit through vp, and the 8 subunit also has been shown to
bind only to the y subunit with high affinity (Glover and McHenry, 2000).


69
Figure 2-6. Architecture of the polymerase III holoenzyme at the replication fork
organized by the DnaX clamp loading machine. The leading- and lagging-
strands are unwound by hexameric DnaB helicase (green), shown bound to the
SSB-coated (yellow) lagging strand. The DnaX clamp loader containing two
t subunits (violet) (8i,Yi, r 2,81,jo,Vi) is connected within the holoenzyme
through the flexible linker regions of the r subunit C-terminal domains that
also dimerize pol III core (a e and 0), and further cement the holoenzyme
through contact with DnaB. The clamp loader % and \|i subunits are not shown
for clarity. Two p-rings (red) clamp the pol III cores to the templates, and a
third is shown bound to the clamp loader. In this schematic, primase would
bind and synthesize a new RNA-primer on the lagging strand. Then it is
predicted that the clamp loader would swing over to the nascent primer,
displace primase through the x subunit, and load a fresh clamp forming a
preinitiation complex. When the lagging-strand polymerase reaches the 5-
end of the previously synthesized Okazaki fragment, it would then release p
and cycle to the newly formed preinitiation complex and commence
elongation of DNA.
The two r subunits cement the array of proteins at the replication fork by dimerizing the
core polymerases in the holoenzyme, binding to the replicative DnaB helicase, and
protecting the p subunit from removal when it is attached to the polymerase a subunit.


70
Through the flexible linker region, these functions of the r subunits are connected to the
y-AAA+ motor region of the clamp loader. In this way, it is envisioned that the clamp
loader associated with the holoenzyme in vivo has a swinging range of motion on the
flexible tether loading at least one clamp on the leading strand and providing continuous
clamp loading activity on the lagging strand for Okazaki fragment synthesis (O'Donnell
et al., 2001).
Duplex DNA is anti-parallel, and therefore the core polymerases of the holoenzyme
must work in some asymmetric fashion to simultaneously complete replication of the
chromosome (Figure 2-6). The single y subunit of the DnaX clamp loader, and
consequential asymmetric distribution of the 5,5,x,and vp subunits gives the holoenzyme
structural asymmetry. The y subunit is the only clamp loader subunit to which \p and
therefore x adaptor proteins bind. This provides the holoenzyme clamp loader both
structural and functional asymmetry through r-DnaB helicase contact, and the x-SSB
interactions necessary for lagging strand synthesis.
Recently it was proposed that the asymmetry in the clamp loader provides
asymmetry to the core polymerases through nucleotide binding to the clamp loader
(Glover and McHenry, 2001). Use of non-hydrolyzable ATPyS was sufficient to allow
the DnaX complex to load a clamp for only one polymerase, presumably the leading
strand polymerase. ATP hydrolysis was absolutely required for clamp loading on the
lagging strand polymerase in their assays, and subsequent addition of ATPyS was able
to disassociate this lagging-strand polymerase. Whether the nucleotide binding status
of the clamp loader, or simply the structural asymmetry of the clamp loader provide
distinct leading and lagging strand functions to the core polymerases will require further


71
investigation. Overall, the structurally and functionally asymmetric holoenzyme has the
means to stay continuously linked to the leading strand while performing discontinuous
synthesis on the lagging strand at the replication fork by the distinct structural and
functional characteristics of the DnaX clamp loader. Polymerase processivity is required
for DNA replication in all free living cells, and nature has provided a consistent
mechanism to provide the means for polymerase processivity though conserved evolution
of these replication proteins across all branches of life.
Clamps and Clamp Loaders of Bacteriophage, Eukaryotic, and Archaeal Organisms
The combined biochemical and structural investigations of the E. coli clamp and
clamp loader have yielded many great details into the mechanism of processivity clamp
assembly on DNA. These intimate details have allowed complementary studies of
clamps and clamp loaders of other organisms to thrive due to the evolutionary
conservation of these processivity proteins across all branches of life, and suggest a
common mechanism of clamp loading in all life forms. It is well known that sliding
clamps contact polymerases with DNA for processive synthesis of complete
chromosomes or replicons in a fundamentally similar way (Ellison and Stillman, 2001;
Hingorani and O'Donnell, 2000). Now, structural evidence is revealing the extraordinary
functional similarities in clamps and clamp loading machines of viral replicases,
prokaryotes, eukaryotes and archaea for processive DNA elongation (O'Donnell et al.,
2001). All organisms through these evolutionary branches of life appear to utilize AAA+
proteins as the energase of their clamp loading machines (Table 2-1). Of those organisms
studied, each clamp loader appears to, or is predicted to utilize a AAA+ clamp loading
machine, like y complex, that contains several ATPase motor subunits, a clamp
interacting subunit, and a supporting stator subunit (Davey et al., 2002). This


72
conservation of sequence and, to a greater extent, structural similarities of these AAA+
proteins illustrate the evolutionary importance of these clamp loading machines.
Table 2-1. Clamps and clamp loaders through evolution
Evolutionary
Branch
Processivity
Proteins
AAA+
protein
Function"
Some Characterized
Organisms
Prdkaryotic
P
-
-clamp
y / T
8
5
X
Â¥
+
+
+
-motor
-stator
-clamp
interacting
subunit"
-AAA+ adaptor
-AAA+ adaptor
Escherichia coif
Bacillus subtilus
Bacillus-
staerothermophilus
Aquifex aeolicusf
Thermus thermophilusf
Bacteriophage
(viral)
gp450
gp44
gp62
+
+(?)
-clamp
-motor (stator?)
-clamp
interacting
subunit"
T4 phage
T7 phage
RB69 phage
Eukaryotic
PCNA
RFC-2d
RFC-3
RFC-4
RFC-5
RFC-1
+
+
+
+
+
-clamp
-motor
-motor
-motor
-stator
-clamp
interacting
subunit"
Homo sapiens
Saccharomyces cerevisiae
Schizosaccharomyces-
pombe
Caenorhabditis elegans
Drosophila melanogaster
Arabidopsis thaliana
Oryza sativa (rice)
Archaeal
PCNA
RFC-S
RFC-L
+
+
-clamp
-motor (stator?)
-motor / clamp
interacting
subunit
Pyrococcus furiosas
Archaeoglobus fulgidus
Sulfolobus solfataricus
Methanobacterium-
thermoautotrophicum AH
by analogy to the y complex clamp loader
b gp, gene product
' has conserved hydrophobic wedge residues of clamp interaction element
d nomenclature used is for yeast replication factor-C (RFC)
e clamp loader structure known
f Thermophile bacteria


73
Bacteriophage T4 Clamp and Clamp loader
When infecting bacteria, the bacteriophage has the necessary components to form
its own replication machinery. The replisome of T4 bacteriophage contains gene product
(gp) gp43 polymerase, gp41 / gp59 helicase and its accessory factor, respectively, gp61
primase, and gp32 single-stranded DNA binding protein. T4 also provides the
processivity clamp gp45 and clamp loader complex gp44/62 to complete the replisome
(Salinas and Benkovic, 2000). The gp45 clamp interacts with gp43 polymerase through a
C-terminal region of the polymerase. This interaction between the clamp and polymerase
is highly conserved across evolution (Ellison and Stillman, 2001), and the structure of a
T4 clamp bound to polymerase on DNA has been modeled based on the nearly identical
RB69 bacteriophage sliding clamp bound to polymerase (Alley et al., 2001; Shamoo and
Steitz, 1999). To date, these are the only structural views of a polymerase bound to its
clamp on DNA.
The gp45 processivity protein is a ring-shaped clamp formed of six similar
structural domains like (3 clamp, but unlike dimeric p, is a trimeric clamp. Although there
is less than 10 % sequence identity between gp45 and p, (and PCNA, see below), the
clamp structure is amazingly similar (Jeruzalmi et al., 2002). The crystal structure of
gp45 shows that its properties are similar to p clamp and PCNA (Moarefi et al., 2000).
The gp45 clamp has an overall negative charged p-sheet outer surface surrounding an a-
helical inner pore of positive electrostatic potential. The dimensions of the gp45 clamp
show an inner pore diameter of ~35 , and a width of ~25 , however, the clamp has an
overall triangular topology compared to the nearly circular structures p clamp and PCNA.
Consistent with data that show gp45 clamp is the least stable of the known sliding clamps
on DNA (Yao et al., 1996), the gp45 clamp is predicted to be slightly open in solution


74
through a pucker which gives it a lock-washer-type structure (Alley et al., 1999). It is
still possible that the clamp loader must open the clamp further for the loading reaction
(Alley et al., 2000).
The T4 clamp loader is a pentameric complex of the gp44 and gp62 subunits with a
subunit stiochiometry of four gp44 subunits to one gp62 subunit (Jarvis et al., 1989). The
T4 gp44/62 complex binds and loads gp45 onto DNA in an ATP-dependent manner and
requires hydrolysis of bound ATP. In earlier work, it was shown that four molecules of
ATP were hydrolyzed by gp44/62 during formation of the T4 holoenzyme, and that some
step following ATP hydrolysis was rate limiting in the reaction (Sexton et al., 1998). A
highly detailed investigation using fluorescent resonance energy transfer techniques with
fluorescent-labeled gp45 clamp, combined the pre-steady-state kinetic analysis of clamp
opening and closing by the gp44/62 clamp loader with analysis of binding and hydrolysis
of ATP (Alley et al., 2000). This investigation showed that hydrolysis of ATP was
required to open the gp45 clamp, and then additional hydrolysis was required to load the
clamp on DNA. A more recent study on the ATP hydrolysis activity of gp44/62 in clamp
loading conflicted with previous work, and showed that ATP binding alone is sufficient
for gp44/62 to bind gp45, and at least one ATP is required to complete the loading
reaction (Pietroni et al., 2001). This study also revealed that ATP hydrolysis was not
required to open the gp45 clamp, and that the rate-limiting step in the loading reaction
was either ADP release from gp44/62, or a conformational change before clamp loading.
This investigation is more consistent with the detailed study of the y complex in p clamp
loading.


75
The gp44 subunits of the complex have homology with AAA+ proteins and contain
the Walker-A P-loop and sensor-1 SRC motifs needed for ATP binding and hydrolysis
(Davey et al., 2002; Neuwald et al., 1999). By analogy to y complex, the gp44 subunits
are expected to be the motor subunits of the T4 clamp loader that undergo ATP-
dependent conformational changes that drive gp45 binding and interaction with DNA in
the loading reaction. The gp62 subunit shares no sequence homology to other AAA+
subunits, and does not bind or hydrolyze ATP. Until its structure is known it can only be
predicted that gp62 is a AAA+ homologue, and the subunit that interacts with the gp45
clamp during the clamp loading reaction (i.e. like the 8 subunit of y complex). There is
no support-like stator subunit in the gp44/62 complex as all of the gp44 motor subunits
are identical. However, even in y complex, the 5 stator subunit has been shown to be
directly involved in the catalytic motor function of the clamp loader through precise
positioning of its SRC motif by conformational changes (Johnson and ODonnell, 2003).
One of the gp44 subunits of the T4 clamp loader may work in a similar manner, and it
may turn out that this putative, distinct, gp44 subunit may also be incapable of ATP
hydrolysis like 8 in y complex. These predictions await further biochemical and
structural analyses of the T4 clamp loader.
Eukaryotic PCNA Clamp and Replication Factor-C Clamp Loader
The essential replication machinery of eukaryotic organisms was originally
identified by investigations using the SV40 DNA virus as a model system. SV40 uses
host cell machinery in replication of its DNA, and requires only its own T-antigen for
replication initiation and DNA helicase activity (Waga and Stillman, 1994). Processivity
proteins, proliferating cell nuclear antigen (PCNA) and replication factor-C (RFC) were


76
identified as the sliding clamp and clamp loader, respectively, for the replicative
polymerases 8 le (Waga and Stillman, 1998). Like the y complex clamp loader, RFC has
DNA-dependent ATP hydrolysis activity that is further stimulated by the presence of the
PCNA clamp (Lee et a!., 1991). RFC is also directly involved in a primase to polymerase
switch, similar to y complex, and influences the length of primer synthesis by polymerase
a primase (Mossi et al., 2000; Tsurimoto and Stillman, 1991).
The PCNA sliding clamp structure was solved from budding yeast S. cerevisiae
(Krishna et al., 1994). Once again, even though sequence homology between the
bacteriophage T4 clamp, prokaryotic p clamp, and PCNA was low, the structure of
PCNA is nearly identical to the other clamps (Moarefi et al., 2000). Unlike the p clamp,
but like T4 gp45 clamp, PCNA found to be trimeric. All three clamps have six
structurally similar subdomains, dimensions (i.e. the PCNA inner pore is ~ 35 ), and
similar electrostatic characteristics on their structurally conserved outer p-sheets, and
inner a-helices. The overall topology of the PCNA trimer is more hexameric or circular
than gp45, remains closed in solution, and is more stable on DNA than gp45, but not as
stable as dimeric p clamp (Yao et al., 1996).
RFC has been extensively studied from yeast and human origin, and has been
identified as a heteropentameric complex consisting of one large subunit and four small
subunits of different mass. The subunit nomenclature is: RFC- (yeast/human): l/pl40,
2/p37,3/p36,4/p40, and 5/p38. For simplicity the yeast RFC nomenclature will be used
here. Deletion analysis of RFC subunits showed that the C-terminus of each subunit was
required for formation of the pentameric complex, and that this complex must form to
allow DNA stimulated ATP hydrolysis activity (Uhlmann et al., 1997). It was also


77
shown that none of the individual subunits alone had DNA binding activity (Uhlmann et
al., 1996). Using surface plasmon resonance (SPR) and filter magnetic-bead binding
assays, it was shown that ATP mediated all interactions of RFC with PCNA and DNA
(Gomes and Burgers, 2001). This study also showed that ATP binding but not hydrolysis
was required for the interaction with DNA, and that use of non-hydrolyzable ATPyS
resulted in a stuck complex of PCNA-bound RFC on DNA. These results are
consistent with complementary investigations performed with y complex. The DNA
structural requirements for PCNA loading by RFC were also tested and showed that RFC
could load PCNA on ds DNA / ss DNA junctions not requiring an annealed 3-OFI
similar to the DNA structural requirement for y complex catalyzed clamp loading
activity.
Recent studies with yeast RFC (Schmidt et al., 2001a), as well as previous
investigations with human RFC (Cai et al., 1998) identified the subunits involved in ATP
hydrolysis activity and those essential for DNA recognition by mutational analysis of the
conserved Walker-A lysine residue in each subunit. The results of these mutational
analyses revealed that RFC subunits 2,3, and 4 were required for ATP binding and
hydrolysis in efficient PCNA loading. Mutations in RFC-1, the large subunit, had no
effect on PCNA binding and loading abilities. RFC-5 has modified Walker-A and B
sequences suggesting that it does not bind or hydrolyze ATP, similar to the 5 subunit of
y complex (Cai et al., 1998; Schmidt et al., 2001a). Consequentially, these Walker-A
mutations of RFC-5 did not effect PCNA binding and loading activities.
As in the T4 clamp loading reaction cycle, the use of ATP by RFC in PCNA
loading is also not fully understood. This may be due to limitations of the SPR and filter


78
binding analyses that were being performed along side steady-state ATP hydrolysis
analysis of these reactions. Despite these limitations, a pathway of multiple stepwise
ATP binding events is thought to be required for RFC loading PCNA onto DNA, and that
this proposed reaction resembles the well studied E. coli clamp loading reaction pathway
(Gomes et ai., 2001). In the yeast PCNA loading reaction, 2 ATP molecules initially
bind RFC, PCNA then binds RFC, and RFC gains affinity for a third ATP.
Conformational changes within RFC are most likely taking place as each ATP binds an
RFC subunit. Finally, RFC-PCNA binds pt DNA, and apparently one more molecule of
ATP binds RFC, perhaps to the RFC-1 subunit. Upon interaction with pt DNA, PCNA is
released, coincident with hydrolysis of up to all four bound ATP molecules. It remains
unknown how many ATP molecules are used for this reaction.
The four small subunits of RFC (2-5) form a functional core, and are used in other
cellular processes. The large RFC-1 subunit swaps with another protein, specific for
functions such as replication termination (Kouprina et al., 1994), and cell cycle
checkpoint control (Green et al., 2000). Differing the nature of ATP binding affinities
and putative conformational changes in the RFC-2-5 core may provide differential
reaction mechanisms for these diverse functions. Perhaps, the first two ATP molecules
bind and cause conformational changes that allow a presentation of general binding site
for PCNA and DNA only when RFC-1 is present, or form and expose different binding
sites when another protein takes the place of RFC-1. Then, other ATPs could bind and
produce conformational changes specific for each function. Another possibility could be
that when a specific protein such as PCNA binds, it alters the affinity for ATP at
additional sites in RFC specific for, in this case, clamp loading.


79
All five RFC subunits are AAA+ homologues, and are related in sequence to the y
and 8 subunits ofy complex (Neuwald et al., 1999; O'Donnell et al., 1993). The
heteropentameric stiochiometry of the RFC subunits predicts that this eukaryotic clamp
loader will have structural similarity to y complex. The RFC-1 subunit is the least
conserved of the RFC subunits, and its specificity in clamp loading with the RFC-2-5
core, and dispensability from the RFC-2-5 core in functions other than clamp loading
suggest that it may be the PCNA clamp interacting subunit of the RFC complex by
analogy to the 8 subunit of y complex, (see Table 2-1). Natural modifications of the
Walker-A and B nucleotide-binding domain of the RFC-5 subunit suggest that this
subunit may not bind or hydrolyze ATP, but it still has a sensor-1 SRC motif that could
interact with the other RFC subunits (Cullmann et al., 1995). Like the 8 subunit of y
complex, RFC-5 is predicted to function as the stator in this eukaryotic clamp loading
machine (Davey et al., 2002). The remaining RFC-2, 3, and 4 subunits, when mutated in
the conserved Walker-A sequence for ATP binding and hydrolysis show the most severe
in vitro defects and in vivo phenotypes (Schmidt et al., 2001a; Schmidt et al., 2001b).
Therefore RFC-2, 3, and 4 were proposed to be the motor subunits of this machine. In
fact, as heterotrimeric complexes, both yeast and human RFC-2-3-4 complexes had
DNA-dependent ATPase activity (Ellison and Stillman, 1998).
This functional analogy of the different RFC AAA+ subunits to the y complex
subunits for prediction of RFC clamp loader structure and mechanism of clamp loading
awaits solution of an atomic structure of the RFC clamp loader. Although, as described
below, the atomic structure of part of the archaeal RFC clamp loader which has


80
significant homology to eukaryotic RFC, has been solved, and closely resembles the
structure of y complex motor subunits.
Human RFC alone and in a complex with PCNA has been viewed by transmission
electron microscopy (TEM) and atomic force microscopy (AFM) (Shiomi et al., 2000).
The TEM results showed that RFC forms a closed-U-form structure in the absence of
ATP. When viewed in the presence of ATP, the RFC molecules appeared as a more
open-C-form structure demonstrating that ATP caused RFC to open. Partial
proteolysis results with ATP, non-hydrolyzable ATPyS or ADP confirmed that there was
induction of a distinct structural change in RFC only in the presence of ATP. Additional
TEM imagining also showed that ATPyS or ADP had virtually no effect on the structure
of RFC (i.e. RFC remained in U-form similar to that without nucleotide). Although the
images were low resolution and did not allow specific distinction of RFC or PCNA
molecules in the imaged RFC-PCNA complexes, the authors concluded that the open
ATP-bound RFC C-form was most likely bound to PCNA in the TEM images.
Archaeal PCNA Clamp and Replication Factor-C Clamp Loader
Biochemical analyses and recent structural analysis of the clamps and clamp
loaders of several archaeal organisms are providing important information that is helping
bridge the gap between the prokaryotic structural and functional clamp loading
characteristics and eukaryotic structural and functional clamp loading characteristics.
Archaeal PCNA clamps and RFC clamp loaders share -30-40 % sequence
homology to their eukaryotic counterparts (Seybert et al., 2002). Archaeal organisms
have a PCNA clamp and a RFC clamp loader complex (Table 2-1). Archaeal RFC and
PCNA have been biochemically characterized for Sulfolobus solfataricus (De Felice et
al., 1999; Pisani et al., 2000), Methanobacterium thermoaulolrophicum AH (Kelman and


81
Hurwitz, 2000), Archaeoglobus fulgidus (Seybert et al., 2002), and Pyrococcus furiosus
(Oyamaet al., 2001).
The RFC clamp loading machine from each of these archaeons is composed of two
subunits, RFC-L (large), and RFC-S (small), and each has AAA+ homology (Davey et
al., 2002). Biochemical analyses of RFC and PCNA of these organisms have identified
several general characteristics, and these characteristics are consistent with the clamp
loading mechanisms of both eukaryotic and prokaryotic organisms. Each archaeal RFC
must form a complex to acquire DNA-dependent ATP hydrolysis activity. This ATPase
activity is associated with the RFC-S subunits, and has shown preference for ds DNA / ss
DNA pnmer-template junctions (Kelman and Hurwitz, 2000; Pisani et al., 2000). For the
Archaeoglobus fulgidus q/RFC clamp loader, it was further shown that the ATP
hydrolysis activity was enhanced 2 to 3-fold by a/PCNA, and that ATP binding, not
hydrolysis was required to stimulate the interaction of q/PCNA with DNA (Seybert et al.,
2002). Each archaeal RFC clamp loader was also shown to support processive DNA
synthesis with its respective PCNA and pol B-family archaeal DNA polymerase. The
major difference in the characterization of these clamp loaders was their subunit
stiochiometry. All have been shown to form pentameric complexes similar to the
eukaryotic and prokaryotic clamp loaders, except the Methanobacterium
thermoautotrophicum AH RFC, which has been shown to form a hexameric complex of
two RFC-L subunits with 4 RFC-S subunits. The general pentameric subunit
stoichiometry resembles the T4 bacteriophage clamp loader and the eukaryotic RFC,
having four RFC-S subunits with 1 RFC-L subunit.


82
About the same time the y complex clamp loader crystal structure was solved, the
crystal structure of a RFC-S subunit complex of Pyrococcus furiosus was solved at 2.8
resolution (Oyama et al., 2001). The RFC-S subunit formed a dimer-of-trimers, not
necessarily the functional assembly of the clamp loader. Each RFC-S subunit showed
significant AAA+ structural homology. Superimposition of a RFC-S subunit with the y
complex 8 subunit structure had a root-mean-square displacement of only 3 for the
two conserved AAA+ domains fl and II), and showed marked overall similarity. The
RFC-S subunits had the Walker-A P-loop and Walker-B motifs flanked by sensor-2 and
sensor-1 motifs forming the AAA+-structurally conserved nucleotide binding site, and
also had the SRC arginine-finger motif. Each RFC-S subunit had an a-helical C-
terminal Domain-Ill corresponding to the circular collar oligomerization domain of the
y complex clamp loader. The subunit interfaces within each trimer of RFC-S subunits
was arranged such that each nucleotide binding site was in contact with the neighboring
subunit, and the interfaces differed between each subunit, suggesting asymmetry among
the subunits in the crystal, a feature distinguishing the y complex structural-functional
characterization (Jeruzalmi et al., 2001a).
The RFC-S subunits are AAA+ proteins, and based on the extraordinary structural
similarity with the y AAA+ subunits of the prokaryotic clamp loader, suggest that the
mechanism of ATP-dependent conformational changes that drive the y complex is most
likely the same mechanism driving the archaeal clamp loader. The RFC-S subunit has
the highest sequence similarity to the eukaryotic RFC-3 AAA+ subunit (Cann and Ishino,
1999), and therefore two predictions can be made. First, the RFC-S subunit of archaeal
organisms is the motor subunit of the clamp loader, and second, that the mechanism of


83
the eukaryotic clamp loader may be closer to the mechanism of the prokaryotic clamp
loader than originally expected. To add to this, the RFC-L subunit generally lacks an
SRC motif, and shows highest sequence similarity to the eukaryotic RFC-1 subunit (Cann
and Ishino, 1999). Thus it has been proposed that like the eukaryotic RFC-1 and
prokaryotic 8 subunit, RFC-L is the clamp interacting subunit of the archaeal clamp
loading machine (Davey et al., 2002).
The functional conservation of the sliding-clamps and clamp loaders in all branches
of life is readily understandable considering the importance of their functions in DNA
replication and therefore in cell division for propagation of any given organism. These
are ancient proteins that have not changed considerably for billions of years. The use of
the E. coli model system for studying the mechanisms of clamp loading for processive
replication has thus given us the fundamental basis for study of the clamp loading
mechanisms in all forms of life. In this dissertation project, the conformational dynamics
of the E. coli clamp loader were under investigation, and it is hoped that the results can
be applied for understanding of other AAA+-based clamp loaders and provide additional
functional insights for other AAA+ motors


CHAPTER 3
MATERIALS AND METHODS
NOTE: The anisotropy binding assays and MDCC-PBP ATPase assays were
performed with both y complex and the minimal complex clamp loader, usually in back-
to-back (i.e. hours apart) assays. Therefore, the term clamp loader will be used
throughout this chapter to describe both, except where different concentrations of each
were used or where only the activity of y complex was studied.
Proteins, Reagents, and Oligonucleotide Substrates
DNA Polymerase III Proteins
DNA polymerase III proteins were a generous gift from Mike ODonnells
laboratory at The Rockefeller University, New York, NY. Proteins were stored in 20
mM Tris-HCl pH7.5,2 mM DTT, 0.5 mM EDTA, and 10% glycerol. Assay buffer for
all experiments contained 20 mM Tris-HCl pH 7.5,50 mM NaCl, 40 pg/mL Bovine
serum albumin (Invitrogen Corp., Carlsbad, CA), 5 mM DTT, and 8 mM MgCb (Table
3-1).
Concentrations of y complex were determined from absorbances at 280 nM in 6 M
guanidine hydrochloride and the calculated extinction coefficient. This concentration
was verified by amino acid analysis following acid hydrolysis of the protein (performed
by the Protein Chemistry Core Facility, Biotechnology Program, University of Florida).
A sample for amino acid analysis was prepared by dialyzing y complex against 20 mM
sodium phosphate buffer pH 7.5 at 4 C to remove glycerol and other buffer components
that interfere with the analysis. The concentration of this dialyzed protein was also
84


Full Text
71
Accompanying the group was a new advisor to the chief, The Reverend Christopher
Schulenburg, of the Hermannsburg Missionary Society of Germany.
The Malete soon dominated the existing Babirwa, BaGananwa, and BaNgwaketsi
tribes of the area (Ngcongco, 1982). The dominance was not without bloodshed, as
conflict over land rights erupted into battle (Ellenberger, 1937). Todays old men of the
village recalled the stories they had been told of the battle. Sitting under a tree at the
kgotla on a quiet afternoon, tales of intellect and bravery emerged.
The young and the women were sent to protect the cattle, with
the men staying behind. Our fathers had never fought with guns before
but had watched the ways the Boers organized battle. For us, battles
were fought in the open but the Boers hid behind trees. Our fathers and
grandfathers copied the white mans way of hiding, with one man loading
guns, another shooting. Only eight of our men were killed. The foes
fought in the open and wore red turbans so they were easy targets.
The heavy fighting resulted in many casualties, with over a hundred dead among
the foe (Ellenberger, 1937). Growing pensive, the men continued. "Yes, our
forefathers were wise men, learning from others and not afraid of to fight for the land
that was ours." This battle coincided in time with the end of the pre-colonial era of
Botswana (Parson, 1984:15).
Early Colonization: 1885-1935
In 1885 the British declared a Protectorate over a wide area known as
Bechuanaland. Each Tswana tribe retained authority under the supervision of the new
European administration. In December of that year the English arranged a treaty
between the local warring tribes, giving the Malete rights to land in Ramotswa and a
limited surrounding area. Although the village belonged to the Malete, it was not until
1909 that the boundaries of the Bamalete Reserve were defined by proclamation
(Ellenberger, 1937). This same time period was also a time of drought. Rinderpest also


82
against us by making too much rain. This was the last time a rainmaking
ceremony was performed in the village!
Sego pointed out how the later years of this time period contained manageable
conflict between church and tribe. He gave the history of this evolving problem as it
related to his bogwera (tribal initiation) in 1929. Sego was a member of the
Matsaakgang, or "Those associated with a dispute."
The National School of Ramotswa, established by the church, was
operating at this time. Many older children went for two or three years
of formal education, independent of church confirmation class. Girls
were more apt to attend than boys, because of boys duties with cattle. A
Malete boy, who had been attending school in the Transvaal, came to
Ramotswa with a letter of introduction for the missionary of the Lutheran
Church. He was admitted to the church classes. The boys new father
removed him from school, as that family did not want him to become a
Christian. They placed the lad in bogwera for his education. The church
and the Magistrate tried to have the boy returned from bogwera, but the
chief refused. A large dispute resulted. It was decided that both types of
schooling was initiation-training and the chief then gave identical age-set
names to the graduates. After that, we worked together as a unified age-
regiment, clearing communal tribal land and building fences.
It was silly for the church to have divided us according to whether
we were a civilized Christian or a heathen In reality, no one was really
different. The heathens were not ruffian people, but good people. As a
village, we were all Tswana. We all followed the Tswana laws and could
understand each other.
Pre-1935: Social Change or Social Continuity
The traditional cultural system incorporated the natural and manmade
environment with laws and customs providing control over the supernatural and natural
ingredients of village life. Gerontocracy and principles of eldership penetrated all
aspects of Malete daily life, directing interpersonal behaviors and social, judicial, and
political structure and function. Extended kinship, based on principles of age seniority,
served to integrate families and establish social cohesion within the community
(Schapera 1944,1953). The result was a communal system of solidarity and reciprocity,
which probably occurred in varying extents between individuals.
Colonialists, aiming to establish Ramotswa as a Christian labor reserve,
interpreted the traditional culture as heathenish and barbaric. The thrust of change was


207
I have presented the family setting in which the aged must function. Changes in
discourse patterns, the loci of power and authority, and the degree of shared functioning
are but a few of the ways in which the doors to the good life have been broken. It is
difficult, if not impossible, for the aged to find access through them. Some of the aged
rely on tradition to keeps the doors working. Other aged modify their approach to the
doors.
These same damaged doors of family structure and function also effect the
behaviors of other generations. Some people believe modernization and Western
thought should build new doors, with the government assuming some of the traditional
family responsibility for care and economic support. Others want the aged to provide
repair by adapting more Western and liberalized ways. For many, the conflicts between
generations prohibit any repair of the damage.
This chapter has dealt with the doors that effect family interactions, There are
other doors that the family, and its aged, must use. These doors operate within the
setting of the community, the topic of the next chapter.


237
wedding as traditional, with the slaughtering of cattle, passing down of laws, and public
announcement through feast and parade. The young see a modem wedding, with
western wedding finery, plates for food and loud radio music..
I wonder if it was so different years ago when the Christian church service and
cloth clothing became part of the rite. Did some of the present aged exalt in "civilized"
weddings to the dismay of their parents? Now, like the Christian prayers with funerals,
change has become tradition, serving to provide unity and state legitimacy to birth,
marriage, and death. Change and tradition has interwoven to provide meaning.
It is much easier for youth to accept the process of modernization over the
performance of much of the traditional. Tradition is perceived as a separate entity, used
by the aged as a method for reasoning and the basis for behavior. Many customary
practices that provide depth and meaning to village life are discounted, such as kgotla
meetings and the role of the aged in preparing feasts. The fragility of valued practices,
such as the dependency of the pre-wedding dancing on the songs, is unrecognized. The
mortality of the aged is not associated with the possible mortality of the presently valued
process for a meaningful, modernized life. Only the "useless" traditional behaviors will
die.
The social situation creates conflict concerning which aspects of tradition have
value, especially in regards to the gerontocratic principles. Schisms between the old and
young abound. This was graphically and publicly proclaimed with the dance between the
young and old men. The symbolic battle drained the energy of both men, as do the
skirmishes in real life. The aged tire first during the interplay of daily interactions.
Independent action for self well-being replaces family and village welfare, as the strength
of youth frequently wins over wisdom of age. Kgotla meetings and funerals, sitting and
death, are for the old. Sexuality and vitality is assumed to be only for the young. The
aged are accepted as having limited learning from past experience, but should be far


APPENDIX B
THE LADDER INSTRUMENT
The Ladder
The ladder is approximately twelve inches high. It is constructed with five rungs,
each about three inches across.
Opening Sentences
This is a ladder. Each rung represents different people. (Point to rungs.)
Sometimes I will hold it so that is sideways, instead of up and down. I will ask which
rung represents you. (Move hand up and down rungs.) There is no correct or wrong
answer. I want to know how you feel about yourself.
Questions: (Hold ladder on its side)
L The rungs are five people. (Point to right-hand rung) This person is the stranger in
the village, he/she is all alone as he/she knows no one. No one visits or talks with
him/her. (Point to left-hand rung) This is the person who knows everyone. He/she has
many friends and people come to visit and to ask for advice. Which rung is you? Why?
2. (Point to right-hand rung) This person has nothing that others want; no food, no
knowledge, no house and no physical body. (Point to left-hand rung) This person has
everything and gives things, and assistance, to other people. Which rung are you?
Why?
3. (Point to right-hand rung) This person can do nothing for him/herself. He/she
cannot walk, must have food and water put in their mouth by someone else and
someone must dress him/her. (Point to left-hand rung) This person can walk as far as
they want without getting tired, can plow or earn money, and is never sick. Which rung
are you? Why?
(Questions 4-7: Say "The questions change a little now". Hold ladder upright. Point to
middle rung to begin each question.)
4. This is where you were at the time of Independence in 1966. Where are you now?
Why?
5. This is where Botswana was at the time of Independence in 1966. Where is the
country now? Why is this so?
6. This is where the people of Botswana were at the time of Independence in 1966.
Where are the people now? Why did this happen?
7. This represents the position old people had in society and how important they were
to others at the time of Independence in 1966. Where are the old people now? Why is
this so? Which rung are you? Why do you put yourself there?
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235
panties show through. The grooms brother is the best man. He is finely dressed in a
brown jacket with a black bow-tie. The short Christian ceremony ends with the couple
scrambling back into the truck, to be driven to the compound of her childhood. Before
guests are able to arrive, the newlyweds, with the wedding party, are cloistered in the
hut.
The feast begins, with the honored guests served rice, potatoes and meat on
china plates. Others use their fingers to eat from paper plates. The fanciest foods of
iced fruit and select beef are served to the cloistered wedding party. The radio,
connected to a car battery, plays the latest hit songs at ear shattering level.
Conversation is impossible as the dancing begins. Women move near the fence,
and sway in soft, rhythmic motion. They dance alone, lost in thought. Men begin to
dance alone, or opposite others, near the huts. Their movement is more forceful and
angular, as they act out their thoughts. The mens dance is as much an expression of
feelings as an actual pantomime.
A few young people snicker as an aged man, dressed in tom and patched
overalls and high rubber boots, begins his dance. Soon he becomes the center of
attention. With movements symbolizing a walk down the street, he catches the eye of a
pretty girl in the crowd. His face, shimmering like instant coffee crystals, portrays
instant love and happiness as he dances in front of her.. He moves on, revolving around
the circle of onlookers. He comes upon a group of younger men. First, his face shows
bewilderment, indicating he does not know what to expect from them. The boys begin
to point and laugh. The dancers features change to fear and repugnance. He quickly
moves away with sadness in his eyes. He retreats into himself, ignoring others as the
dance transforms itself into a reflection on his life. He starts off as a care-free child,
grows into a masculine adult and ends up as an feeble old man, always expressing lifes
hurdles and enjoyments though movement and countenance. His eyes become aglow


CHAPTER 3
ORGANIZATION OF THE STUDY
In Botswana, families give good care to their old parents. That is one
tradition that everyone believes in. There is no need to study old people,
as they are taken care of by their family, (a middle-aged government
employee)
I am old. Tradition has changed. You should learn about the old people
and tell the world how badly our family forgets us. It is alright that you
are white on the outside, but to learn about the hidden parts of being
old, you must become a Tswana on the inside. (67 year old woman)
The democracy, social freedoms, and racial equality of Botswana provided a
research locale where I could investigate the impact of modernization on aging while
blending into village life for research depth and breadth. This chapter presents the
formal steps taken for such work, beginning with permission for research and village
selection. Methodology and analysis reflect the incorporation of anticipated and actual
difficulties. The chapter ends with a synopsis of village population demographics. The
need for adaptability in, and during, research is stressed, as I had to unlock my
American door in order to find the Tswana doors and develop open and meaningful
communications with understanding.
Formal Permission for Research
The myriad of necessary steps required for formal research were undertaken
during my initial visit. Rigid regulations dictated the order of these steps. The first
action was to gain affiliation with a ministry or governmental research institute.
There is no specific governmental unit that deals directly with aging nor a
specific government program targeted to the old. Policy emphasis was placed on such
matters as maternal child health, increasing rural employment and decreasing the public
42


78
three hectares, but" 100 kilograms of Kafir corn {sorghum} could be grown in a good
year."
Crop land, like land in the village, was assigned by the chief. This was not done
haphazardly or without consideration of the potential user. The selection would involve
rationality. John chose his land carefully.
As a newly married man I had one piece of high ground. My
growing family required more land for sufficient food production. I
requested a second parcel about 20 kilometers from the village. I saw
that this piece of land was in a gully that carried water. I chose it
because it would produce in drought, when my other land would not. Of
course, if there are heavy rains, I lose out with flooding. This land may
seem far away but other people walked even further.
Daily Life
The above is the setting of the present day aged when they were children, or as
children who had become young adults. I will now present the early life-histories of
three individuals. Each had different family and social experiences.
Elizabeth. Elizabeth was born in 1888. Other than her cropped gray hair and slow gait,
her body belies her present age of 101 years. Quick in wit and tongue, she relates the
following experiences while breaking up wood and feeding the open cooking fire.
When I was born I was given my Tswana name of Botlhale,
meaning wisdom, as Elizabeth is my Christian name. My childhood was a
time of pleasure, working with my family at the lands and being
responsible as a very small girl for sweeping the hut and gathering fire
wood. My father reared sheep, and we used the skins as blankets. These
mmaselekwana were used only at night for they were very warm and soft
to sleep with. My father and mother made us children clothes from the
sheep skins. Girls wore fringed leather skirts that covered our lower
front. Boys, like the men, covered their private parts with a animal skin.
Everyone wore clothes to hide their nakedness.
Sometimes my father would travel to trade with the men of the
Kalahari. He would give them our home grown tobacco in exchange for
tanned hides. The best ones were from the bucks of the antelopes. They
were smoother than sheep skins, as the San had a special talent for
making hides soft and strong. We used these skins for special clothes.


178
hand from one to another. "People think I am this very old woman. I would like to be
regarded as this young one. I will take this lady as she is like me, not too old and not
too young. The young one will be my daughter." These pictures are placed by the
house. "My daughter is cooking, while I am supervising." She select more people.
"Here is my son, plowing the garden, and my grandson is hauling water. The
granddaughter is sweeping and the baby is happy." Suddenly she shakes her head and
jerks away all the people.
Who will take care of me when I am old. Family just causes
problems, too many problems. They argue and disobey the laws. They
spend money on themselves and forget the old lady. My children do not
obey. Having many people in the house causes fights. I want to be
happy in my house. By law I must have someone to take care of me, but
I would really be happiest if there was no one.
Slowly, she looks at the scattered facsimiles of people. She replaces the
granddaughter and herself. 1 will only have my granddaughter. She will obey the law."
The other pieces are picked up. "I also want Leru and Kaizer in the picture. I will
scream back when they yell at me, but that is breaking the law. I do not want to break
the law."
The caustic finish to the picture creates nervous laughter. Leru and Kaizer assist
Happy Sounds off the ground while commenting on her honest picture. The smiles
become more genuine as we gather up the assorted game pieces. Happy Sound speaks.
"Thank you; Thank you; Thank you. That was hard to do, but it was good."
On the way to the gate, both Kaizer and Leru comment on how little they
understood their mother before I began my visits. After extending an invitation to
return, Kaizer reflects on the afternoon. "I liked the picture. I never knew mother
thought that way." I leave with thoughts on how my data collection may influence
future family interactions. Do researchers have the right to interfere and make private


CHAPTER 9
FINDING THE GOOD UFE
I am happy as my family takes care of me and respects me. What will
happen in the future no one knows. (A 78 year old man)
I live with my family but they act like they dont care. Who will take
care of me when I am old? (A 68 year old woman)
Not a day passed when I did not hear the serious asking of the rhetorical
question of "Who will take care of me?" The presence of family and riches appeared to
have little to do with the asking. The question seemed to be more of a pondering of
the continuation of the present degree of life satisfaction. No one wanted to lose
whatever segments of the good life were presently available.
The components of the good life involve freedom from physiological want,
security in care and service, and being regarded as a worthwhile person. This chapter
delves into who finds the good life, and the role of the gerontic fund in success or
failure. First, time must be devoted to clarifying basic principles of the good life in
Botswana.
Core Elements in The Good Life
Historically, the basics of the good life during old age in Africa have been
thought of as respect and power. An additional right of elderhood was physical care
when needed (Schapera, 1955). The good life appeared guaranteed. The social and
family emphasis on seniority with aging gave the aged undivided power and authority
(LeVine, 1965; Alverson, 1978; Meillassoux, 1981; Turnbull, 1983).
242


276
to be somebody." The problem lies in the new social structure, which has altered the
overt expression of some the traditional behavioral laws, not the elimination of them.
Western education has given the younger generation knowledge and desire for achieving
through skilled employment rather than participation in agriculture. This employment is
limited in the cities and more so in rural areas. Adult children now want to live in
nuclear families. This means that the daughters must serve husbands first, rather than
fathers and mothers. The desire is present, the opportunity to fulfill valid desires is not.
The generalized poverty and unemployment means life satisfaction if difficult for
anyone to find. Younger rural men turn to alcohol, and rural women turn to single
motherhood in order to find satisfaction. These younger adults do not perceive their
behavior as deleterious to their future, as they maintain the belief in traditional
gerontocratic principles. "All good things come to those who wait." "One has children
to care for them in old age."
This may never be. What used to be advantages of the gerontocratic principles
are becoming liabilities. Some behavioral laws that remain intact work against the aged.
Valued items, once controlled by the aged, are now controlled by adult children and
grandchildren, and are not available for use. Social and economic demands on care-
providers have taken on new dimensions. They are not always available to provide food,
water and companionship. Service and security are frequently lost because conflicts
between time and duty cannot always be resolved by the care-provider. The care-
provider is also limited in performance, as other laws regulate the manner in which
service can be provided. The care-providers often prefer to maintain household unity by
upholding these laws rather than disregarding them and alleviating discomfort. All these
things place additional cracks in the doors to old age rights and security. Both the care-
provider and the aged care-receiver are trying to balance the precarious positions of
these doors.


156
TABLE 6.4 FIDUCIARY ASSETS. (N = 105 unless noted)
A Income: (Mean = 3.0)
1. Registered destitute. (11%)
2. Irregular remittances from others. (36%)
3. Irregular employment and irregular remittances. (8%)
4. Regular remittances. (32%)
5. Regular source of income (employment, rental, pension). (12%)
B. Investments in Cattle. Other Livestock. Animals: (N = 73; Mean = 1.7)
1. Owns no animals. (63%)
2. Owns chickens or a dog. (21%)
3. Owns less than 5 goats or pigs or has one cow. (4%)
4. Owns 5+ goats or pigs or 2-4 cattle. (10%)
5. Owns 5 or more cattle. (2%)
C. House Ownership: (Mean = 3.5)
1. Temporary housing provided by government. (0%)
2. Housing owned by other family member. (31%)
3. Owns house, major repair needed. (16%)
4. Owns house, minor repairs needed. (26%)
5. Own house, in good repair. (27%)
D. Furniture and Household Goods: (Mean = 3.1)
1. No bed, 2 or fewer blankets. (19%)
2. No bed, 3 or more blankets. (24%)
3. Owns bed, no furniture. (12%)
4. Owns bed and either wardrobe or chest of drawers. (13%)
5. Owns bed, wardrobe/chest and one other piece of furniture. (31%)
F. Agricultural Land: (Mean = 2.5, N = 103)
1. No control over land. (42%)
2. Controls one or less hectors. (7%)
3. Controls two to three hectors. (26%)
4. Controls four to five hectors. (9%)
5. Controls six or more hectors. (17%)


158
that, unless the aged generated their own money, none was available for personal
spending.
Men, compared to women, were more apt to be employed and to have pensions.
The four men who held pensions, (20% of males) had experienced forced retirement
from civil service or formal industry. With the females, which made up 80% of the
study groups, only two held pensions. This discrepancy is due to, in part, the numbers
of women in agriculture and past intermittent employment. In contrast, women were
more apt to have irregular money-making schemes. The most common were making
and selling bajalwa, and cutting purchased smoking tobacco into snuff.
All traditional beer in the village is made and sold by women, usually older
women. As a group, women provide a constant supply, yet individuals use this money
making scheme only once or twice a year. The making of bajalwa is labor intensive and
requires large amounts of either purchased or surplus grain. Frequently the initial cost,
labor and profits are shared between two or more women.
Snuff is popular with women of all ages, with men preferring smoking. The
making of snuff is an individual endeavor, usually performed by much older women on a
routine basis, processing one or two bags of tobacco a week for a small profit. Smoking
tobacco is purchased, then chopped at home with a large knife. Buyers bring their own
containers.
The Tswana have long regarded investment in cattle as the major form of savings
for old age (Schapera, 1954; Arntzen, 1984a). It must be noted that my initial
questioning on livestock was not satisfactory, as the aged included cattle owned by
extended family members in their answers. Hence, cattle ownership was omitted as a
gerontic fund component for the first 32 respondents. The revised question of "Some
people never owned cattle. Did you ever own cattle?" was more satisfactory. The
implied assumption was that none were held at the present time, and there was no need


186
A pick-up truck arrives from the mortuary. Women, sitting in the side yard,
watch as men in the center yard step aside to made an aisle leading towards the hut.
Jacobs closest male kin carry the coffin to a place of rest at the partially open hut door.
Over a hundred people join in the service of prayers and songs lead by the Christian
preacher. Afterwards, the pallbearers slowly move the coffin into the rondaval. Silently,
the group departs.
Betty and Jacob will spend his last night on earth together, alone, and without
interruption. The cooks will return to the open fires, some staying all night. The
brother will sit by the gate long into the evening. All will be quiet with thoughts only on
Jacob.
March 21.1991
The final preparations for the burial feast begin with sunrise. The meat, already
cooked, deboned, and shredded by male relatives, is made into Tshwaiwa. Mosokwane
(sorghum boiled to a solid state) and samp (corn mixed with butter beans) are prepared
by younger women. Older women are making tea and bread. The mourners arrive,
slowly at first, and then in larger numbers. Many stand outside the hedge, as no room is
left in the yard. Tobie and the babies are not seen.
The coffin is carried from the hut to the center of the courtyard. Ministers
conduct a second, more elaborate service. Bouquets of vividly colored plastic flowers,
wrapped in thick cellophane, are handed, one by one, from a table to the brother. He
reads the attached cards. "Your passing leaves much pain in our hearts, your nephew.
"You remain dear to us, rest in peace, your cousins. Men slowly circle the coffin for a
final, somber glance through a small window over Jacobs face. Women slowly enter the
circle as men return to their places. No sounds are made as Betty is escorted from the
house by Auntie and Dorothy. As her weak body slumps down, she leans heavily on the


152
household established by the old person. Another 32% have a person they could talk
with, but did not see this person daily. Usually this relationship is with extended kin in
the village seen weekly or monthly, or with a migrated child seen monthly or yearly.
Over a third (38%) have no intimate relationship. Spouses, if present, are seldom
mentioned in the context of intimate relationships. Married couples converse and rely
on each other but it may be that the "master-wife" interpersonal relationship prevents
intimacy.
The majority of men have a daily relationship that included intimacy. In
contrast, the mode for women is to have no intimate relationship. The discrepancy
between the genders is most probably related to the social laws. The elder man is
generally master of any compound. Only this authoritative figure is normatively
permitted to express dissatisfaction. This creates an ideal milieu for males establishing
what they interpret as intimacy. In contrast, women are expected to be submissive and
are confined in expressing thoughts. Female authority is limited to being the "woman of
the house." This is not necessarily the eldest female, for with the assumption of chores,
contemporary young women have also assumed control. Without authority, older
women become more restricted in permissible expressions of desires and reactions. This
thought is supported by the finding that both female intimacy and household control
decrease as age groups progress upward.
At one time I thought tribal affiliation and length of village residency should be
part of the fund. This was eliminated as all but two were Tswana, and all but four were
either born in the village or married into the community as a young adult. These
people were similar to other aged in assets and outcomes. This factor should not be
belittled in cases where it may have significant importance, such as research with urban
African aged where the custom of aging in place of birth is disrupted.


49
conversation, however minor, with a greeting and introductions. The discovery and
application of laws were relatively easy in regards to asking questions. Others laws, as I
found later, forced me to open myself to the existence of a new value system, and
temporarily disregard some tenets of my own.
Insight into potential research questions began with service providers, such as the
clinic and village leaders. The most valuable insights into the differences between
research design for aging in developed and developing nations came from the aged.
Participant observation in their household activities, long talks on the meaning of life
and aging, and the simple joining with aged in various activities allowed me to see
differences in life style and world view. Potential key informants were identified,
including John, age 75, and Monate, age 73.
John and I first met when he was walking home from the kgotla after attending
a planning meeting for the annual "United Nations Childrens Day." John was jubilant
about his old age. He thought that maybe some old people had problems, although he
was not sure as, "such things are not discussed with other people." He considered
himself exempt from difficulty, as he had his involvement with kgotla, a caring wife and
family, and good health. I offered to walk with him, being careful not to mention the
destination of "house."
Monate, his wife, was waiting for him on the porch. We shared a delightful
afternoon. They answered my questions. They asked their own. "I have often
wondered, where does the sun go at night?" "I heard America sent a man to the moon.
If the moon is up, why didnt he fall off?" "How long does it take to get to America?
Only 29 hours! Why I can get to Johannesburg on the train in 23 hours so it must not
be that far."
These questions were not a mark of stupidity but indicated two reflective minds
at work, searching for answers within their own realm of knowledge. In this way the


21
participation may be narrowed to only those role relationships and activities that are
most necessary or rewarding. This withdrawal accompanies a preoccupation with self,
leading to a new equilibrium characterized by a greater distance from social
relationships. In this manner an optimum level of personal gratification may be found
(Cummings and Henry, 1961).
Society also retreats away from the aged person. Society retracts because of the
need to fit younger people into the positions occupied by older people, who are thought
to be no longer as useful or dependable as they once were. Difficulties may occur when
either society or the individual is not yet ready to begin the disengagement process. It is
this lack of synchronization that leads to adjustment problems on the part of the
individual. High morale is reestablished as the gaps close and a new orientation to life
occurs (Cummings and Henry, 1961).
This model sought clues to both personal and social stability with the process of
social aging (Atchley, 1987a:186). Adjustment, leading to the attainment of the good
life, was based on the ability of the individual to separate self from the social
environment. The roots of generational and social conflict were perceived as stemming
mainly from the individuals failure to withdraw interest and commitment to others
(Cummings and Henry, 1961). The social contributions of the aged to themselves, the
family, and society were disregarded. The presumed inevitability and inherent nature of
the process left no room for active participation in life. Self-centered activity and social
idleness become the "normal and inevitable" rewards of aging (Cummings and Henry,
1961).
Disengagement Theory challenged the conventional wisdom that activity and
contribution were the best way to adjust to aging. This challenge led to an opposing
Activity Theory. Activity Theory held that older people are the same as middle-aged
people with each group having the same psychological and social needs. Activity


128
interpersonal behavior involved with income production, and the use of this income, is
negative. Social opinion, combination with her age, make her as a child, best to be
avoided if possible.
Sego portrays the Western picture of dependency and lack of autonomy. His
urban son also pictures him in this manner. In the village, Sego is one of the few highly
esteemed, social elders. What we perceive as dependency is actually the dynamic social
process of interdependency. His actions involve others, both in the extended family and
in the community. Not only does he gain from relying on others but others gain through
serving him. His remittances, similar in size to Dianes income, are used to help others.
Decisions are seen as wise, as they are made with group input. The high priority given
to his extended family with much give and take is admired, and no one counts the
lopsided directional flow of assistance with his physical decline. Although similar in age
to Diane, he is not feared.
Autonomy extends beyond doing what one desires to include the maintenance of
the integrity of the self (Munnichs, 1976:5). Society defines the values that should be
maintained. Sego is autonomous, expressing integrity in his actions that are in harmony
with the social context. Because of this, Sego is seen as the true elder, set apart from
most others. He admits that he has to purposely continue in the process of achieving in
order to maintain his status.
The term motsofe, once a positive term for old age, has now become pejorative
and carries degrading connotations. The aged see living to sixty and beyond as a reason
to be proud. Yet, in the public eye all aged are nearing or in childhood. It is better to
avoid them than risk danger, now that no one knows who can be a witch. The resulting
withdrawal of opportunities to engage in meaningful behaviors, and the avoidance of the
aged from fear, places the aged in a precarious position. The active and the physically
limited yet mentally alert, although viewed as useless, see themselves with much to give


91
Monate joined her husband in South Africa a few years after his initial
departure. She found work as a maid to an English family in the area. The couple
openly talk of their approach to migratory labor.
We knew our success was dependent on the way God wanted our
life to go. People have no control over such outside forces, or can one
make definite plans for the future as God brings that, but one must try to
better themselves. All men are good and without evil, so we were not
afraid of what would happen if we left Ramotswa. To better ourselves
we learned English as we worked. When times changed, we knew enough
English to find work in the cities.
In contrast, Elizabeth remained in the village to work the lands while her
husband worked at the mines.
Before he left, we made sure all his possessions were in place,
where no one would touch them. Just to touch the belongings of
someone working in the mines would cause his death. Even a shirt left
on his bed must stay there until he returns. Our house was on my in
laws compound. I had to obey my mother-in-law as she taught me to
take over chores. That wasnt so bad, as all things come to people who
wait. Eventually I was in charge of the house.
World War II brought new economic demands to the village. The colonial
administration increased taxes for the war fund and worked through chiefs to increase
migration to the Rand mines for gold production (Bhila, 1984). It was estimated that
40% of adult males and 5% of the females of Ramotswa were absent at labor centers in
the Union of South Africa during the 1940s (Schapera, 1953:30). Other men were
enlisted in labor battalions to serve the British Army in northern Africa and southern
Europe (Bhila, 1984).
The Money Market Economy and Family Life
The effects of early massive labor migration on family welfare differ between
British ethnographers and oral history. Writings emphasize the lack of economic
support from absentee workers, including defection from family responsibility and
complete desertion. Many aging parents left unprovided and had to "fend for
themselves" (Ellenberger, 1937; Schapera, 1944; 1953).




247
on four items for both of the two categories, using a five point scale per item.
Achieving is the most subjective of three variables. It is also the area where the
confines of language and world view affected data gathering the most. The respondents
were unable to provide gradations for responses. This resulted in specific yes or no
answers for the five tested items. The total score is the addition of scores for the three
areas, as differences in question design among the areas prevents valid weighting of
categories. Hence, the total scores de-emphasizes the area of personhood. I limit the
use of total scores for this reason.
Freedom From Physiological Deprivation
Freedom from physiological deprivation encompasses the areas of having
adequate food, the necessary expendable goods for life maintenance, health provisioning
and protection from the environment. (See Table 9.1.) The aged stress, that while
there can be disappointment in not having advantages of living a middle or upper class
life style, it is not a necessary dimension of success. There is no self-judgement or
shame involved with the lack of material goods. The aged define the minimum as
having enough freedom from deprivation to allow adequate calories for health and
activity in a safe, health-promoting environment.
Freedom from hunger is mentioned repeatedly as a prime component in the
good life. The frequency of meat and vegetables intake is not included as a variable, as
the group considers obtaining any food the prime objective. Three solid meals a day are
obtained by one/fourth of the aged. Another fourth are at the other polar end, having
one meal a day, either with or without milk. Milk is the foodstuff singled out by the
aged as it balances the grain and prevents nausea with tea. Approximately half eat
twice a day, with slightly over half of this group having milk. Eating between meals is
very rare, as snack foods are not generally affordable.


224
health care and medications do not seem to guarantee availability, as other indirect
expenses are too great.
Earlier, I supplemented Sepias expense of illness through my desire to provide
comfort with a blanket. The error was acting on my cultural care-giving heritage instead
of his, bringing shame to me and disquietude to Sepia. Similar unconscious errors were
observed repeatedly with foreign health-care providers. The difference was that the
cultural conflicts were not recognized. Instead of embarrassment, there was anger for
impropriety of behavior or refusal to comply. This anger was directed towards the
Tswana patient, adding timidity to already present trepidation.
Health-care workers also make errors in failing to seek depth for the
interpretation of symptoms. Monate knows that total wellness does not include
coughing, but similar to undernutrition, malady is an accepted inclusion in everyday life.
Coughs and weight loss are to be expected with aging in poverty. In Monates case, arm
pain is the major concern, as it prevents work. In terms of health, the cough is the most
important symptom to treat. In the same vein, individuals seek help for dizziness and
headaches during the hunger season, without mentioning weight loss or a lack of food.
Yes, the proper treatment of the presented symptom is pain-relief. Although correct in
theory, such treatment falls short from alleviating the true cause of symptoms and
complaints. I openly admit providing comprehensive medical care based on what
patients say, and how they perceive health, can be a challenge. The reality of the
effects of poverty, and belief in sorcery, should be incorporated in meeting this
challenge.
In opposition to misinterpretation, the Tswana frequently fall overboard in
interpreting the path for health. This too must be considered in the total scheme of
health care. The drive for health unnecessarily limits the ability to meet the labor
demands of poverty. The life-long washing of surgical scars, and life-long avoidance of


194
to be taken literally. The daughter realizes she skimped in cooking time, the son thinks
of the money spent on himself instead of the family. Happy Sound, with repeated
questions on future care, was not implying a fear that she would be deserted. Instead,
she was making known her familys failure to serve and defer, according to her
perceptions of rights and proper treatment.
Disagreement, separate from complaint discourse, follows a different course.
The aged report that, based on tradition, the eldest person rules with no back talk
permitted. They say argument was unknown in the days before civilization. If this held
true, why the reported use of uncles, headmen, and lastly the chief to settle household
differences (Schapera, 1953:40)? No doubt dispute has always been present in
household. The question is how verbal did such differences become.
Todays open expression of conflicts between siblings, and between children and
parents, creates obvious anxiety in aged parents. To the aged, acts of yelling extend
beyond the purposeful breaking of the laws. Open arguing between any family members
presents a threat to their status as elders, as it overtly shows that the aged member lacks
control over the situation. The yelling and fighting between adult children is the most
mentioned cause of unhappiness by the old. Participation in yelling is perceived by the
aged as an undesirable source of power, as it indicates a lack of self-control. In
contrast, the discarding of other discourse regulations does not create as much anxiety.
Many of the aged break the rules themselves, as they talk out of turn, interrupt, boast,
accuse, and praise directly.
On the other hand, the younger generations accept open and loud disagreement
as a valid method for anxiety reduction in times of conflict. To them, fighting between
siblings is not associated with respect for elders, and argument with parents is viewed as
a necessary a step for the now desired independence. It is not used as a purposeful


149
TABLE 6.3 SOCIAL/FAMILIAL ASSETS. (N = 105)
A. Adult Children: (Mean = 4)
1. No adult children or has lost contact. (10%)
2. See adult children less than once a week. (7%)
3. Adult children in village, sees once a week or more. (11%)
4. Contact with adult children in household only. (19%)
5. Contact with adult children in and out of household. (53%)
B. Grandchildren: (Mean = 4.1)
1. No grandchildren. (10%)
2. Has grandchildren but not in household. (3%)
3. Only children 14 years or below living in household. (11%)
4. Has only children above the age of 14 in household. (13%)
5. Has children of all ages in household. (62%)
C. Other Meaningful Family: (Mean = 3.7)
1. No other meaningful family. (15%)
2. Sees less than once a week. (6%)
3. In village, Sees once a week or more. (9%)
4. In household only. (34%)
5. In household and village. (36%)
D. Intimate Relationship: (Mean = 2.9)
1. No intimate relationship. (38%)
2. Has intimate relationship, sees person less than once a month. (7%)
3. Has intimate relationship, sees at least monthly. (10%)
4. Has intimate relationship, sees at least weekly. (14%)
5. Has intimate relationship, sees daily. (30%)
E. Known to Service Agencies: (Mean = 3.5)
1. Has no contact with service agencies. (7%)
2. Is known to one service agency. (18%)
3. Is known to two service agencies. (22%)
4. Is known to three service agencies. (26%)
5. Is known to four or more service agencies. (28%)
F. Access to Transportation: (Mean = 2.7)
1. No access to transportation. (18%)
2. Access to bus only. (13%)
3. Access to bus and car outside of village. (55%)
4. Access to bus and car in village. (5%)
5. Access to bus and car in household. (9%)


TOTAL NUMBER
63
600
550
500'
450-
400'
350'
300'
250-
200
150
100
50
O'
Wmm
m,
Children
Adults
AGE GROUP
Aged
Figure 3.2: Population Breakdown in Census Areas by Age Groups and Sex


TABLE OF CONTENTS
page
PREFACE iii
ABSTRACT xi
CHAPTERS
1 INTRODUCTION 1
Overview of The Study 3
The Good Life 15
2 THEORY 20
Early Gerontological Theories 20
Modernization Theory 23
Social Environmental Theory 27
Research Hypothesis 37
3 ORGANIZATION OF THE STUDY 42
Formal Permission For Research 42
Research Design 50
Becoming a Village Member 59
Village Demographics 61
4 THE EVOLUTION OF THE VILLAGE 69
History of the Malete Prior to 1885 69
Early Colonization: 1885-1935 71
"Becoming Civilized": 1936-1966 85
"Being Modem": Post-1966 97
5 GROWING OLD: THE LIFE CYCLE 102
The Attributes of Aging 102
The Life Cycle 105
Variables Underlying The Life-Cycle Phases 121
6 THE INDIVIDUAL: ASSETS FOR USE IN DAILY LIFE 130
Priscilla and Alfred 130
Maria and Martha 132
The Gerontic Fund 133
A Comprehensive Look at The Gerontic Fund 167
7 THE AGED AND THEIR FAMILIES 170
The Kerengs 172
The Mosokos 179
IX


171
wage earners. (Goldstein, Schuler and Ross, 1983). In turn, wage migration further
thwarts intergenerational ties and removes family as an asset (Shostak, 1983; Khasiani,
1987).
Many African rural aged do reside in extended families, which have been diluted
in size through wage migration and thinned in ability to provide adequate care due to
economic pressures (Hampson, 1982; Hampson, 1985; Hay et al., 1985; Tlou, 1986;
Khasiani, 1987). These new social conditions exist side by side with continuation of
familial piety, thereby providing protection to the majority of rural aged (Biesele and
Howell, 1981; Colson and Scudder, 1981, Shostak, 1983, Khasiani, 1987). As mentioned
earlier, this pollyannish conceptual framework, and the assumption of normative care
provisioning tempered with economic constraints, can have faults. No situation
automatically fulfills the needs and expectations of the aged.
Thought needs to be given to the communications and behaviors found within
the locally existing families of the aged, far beyond the economic angle. A hard look at
the Tswana family will be taken in this chapter. The intent is to present the family of
today as a setting for social exchange, asking if the multiple changes in family life have
sealed or broken traditional doors and/or created new ones and thus effecting the value
and use of the gerontic fund. I do not stress the gerontic fund as such. My intent is for
the reader to realize how the individual and family interact. This includes the
generalized give and take, the various options for exchange, and the way decisions are
made within the family.
I have chosen two typical households and their extended kin to illustrate
common threads reflecting culture and historical developments. Factors behind kin
interactions, and behaviors, are discussed afterwards in terms of cultural continuity and
social change. The chosen events and behaviors described are not unique, although I do


296
Gage, Timothy B. 1991. Human Variation in the Age Patterns of Mortality. Association
for Anthropology and Genontology Newsletter 12:3:6-7.
Glascock, Anthony P. and Susan L Feinman. 1981. Social Asset or Social Burden:
Treatment of the Aged in Non-Industrial Societies. In Dimensions: Aging,
Culture and Health. Christine L. Fry, Ed. Bergin & Garvey Publishers, Inc.,
South Hadley, MA
Goldstein, Melvyn, Sidney Schuler and James Ross. 1983. Social and Economic Forces
Affecting Intergenerational Relations in Extended Families in a Third World
Country: A Cautionary Tale from South Asia. Journal of Gerontology 38:6:716-
724.
Goodenough, Ward Hunt. 1963. Cooperation in Change. Russell Sage Foundation, New
York, NY.
Gubrium, Jaber F. 1973. The Myth of the Golden Years, A Socio-Environmental Theory
of Aging. Charles C. Thomas Publisher, Springfield, IL.
Guillette, Elizabeth. 1990. Socio-Economic Change and Cultural Continuity in the Lives
of the Older Tswana. Journal of Cross-Cultural Gerontology 5:191-204.
Guillette, Elizabeth A. 1991. The Impact of Recurrent Disaster on the Aged of
Botswana. Paper presented at the 50th Annual Meeting of The Society for
Applied Anthropology. March 18,1991. Charleston, SC.
Hampson, Joe. 1982. Old Age: A Study of Aging in Zimbabwe. Mambo Press, Gwere,
Zimbabwe.
Hampson, Joe 1985. Elderly People and Social Welfare in Zimbabwe. Aging and Society
5:39-67.
Hansen, Art. 1990. Dependency and the Dependency Syndrome. Paper presented at the
American Association of Anthropology. November 28 December 2,1990. New
Orleans, LA
Harrison, Paul. 1987. Inside the Third World, The Anatomy of Poverty. Penguin Books,
London, England.
Havighurst, Robert J., Bernice L. Neugarten and Sheldon S. Tobin. 1963. Disengagement
and Patterns of Aging. In Middle Age and Aging. Bernice L. Neuggarten, Ed.
The University of Chicago Press, Chicago, IL.
Hay, Roger W., Susan Burke and D. Y. Dako. 1985.^4 Socio-Economic Assessment of
Drought Relief in Botswana. Botswana Co-operative Union, Gaborone,
Botswana.
Hendricks, Jon. 1982. The Elderly in Society: Beyond Modernization. Social Science
History 6:3:32-45.
Hendricks, Jon and C. Davis Hendricks. 1986. Aging in Mass Society, Myths and
Realities. Little, Brown and Company, Boston, MA


The Family as a Setting for Exchange 189
8 THE AGED AND THE COMMUNITY 208
Social Interactions 210
Social Concurrence and Collision 240
9 FINDING THE GOOD LIFE 242
Core Elements in The Good Life 242
Finding The Specifics of The Good Life 246
10 AGED CHILDREN: SOCIAL ELDERS 261
The Demand for Innovation 261
Aged Children Become Wise Elders 265
11 CONCLUSIONS 270
The Aged 271
The Context for Exchange 272
A Comparison to World Aging 277
APPENDIX A: THE GERONTOLOGICAL ASSESSMENT FORM 281
APPENDIX B: THE LADDER INSTRUMENT 291
REFERENCES 293
BIOGRAPHICAL SKETCH 303
x


44
Village Visitations
I now had to leave the familiar safety of the capital city of Gaborone and
venture out into the unknown. The first unknown was the bus station. In spite of dire
warnings of personal danger and the dictum of "Dont go near the bus station" from
European and American workers, I had no other transport. Eventually the bus station
became one of the highlights of my trips to Gaborone, meeting new friends or sitting on
the curb and sharing an ear of boiled maize with old friends. Masses of travelers, street
venders, trash and aromas generated a sense of accomplishment when boarding the
correct unmarked bus.
I must admit that ambiguous thoughts and anxiety dominated that first bus ride.
I was meeting the unknown and anticipating differences and similarities with the known.
I planned to base the wording of my study on the local reflections toward the aged.
This seemed very logical for meetings with local government officials. My key concern
was how I would approach the chief, as he was the protector of his people and culture.
In Ramotswa, this person was the "paramount chief of the Malete," in contrast to a
village chief. His approval was mandatory! The stereotypical image of a tribal
authoritative figure, gained from old pictures and ethnography, was firmly locked in my
mind. I knew the image was not reality but I could not shake my expectation of
required submissiveness under his power. My reverie was cut short with numerous
nudges and shouts that I had arrived in Ramotswa, and the South-East District Council
was on my right.
Contrasting with the earlier central government presentation of nonchalance
toward the aged, local district government was open and frank regarding the necessity to
address social problems relating to old age. Some officials placed the problems on the
shoulders of the aged, claiming that laziness and adherence to the old ways were the
sources of their difficulty. Others blamed social change with the refusal of adult children


180
the brain is different between men and women. The mans brain is made
so he can be Master. He can see the important things in life and tell
others about them. The womans brain is more soft, so she can tend to
children. She knows all about the house and how to make people happy.
This doesnt mean she is less intelligent. It takes both kinds of brains to
have a good family as decisions in the house are often made together
with both man and wife saying what they see should happen.
An eight year old boy enters the gate in the hedge and quickly disappears behind
the house. Jacob beams.
That is my grandson, Tobie. He is coming home from school. I
wish my granddaughter was as trustworthy as Tobie. I have two sons,
both live in South Africa. One was divorced three years ago, It is the
law that with divorce all children go to the father. That is how we got
Tobie, as my son could not take care of him. The other son was
divorced a long time ago. At that time he gave us his infant daughter,
Dorothy, to raise while he worked. She is out having fun in the village
with her friends while her baby is inside. She thinks Betty should care for
the baby. Betty is old and is raising Tobie so she says Dorothy should
tend to him. Dorothy is too young for me to give her responsibility for
the household. Like other young adults, she gets caught up with people
in the village and thinks of herself. She is worthless as a grandchild. She
does not try to achieve, but lets nature take its way.
Tobie comes out with a bucket to get the evening supply of water. Betty pokes
her head out the door, a four month girl in her arms. She asks if Dorothy is home yet.
Jacob shakes his head. As we disperse, he calls out "Come back next year.
December 19 1990
I have seen Jacob and Betty many times since returning to Ramotswa. Both
have lost weight and have become disillusioned with old age. The pension was only for
twelve months, not life. Tobies father has not sent money for three months. Dorothys
father has not been heard from for seven months. He still does not know Dorothy had
a second baby in November. Both of her babies receive free supplemental formula, as
Dorothy is unemployed and the children are underweight. Tobie gets a meal at school.
Jacob and Betty applied for destitute funding last month but were turned down.
Betty said the social worker entered the house and saw the table and dresser. "This
made her think we are rich, without thinking that we could have had money before but


12
advisement is seen as based on uncomprehending thought. Even the chronologically
old who are recognized as active and valuable social elders, with multiple resources and
vigor, are constantly and actively working against the label of "child."
Tradition and Cultural Continuity
Modernization of village structure has not supplanted tradition and cultural
beliefs in the varied aspects of daily life. Even the most modern of people exhibit
actions and thoughts reflecting cultural continuity. Traditional ideology still dominates,
although frequently in a modified manner. Underlying all behaviors are two cultural
traits: the continuous "process of achieving" and a following of what the Tswana refer to
as "the laws." These will be defined and discussed separately.
The Process of Achieving
Achieving is an on-going, perpetual process. One never achieves, as there is
always a need for continuing participation in the "Great Works" of supplying food,
building a family and keeping community commitments. The person is not judged by
past achievements, but the continuation of them (Alverson, 1978). The focus is on the
present. The past is completed and to be forgotten. The future is unpredictable and
should not be the foundation for worry. There is no such concept as self-caused
personal failure with achieving. It is external circumstances that get the upper hand and
prevent progress within the on-going process It is the failure to strive for betterment
under hardship makes one useless, for it is the "doing" that provides social value, plus
contentment and fulfillment in life.
In todays world, the process of achieving continues to emphasize family
corporacy, generational symbiosis and social solidarity. Actions are important in that
they not only accentuate ones own welfare but help others. Family roles and a
meaningful place in society are paramount. These attributes direct the group-centered
approach to achieving. The process should involve interdependency between family


33
individual is less a function of age than one of social values. Continuing adherence to
middle-age values with visible productivity is the only means for acceptance (Hendricks,
1982).
Social Environmental Theory, as an adaptation of social exchange, provides the
basic framework and methodology for this research. Both the aged individual and
society play an active role in the process of aging. Exchange behaviors, reflecting
resources, action and attitudes, affect the process and individual outcome. This provides
the essential structure in evaluating the aged as individuals, and as a functioning group
within a society.
Social Exchange Theory, involving resources and success in trade, has been used
in famine research (Sen, 1981). As far as I know, the gerontological Social
Environmental Theory has never been applied in total, with the delineation of actual
resources, the reception of others in accepting resources in trade, and gaining the good
life. I attempt to apply the complete theory to the Tswana aged as they experience life
in Botswana, a contextual setting very different from America. Therefore, I must take a
critical look at the concepts, including the asking of some questions without answers.
Many of the references cited for the actual Social Environmental Theory reflect
thoughts on its application to political issues and the generalized reaction of society to
the aged. Much of the background material for theoretical claims came from relatively
stable Western societies. Gubrium (1973) provides much data for the reasoning behind
concepts but provides only brief categories of resources in the resource dimensions. I
must take these fragmentary categories, adjust them for the Tswana and make them
complete.
The theory, as a reflection of Western thought, definitely presents challenges
when applied across international settings. Both social exchange and social
environmental theory are set in a money market context, stressing that the basics of


227
she reaches the footpath. Elisa and her cousin, at first startled, now laugh heartily. The
cousin is asking more about the teeth, as I quickly close the interview.
Back on the footpath, the trembling Agony refuses to let me explain. I see how
the removable teeth has only reinforced her earlier learning about life.
That woman is a witch. I always thought she was a witch. I can
tell because of the look in her eyes. This just proves it! There is no way
a person can take their teeth out! Old people are the worst kind of
witches. They go to church to get Gods blessing to perform their crafts.
That old woman is a witch, and Ill never go back there again.
Agony never allows me to speak of the miracles of modern dentistry.
New technology, from fancy earth movers to computers, is constantly introduced
to the village. The aged have extensive experience with new and unusual objects
entering their lives. Dentures are but one more thing the modem world has created for
better living. Much to the new originates from outside the area. Elisa, in a position to
obtain dentures, did so. Surprize to her cousin arises from the uniqueness of this
marvelous new appliance, as it is just one more item she had not seen before.
For those young adults, who have never migrated, familiarity with technology
stems from what has been in the village for some time (telephones, radios and
electricity), or from the things their age-group has brought in (portable tape recorders
and digital watches). The aged are believed to be "out-of-date" and "out-of-touch" with
material goods symbolizing progressiveness. In Agonys eyes, this belief is particularly
applicable to Elisa, an old, child-like woman. Explanation could only come from what
Agony already knew: the metaphysical world. The metaphysical provided a sound and
valid basis for understanding the event. How else could a woman remove teeth, other
than being supernatural herself? The explanation is that much more plausible as Agony
knows that aging and the powers of witch-craft coincide. Tradition, which the young try
so hard to evict from their life style, continues to intersect their daily lives. Old beliefs
infiltrate thought, sway reasoning, and tinge the direction of behavior.


110
segment of work. At all times, the person is learning about life. Respect and deference
increases as one ages, and is greater for males than females.
Elderhood: The Mopomo
As the years pass, a person gains recognition of being a social elder, or mogomo.
A social elder is one who is still growing-up but knows much. The woman becomes a
masadi mojolo and the man a manna mojolo. These terms do not parallel our terms of
grandparent for social aging. The elder may have had grandchildren but people may
have grandchildren when relatively young, and the sixty year old woman may have a
teenage child. Strands of grey hair or laugh lines around the eyes are frequently named
as a marker of elderhood but a good number reach their seventies or more without
dominant greying or massive wrinkles.
Some fifty year old women proudly consider themselves to be masadi mojolo.
Men tend to wait until they are much older before labeling themselves as manna
mojolo. Basically, the terms signify that the person has many years behind them, enough
years to be proud of living a long time in a village where many continue to die young.
Social elderhood has been reached, which reflects the presence of judgmental over
physical strength. Many duties have been assigned to younger household members, but
elders are not idle. Available time, not age, provides opportunity for leadership; the
growth of wisdom contributes to the quality of leadership. The household is under their
control. The village considers their decisions concerning social and service club matters.
Romanticism needs be avoided in describing this period of life, as all is not ideal.
Conflicts exist between social fact and its application. Elders must actively strive to
maintain their position, as deference is no longer automatic. The right to control
through seniority in age must be proven with demonstrations of proper knowledge and
contributions. No one claims special privileges or taboos associated with elderhood.


268
stored in the silos, maintained but empty since 1935. The South-East District Council
agrees to transport the food on the weekly service truck coming from Gaborone. The
Embassy of the United Sates of America in Gaborone agrees to provide limited funding
for the initial purchases, through its special Development Assistance Program .
A short but relevant note must go here. Several externally-based volunteer and
grass-root organizations denied the group financial assistance. Careful presentations of
needs and environmental constraints could not overcome ethnocentrism. "Senior Citizen
Programs should provide dances and parties so the aged can have fun." "Why arent you
including field trips in the program?" "You should establish Meals-on-Wheels to provide
cooked meals."
Excitement about the program increases during my remaining days. The aged
see The Wise Elders as a means of achieving in their own right. Through the
purchasing program, the aged can obtain food economically, for the benefit of the
family. Families begin to think of the aged as providers of food, not an empty mouth to
feed. The public makes comments that imply that the aged are becoming recognized as
capable of valid and valuable contributions. More remarkable to the public is the fact
that the activity of the aged is being supported by the government and tribal
administration.
Repairs To The Broken Doors
The Wise Elders purposely overlooks the often stressed Western goal of creating
independence and stimulating self-reliance through self-help programs. The desired
outcome is built on the traditional goals of family interdependency and cohesiveness.
Program design emphasizes that the aged are not the recipients of service or care, but
are the providers for enhanced family nutrition and contributors for economic savings.
The Wise Elders program is unique in that it placed the aged in a position to
begin repairs on the broken doors and thus increase their access to sources of social


151
them. If grandchildren live locally they tend to reside with the grandparent, as only 3%
were with local but nonresidential grandchildren. The 10% without any local
grandchildren lamented this fact.
Adult grandchildren, with adult roles and duties, were considered as extended
family members. Spouses, siblings, parents siblings, even more aged parents, and Active
kin, were also classified as extended family members. They must have had positive
significance in some manner to be considered an asset. The relative who was not
desired to be seen, thus never seen, was excluded. Some extended family members were
older than the individual under study, some were younger, but all the significant others
mentioned by the aged were adults. Again, proximity was the key in determining the
strength of the asset.
The aged are evenly divided between having significant others in the household
and not in the village (34%), and significant others in household plus weekly contact
with relatives in the village (36%). Very few (8%) have weekly contact with family
members residing in the village with no extended family living in the household. About
one/fifth of the aged (21%) are without any extended family or have less than weekly
contact with them. It may be that such kin were never significant. More likely, in
consideration of the social context, having kin in the household increases the likelihood
of maintaining meaningful relations with other family members.
Age, gender, kin relationship, and social norms enter into the degree of
permitted emotional intimacy. Intimate relationships are also affected by geographical
distance. As Maria says, "Even though I have two daughters and many grandchildren,
there is no one I can talk to, I mean tell my feelings and share the good and the bad."
This type of intimate relationship is included as an asset.
Slightly under a third (30%) have an emotionally intimate relationship with
someone seen daily. The person is usually an adult child of the same sex living in a


FREQUENCY
67
40
35
30
25
20
15
10
5
0
60-69 70-79 00-89 90-99 100-109
AGE GROUPS
Figure 3.3: The Interviewed Aged by Age and Sex


24
age groups. All ages would be involved with this change as critical shifts in attitudes,
ways of life, and social structure occurred. In turn, this change influenced the experience
and interpretation of aging (Achenbaum, 1987:453). It was believed that economic and
technological innovation transformed the roles of the elderly. The aged lost ascriptive
status and could not compete with others in the new environment. The assumption was
that modernization marked the beginning of the end for old people.
Leo Simmons presented this assumption in The Role of the Aged in Primitive
Societies (1945) and later argued that the role of the aged in a given society was
inversely related to the level of technological development and occupational
specialization (Simmons, 1960). The premise was that the structured framework of
primitive societies favored the elderly as advisors, carriers of culture and controllers of
civic and political powers. With introduced technological change, seniority rights
disappeared as new power-producing roles were allocated to the young (Simmons, 1960).
Cowgill and Holmes, the leaders in the application of Modernization Theory to
the aged, conclude that all factors of modernization lead to social change that is inimical
to the status of the aged (Cowgill and Holmes, 1972). Society moves away from giving
beneficial ascriptive status for the old and institutes achieved status based on a money
market economy (Parsons, 1964). Whatever seniority rights and authority the aged
possess are undermined and eventually eliminated as the young usurp power (Cowgill
and Holmes, 1972). The speed or intensity of change, either in the present or future,
cannot be predicted (Achenbaum, 1987).
Modern health technology, economic technology, urbanization and rising levels of
education were identified as the prime movers in the devaluation of old age. These
factors unite in a functional manner to deprive the aged of their central importance in
the affairs of daily life. In any event, status for the aged inverts as the old are


129
as good people. This contrast of perceptions influences the availability of doors, with
age-children having the most difficulty in gaining access, regardless of its condition. If
access to doors can be made, the door of acceptance, which has always had cracks and
damaged areas, is now more severely broken. The following chapter will examine the
assets the aged control to use in their attempts to deal with the existing doors.


160
Table 6.5: History of Cattle Ownership and Herd Loss with Drought by Age Group.
Year
Herd
Lost
Age
Group
N
# Never
own
cattle
# Owned
Cattle
1930
1960
1980
Retain
Cattle
60-69
31
8
(26%)
23
(74%)
0
7
(30%)
12
(52%)
4
(17%)
70-79
19
4
(21%)
15
(79%)
0
10
(67%)
4
(27%)
1
(6%)
80-89
19
3
(16%)
16
(84%)
0
13
(81%)
3
(19%)
0
90+
4
1
(25%)
3
(75%)
3
(100%)
0
0
0
Totals
73
16
(22%)
57
(78%)
3
(0.5%)
30
(53%)
19
(33%)
5
(9%)


Traditional ideology continues to influence expressions of thought and behavior.
Social change alters the directions flow of benefits from the aged to the younger
members of the village. Families, which continue to be thought of as insurance against
old age, usually provide basic physical care but most aged are excluded from roles of
authority and self-expression. A community disregard of the social contributions made
by the aged adds to their disenfranchisement from a meaningful life.
xii


23
quantity, of activity that determines if satisfaction exists (Hendricks and Hendricks,
1986). Second is that the availability and performance of meaningful interactions are
determined through the offerings of society. It is now clear that psychological
disengagement, or continuous meaningless behavior, is neither natural or inevitable, and
that most cases of poor activity selection result from a lack of opportunities for
continued involvement (Atchley, 1987a: 186).
Modernization Theory
Modernization Theory began with the social sciences as a means to analyze social
change. The theory assumed unilinear evolution, with universal patterns in structural
growth that developed along set lines, regardless of context (Parsons, 1964). As
technology was introduced, all aspects of a society were thought to move away from the
diffused activities based on the closed ascriptive status systems that were associated with
extended kin networks Eventually, industrial-intensive societies, based on achieved
status, would evolve. The need for integration of modem bureaucracies and money
market economies would create convergence of modem structural characteristics in all
societies. New cultural values to justify the emergent dimensions of human society
would evolve from changing language, religion, and other belief systems (Hendricks and
Hendricks, 1986:100). These values would then be incorporated into human
relationships and reflect secular and instrumental rationality.
Gerontologists perceived within Modernization Theory a useful framework for
describing and explaining continuities and changes in the social position of the aged
across space and over time. The theory had drawbacks because of its generalized,
political nature and its emphasis on the reliance of developing nations on established
countries for socioeconomic direction. Instead, communities became the focus, rather
than nations. The interdependencies in the local socioeconomic-political arenas directed
the aging process. Social change was the cause of change in relationships among various


251
earlier research suggests employed adult daughters do not intend to return to the village
to provide care for ill aged-parents (Guillette, 1990).
Assistance and care-giving begins long before the onset of frailty, as duties are
delegated to growing children by parents. Later responsibility for household
management is given to adult children who remain in the village, thus establishing the
concept of family acceptance of old age disability and provisioning for more complete
care when the need arises. The care the aged seek includes emotional support, as well
as physical care and normative assistive behaviors. (See Table 9.2.)
Within this category falls the earlier mentioned norms of being served cooked
food, having water supplied for personal use, assistance with chores, and home
maintenance. Reliance on others is something to be proud of, contrasting with the
negative judgment of dependency found in America. Many aged can, and do, perform
these acts. Performance is viewed as part of achieving, but only when such services are
available. For example, Happy Sound did not see herself denied service although she
did the majority of household cooking and other chores. Many were not constantly
"served", but approached the problem like Happy Sound and her chores. Outside
forces, such as employment and school created the need for performing the act oneself.
Other aged are sometimes forced into doing these activities, which should be
provided as a right of aging, when assistance is not available or refused. This lack of
assistance and/or refusal brings shame. Idleness, play, or self-centered activity on the
part of service provider are not justifiable grounds for omission of service. This is
frequently the case with the Mosoko family, where Betty resents having to clean and
cook.
One/third of the group consider themselves as receiving all the rights to service
on a regular basis. Availability and access to prepared food is the most common missing
service for all those not receiving full rights. Of the 67 aged who were hungry in the


191
created personal hardship in a household where it could have been avoided. This self-
denial was part of the Tswana way of finding integrity in achieving.
No one denied the aged food if they asked during meals, or housing when a roof
was blown off during high winds. I found that sick or extremely hungry aged, including
those who lived alone, always found temporary assistance from kin if help was actively
sought. (Hesitancy to ask was from the awareness that others also live in need, and/or
from fear of sorcery.) It is not polite to constantly ask a particular individual or
household for food or money everyday. A few of the very poor say they "have run out of
kin to ask." Somehow, even these individuals find meager, but sometimes life
supporting, food.
No sexual misconduct towards the aged is ever mentioned. I also doubt if it was
present, as laws regarding sexual activity were strictly adhered to. Rape is virtually
unknown. No aged acknowledge ever being beaten as an old person. One womans
complaint is that the new law regarding beatings prevented her from knowing where she
stood with her husband!
The Tswana perceive situationally-induced deprivation as quite different from
abuse. Suffering induced by such lacks as material goods is seen as the usual way of
life. Being cold from the lack of a blanket, or uncomfortable from a lack of a bed or
chair, is interpreted by the aged as a result of self-poverty. Adult children and other
family are not faulted. Additional goods may be desired, but they are not expected as
gifts. Only the most affluent households may give the aged a blanket or cloth slippers at
Christmas. Usual gifts are a bar of soap or a box of tea. Migratory children, returning
for a visit, usually bring a a few food stuffs to be shared by the household. Such items
are seen as gifts, not as part of the obligation of support.
Many people, of all ages, suffer from some sort of economic shortages.
Whatever extra money is available is spent by the producer of that money, usually for


52
As I have demonstrated, many facets of the research could not be approached
with standard instruments or Western concepts of personhood and aging, without
comprising validity. Some conventional questions were used with revision. Other
question were devised for this particular ethnic group. I sought additional validity with
pre-testing and evaluation of instruments with 20 aged villagers before use. This pre
testing stimulated adaptation of some questions and the elimination or addition of
others. Throughout the study, the aged continued to identify the more elusive segments
of culturally specific domains while answering ubiquitous questions regarding life in
general. Areas that were identified early in the study were added to the assessment
form. Other areas became topics for conversation. The final constructs should not be
considered valid for all Tswana age groups, nor should they be used to say the aged are
more or less happy than other generations.
Linguistic patterns and the absence of ranking as part of the thought process
made it necessary to capitalize on innovative research techniques. The use of the
"ladder technique" (Hansen, 1990) allowed for placement on a continuum. While
individuals cannot perceptually rate themselves in an abstract manner, they can see
themselves as individuals in a particular setting. Two opposing situational occurrences
involving people were placed at opposite ends of a small, five-rung ladder. (The
situational questions are described in Appendix B.) The individual was then asked to
place him/herself on the ladder, according to self-perceptions. (Holding the ladder on
its side eliminated ideas of worst, bad, good, better and best or hierarchy among
people.) Discussion revolved around the reasons the individual selected a particular
rung. This brought forth thought on how the individual perceived similarities and
differences between self and others and what they thought was important and/or
acceptable and what was disliked.


190
Particularly in this chapter, the observers emotional reactions must be put aside.
Tswana behaviors cannot be judged by Western tenets and called abusive.
Abuse, as perceived by the Tswana aged, is the purposeful withdrawal of
available food, care or shelter. An unserved meal or being left alone occurs in all
households and includes all family members, not just the aged. Usually it is attributed
to the impact of outside, uncontrolable forces, as with Kaizer failing to repair the roof.
In these cases, any accompanying suffering is not perceived as wrongful abuse. It is
acceptable deprivation due to nature getting the upper hand over good people.
This does not mean people do not react when others behave inappropriately.
Dorothy felt put-upon with the pantie incident. Betty felt put-upon being expected to
tend to Dorothys infant. Even so, these two individuals lacked what we consider
feelings of abuse. There was no assigned guilt involved with the doing of wrong. Again,
wrong is nature controlling life.
Ideally, suffering from wrong should not occur. When suffering does occur, it is
because of the way things are. Laws are broken, such as with yelling. That is wrong,
but the results of broken laws do not appear to be considered directed abuse. It is
more like uncontrolable suffering for which no one is really at fault. Dorothy believed
Betty had no control over the discarding of the panties, and Betty saw external events
controlling Dorothys time away from home. A person can try to achieve, but nature
determines the outcome (Alverson, 1978). Dorothy was taken to the headman to create
shame and stimulate her to work harder at the process of achieving, not as punishment
for psychological or physical abuse.
My own concept of abuse had to be temporarily set aside as I learned more and
more about the Tswana value system. When using the Tswana concepts, I found no
culturally-defined abuse. Some of the observed deprivation resulted because the aged
refused to alter laws to their own benefit. The refusal to use anothers belongings


289
71. What do you think would happen if you were dying?
5.Anticipates full time care and support in desired locale.
4. Feels that family will respect wishes and be present most of time.
3. Feels family will not respect wishes for locale but will be with them.
2. Feels family will not respect wishes about locale and will not be present.
1. Feels dying will not be recognized by others, and death will not be known.
I would like to ask you questions about your work and money. Please remember I am
not from the government and this information is between you and me.
72. Type of work (include agriculture): Past Present
73. In terms of work, what are you doing now?
1. Employed full-time by someone else.
2. Employed part time by someone else.
3. Self employed full-time.
4. Self employed part-time.
5. Employed at a temporary job.
6. Earns limited money with crafts, snuff, bajalwa.
7. Not producing income.
74. Where does your money come from? (Check yes or no for each of the following,
and if yes, enter amount if known, frequency and regularity.
Yes/No Amount Frequency Regularity
Present employment
Spouse employment
Money from family (Who)
Agriculture
Cattle
Destitute Program
Investments
Retirement Pension
Savings account
76. When was the last time you had money in your pocket?
77. How did you get the money.
78. What did you do with the money.
79. Some people have never owned cattle. Did you ever own cattle?_
80. When was the last time you had cattle?
81. What happened to your cattle? (ask reason for deaths)


57
Dirt Road
Paved Road
Census Areas
2 -Catholic Church
3 -Lutheran Hospital
&Church
4 -Rock Pile
6-Kgotla (Tribal
Administration)
7 -Old Kgotla area
8 -Community Center
Figure 3.1: Map of Ramotswa indicating Census Areas (not drawn to scale)


232
the family economic status. Mmanato is arranging for one of his finest cows to be
brought into the village. His brothers, John (Monates husband) and Masimi, are
assisting in planning. Monate, Priscilla and Happy Sound are helping Ida prepare
bajalwa. Betty, still in mourning, sends Dorothy in her place. The older women are
happy to have her strength and thoughts. It is a scene of mixed ages, with the broad,
extended family working as one.
The ceremony begins early Thursday morning. Idas daughter is taken in tow by
a group of women dressed in finery. Led along the village paths, the bride-to-be sings
out "I need a man to cook and clean for. I need a master to take of me." The women
search every nook and cranny of the village, looking for the proper man. The lack of
success does not deter them from parading the bride to her mothers house. Here she
receives the laws of marriage, in the form of secrets passed on by the elder married-
women kinfolk.
The fifty or so women from the mornings parade gather for the brides party.
Mmanato, John and Masimi sit, as guards, outside the compound gate. Only adult
women may enter. Children play in open public area, going to the men if assistance is
needed.
Calabashes (gourds) of bajalwa are passed among and between women. Soon
the mortar, used for pounding grain, is turned upside down. It is carved from a tree
trunk, with a flat top tapering to rounded-oval base. Monate giggles and squirms her
hips as she points to the mortar, now a 2 foot tall phallic symbol. The turning of the
mortar signals it is time to begin the dancing.
The women raise their voice in song. A line is formed, each woman holding the
chest of the person in front. Intricate foot work keeps time with the songs beat.
Winding between the mortar and compound buildings, the women dance until they tire.
New songs are started. Each woman takes a turn dancing around the upturned mortar.


260
does it make a difference if the household contains a second aged individual, as I
thought that another aged household member would assist in keeping doors open.
Living in a three generation household slightly increases the probability of obtaining the
good life (p. = .048), but the number of household members make no difference.. The
failure to find a good life probably exists because the setting prohibits affordable trade,
either with the present gerontic fund or the calling upon past debt for rewarding
benevolence among family members.
I believe much of the diversity in finding life-satisfaction occurs from success or
failure in the exchange process is related to access to doors, their condition and sway.
Household relationships have become competitive in nature (Comaroff, 1953). Social
relationships involve mistrust and antagonism. Success in finding the good life depends,
in part, on the success of finding willing and cooperative significant others in a
supportive setting. Only then can the aged find esteem through service, security and
personhood. as status cannot be self-generated.
Why do fund-rich score high in obtaining service and security and low in
personhood. One must keep in mind the division between esteem or respect (holding in
high estimation with the ingratiating regard for anothers wishes) and deference (the
yielding or submitting to superior authority with the possible absence of esteem). Power
breeds deference but not necessarily respect. The fund-rich obtain service and security
from the power that the fund generates for trade. This does not automatically provide
respect for the individual as a person. Many of the doors for respect have been broken,
and can only be transcended with the retention of middle-age values of activity and
economic contribution.


19
care is, the better. But such aspects of family life alone do not represent complete
success, for with service must come respect. Household members should not show
disagreement with the wishes of the old concerning proper behavior or action. Even
migratory children must show respect with visits and with the remittance of money. The
showing of respect does not represent power, but the value of the old person to
members of the family.
The successful aged continue meeting the moral obligations encompassed in the
laws. Like the successful adult, visitations and socializing are mandatory. This includes
maintaining proper relationships with multiple kin and contributions to the welfare of
the village. For the man, this may be participation in the Kgotla (tribal meetings). For
the woman, it is participation in village organizations, such as a modern burial society or
committees for community improvement. No true division exists between leisure and
work. The successful aged are always at work. To sit idle is to be worthless.
In summary, the aged of Ramotswa base their goals on a continuing but
changing society. Their expectations for life come from tradition and from life
experiences with the new and old ways. Doors should be open to keep a constant flow
among self, others, and the world about them. The following chapters present the doors
and degrees of success the aged have in obtaining the good life.


135
contribute to the funds strength but are not unique components in themselves. I
assume necessary components are the same for both males and females, although they
may use them differently during exchange.
Each aged person controls a gerontic fund, although contents vary in amount.
Self and others place a value on the fund, and the congruency of values influences its
purchasing strength. In turn, the contents and their value to others influence the degree
of flexibility in deciding on a course of action for negotiation and expediency for
spending, saving and budgeting. A large gerontic fund, in itself, can generate power
without expenditure (Dowd, 1980:53). This power is not available to the fund-poor
(Sen, 1981). Thus, a person is influenced by the knowledge of their own gerontic fund
and how it may be used for interactional activity to promote well-being. The actual
number of exchanges is not as important as the amount of resources required to meet
physical and psychological needs. The division into dimensions and the necessity of asset
ownership during the exchange process allow me to later specify where assistance for the
aged is needed. It also allows for investigation into the impact of time on reaching goals
through fund use.
The gerontic fund can be altered in shape and size with time. It may be
invested to further increase its strength, such as improving a room in an owned house
for rental income. It may be spent, as with the transfer of land control in exchange for
part of the crops. At other times, parts of the fund are simply lost within the
environment, as with movement of a family member or livestock death during drought.
The ease of rejuvenation and the potential of permanent loss are considered in its use.
As the Tswana so aptly commented in terms of physical actions, family communications,
and monetary spending, "I can do it but I cannot afford to do it."
Meaningful research regarding the application of the gerontic fund concept
requires that its contents, and the determination of its strength, be specific to the group


273
Cultural Duality
Rebellion against traditional behavioral expressions of the law is not new. It was
noted when the present aged were children and young adults (Schapera, 1944:265-266).
Behaviors arising from laws continued to be questioned, mitigated, and sometimes
discarded, throughout their lives. Each person attempted to pick out what was
materially and morally superior when faced with two distinct modes of life. This has
resulted in a cultural dualism (Harrison, 1987:46).
Cultural dualism is found among the aged. There is also a generational duality
as each succeeding generation digs deeper into the traditional culture. The rebellion of
the youth of today is towards what is left of the original culture, that which was
originally deemed worth saving (Harrison, 1987:48). The self-identity of each generation
is now threatened with the conflicts involving moral obligations between self and others.
Contradictions also exist between self and society.
This is the area where the aged feel the pain of social change the most. Many
of the doors to self-identity, and personal integrating with aging, have been severely
broken and are difficult to manage. What few material resources that some aged use to
promote self-identity, such as agricultural land and housing, are not valued by the job-
centered young. Acts of achieving for the betterment of others do not always generate
social recognition. Traditional definitions of a social elder have all but disappeared.
For some aged, the doors to self-identity are too damaged to move. Jacob is not alone
in feeling ancestorhood is the last open door.
Cultural Continuity
The flow towards cultural duality is counteracted with a strong current of cultural
continuity. The diversity in obeying the laws and applications of the seniority principles
does not prevent a generalized consistency in shared beliefs. Traditional philosophical
values continue to influence the direction behavior should take. Mankind is good.


11
anyone who is older than the speaker (Schapera, 1953:38). Such a definition does not
limit elderhood to the chronologically aged. There has been a tendency to unite the
two, resulting in a mix of truth and myth (Diouf, 1985).
Schapera only hints at a division between the socially defined aged and the elder.
This division can be very strong, with a removal of status and supportive care when
decrepitude occurs (Glascock and Feinman, 1981).
The Aged
In contrast to developed nations, old age occurs earlier in developing countries.
Harsh environments and working conditions, limited health care and malnutrition result
in a physiologically aged body long before age 65 (Diouf, 1984; Tout, 1989). The
United Nations (1985:98), in recognition of premature aging, demarcates old age as
beginning at age 60. Although chronological age alone does not present an undisputable
representation of biological and social age, it is used in this research to provide
consistency, and for cross cultural comparisons.
Chronological aging is distinct from social aging. Some of the Malete are socially
old, or designated as "children," before the chronologically assigned date. Other
chronologically old are perceived as modern "adults." Imposed social labeling does not
remove the cherished traditional values and behaviors held by the aged, which previously
promoted them as elders. Some are treated as elders in the true spirit of the word,
leaders with meaningful family and social roles. The majority of aged are regarded as
children.
I questioned the aged about their own parents. Once an old person could no
longer function as an active social elder in society, status and power were taken away
and replaced with the care and supervision needed by a child. They were treated as a
child incapable of meaningful thought and action. The same is true today, except the
timing of childhood occurs much earlier. Any contributions are seen as valueless. Their


234
The wife has learned the laws of marriage, such as where to go and when
to be home, so the man just has to remind her. She already knows what
will happen at night. In the old days, people didnt know about sex. The
man had to teach the wife, and if she refused, he threatened to tell her
parents. Sex is always important, with any age.
One of the younger women is leaving in my direction along the path. She, too,
talks of marriage.
Few girls today want to get married. Most husbands dont want
their wife to work, but farm the lands. The food you get from the lands
is not worth the effort. I refuse to plow. It is a silly custom, to do as
parents did. I want to get married after I do all I want to do: make
money and build me a house.
Also, women wont spend many years with a husband as one or
the other leaves. You can leave at any time. Sometimes the woman
leaves if she makes the most money. Sometimes divorce is over love, like
having a boyfriend or girl friend. It is okay to have boyfriend when you
are married, but it can be a menace. As long as the man doesnt see
him, he doesnt care. Keep lovemaking a secret and all is well.
A parallel event for the groom occurs the following day. He, too, must call out
his need for a woman to hunt and provide food for, a woman to take care of his house.
The male laws of marriage are passed onto him on top of the rock pile, not to far from
the kgotla. His party is for men only. Meanwhile, the brides family slaughters and
butchers the cow. John and Masimi, as close relatives, hang their choice parts in a tree,
to be taken home after dusk and the completion of chores. The women pause in the
cooking when Monate arrives in a huff.
As I was walking, some teenagers called me a worn-out, old
woman. They laughed at my clothes and said I wasnt fit to be seen.
They told me go home where I belonged. I wanted to turn around and
go home, but I held my head high and kept walking. Why shouldnt I
come to help? It makes me mad that those same teenagers will come
tomorrow and eat the food I cook.
Saturday brings the wedding. The groom, dressed in a new suit with the labels
still attached, and the bride, regal in a white wedding gown and carrying a bouquet of
white plastic flowers, climb from the open pick-up truck at the church. The brides
maids assist. Dressed in sheer yellow dresses, their tiger stripped bras and bright red


217
sisters hands reach out to massage the sick mans back. She explains that her visit must
be short, as she must return home to her own family. She then continues:
it is awful the way my brother and mother live. There is much suffering.
I love my mother and brother even through they are useless. I bring
food when I have extra. I help with chores when I have I time. I try to
help, but there are limits on what I can do. I must serve my own master
{husband} first.
The food is gone. The mother complains how the neighbors do not give her sugar or
tea, although she is sure they have some. Sepia nods and says:
today, when children go for a walk, all the think of are paths leading to
fun with other young people. No one came today. I went out, but no
one had coins that they would give me. I walked to the hospital. The
walk took a long time and when I got there I had to wait. They told me
I was just worn-out, that nothing was wrong, and there was no need to
come back. I was given some pills for pain so I do feel better.
There are many diverse elements that contribute to the feeling of personhood.
Sepia lost two of these elements: fatherhood and manhood. He describes the loss in
terms of events. I agree that it was a time when his particular needs did not blend with
the offerings of the employment and medical systems, but more is involved than the
breaking of a labor contract and the insertion of a catheter.
Sepia does not feel guilt or the pain of sin with his return to the village, as
neither exist in his culture. He does feel shame in that he did was unable to continue in
the process of achieving for the good of his South African and Tswana kin. He also
feels agony for the way his mother must now live. She must give him care, whereas he
should be providing for her. This inner turmoil clashes with his concept of personhood
and produces a loss that cannot be overcome in conditions of poverty and poor health.
Sepia is experiencing other unexpected loss. He can accept the poverty. He
knows he is useless, and that is acceptable for others to call him so. It is the loss of
acceptance that bothers him. No Tswana cared that he was in extreme need and
deserving of food and bus fare. He had been treated as a undeserving and unwanted


145
Visual problems are much more common than hearing problems. Cataracts and
past eye damage from flying dust are common. Corrective lenses, which have to be
purchased, are owned by very few, and are not updated with changes in vision. Like
hearing, the measurement of acuity does not involve technological tests. Judgements are
based on what the person said, which closely corresponded to observations on my part
during the interview.
The Tswana view vision as "the core of being," the major ingredient of
personhood. "The eyes make you a person. Without vision, one is no longer a real
person. He does not see others and others do not see him." Poor vision, although
increasing in intensity with age, was not thought of as a direct function of being old. It
was interpreted as due to other long-standing, uncontrollable circumstances. Women
attributed failing eyesight to a weakening in the uterus following menopause. "The
womb controls all the ligaments of the body. My womb is now old and dropping out of
place. It has pulled the ligaments to my eyes down so that I can no longer see." Men
mentioned past abdominal surgery or injury to limbs as the cause. Blindness, in turn,
created a "failure within the being" that tended to abort the process of achieving and
hence, personhood.
Most 60 to 69 year olds (90%) have minimal problems; are able to recognize
passing people on the lanes and identify pictures held at normal range. Seventy to 79
year olds retain the mode of good vision, although increasing numbers have difficulty in
the sense that vision "wasnt as good as before." They may need help threading a
needle, or have difficulty in seeing at night. Half of the 80 year olds do not visually
recognize me before entering the yard, and also have severe problems with sewing or
other fine handwork. All of those 90 and above are either totally blind or can identify
only the basic shape of large objects. The visually impaired person has learned, in many


283
14.I will read a list of items found in houses. Tell me if it is in your house, and if you,
or someone else owns it.
In the house Owns item Number owned Condition
Shoes
Coat
Blankets
Bed with mattress
Mat or Foam bed
Watch or Clock
Radio
Chair
Table
Inside Stove
Dresser/wardrobe
Working Car
Other
15. Do you have soap, matches, and food in the house now?
5. Has soap, matches and food for next meal.
4. Has food for next meal, either soap or matches.
3. Has soap and matches, no food.
2. Has either soap or matches, no food.
1. No soap, no matches, no food.
Lets talk about your health now.
16. In general, how is your health?
17. Is there anything about your health that worries you? Explain
18. What sort of health care is available that you can use? What would your like to be
available, including help from your family?
5. Can get all preferred services.
4. Can get general health care but not specialized care, (eye glasses, traditional
medicine).
3. Can get regular assistance/care at home, denied aspects of formal care if ill.
2. Difficult to get formal and informal care.
1. Health care of any sort is not available.
19. Which of the following people did you get help from in the last 6 months.. If you
saw more than one person or clinic, tell me. Also tell me if there are any you would
like to see but cannot.
Times seen Reason
1. Western doctor
2. Clinic nurse
3. Family Health Educator
4. Ngaka ya Setswana
5. Moprofiti
8. Other
9. None (Why)
20. In what way does your health prevent you from doing the things you want to
do?


56
These areas included sections at, or near, the major shopping area, the hospital, one
church, the kgotla, the recently developed areas on the village outskirts and the
immediate eastern side of the government council buildings, as shown in Figure 3.1.
Each census tract was approximately half a square kilometer. I omitted distances over
three kilometers from my residence, because of walking time. These were the hilly
section to the far east and the area containing the high-school and government
employee housing west of the council buildings. These two areas were used for pre
testing, with findings comparable to the randomly selected areas.
Each house within the identified areas was visited to obtain a census of ages and
gender. (Only one household was not directly interviewed, as no one was ever home
during multiple return visits. Three other households provided identical data on that
households residents) This census varied somewhat from the national census as only
household members residing in the house on a regular basis were included. Migratory
workers returning only on weekends or less frequently were excluded. Individuals who
were 60 years or over were interviewed, either at the time of census or with a return
appointment. Three attempts were made, during different periods of different days, to
contact the aged who were not at home.
The selection of 30 individuals for in-depth study was made with stratified
random sampling. These individuals were selected to represent one of each of three
categories reflecting level of activity. These levels were the active-old, the limited-active,
and the decrepit-old. Division was based on initial high, medium, and low scores of
physical functioning obtained from the Gerontological Assessment Form. One person
from each category was selected in each census area, being sure to include at least one
male and one female from the sample. As sometimes only one male and only one
decrepit-old resided in a census tract, absolute randomness was difficult to achieve.
(The only two decrepit males who resided in the areas were automatically included.) All


127
educated adult son lives in Gaborone. Monthly remittances, Segos only source of
income, provide for necessary purchases.
Sego is actively seeking a new wife. He firmly believes a man should be served
and tended to by a young woman with him as the master. Until he finds the right lady,
Sego relies on the woman who is living in one of the three rooms of his house. She
cooks and cleans in exchange for room and board. When she is gone, Sego goes
without food or seeks it elsewhere even though he can cook.
Sego talks about wanting the security and assistance with decision-making that
come with marriage. To compensate he frequently visits various relatives in the village,
seeking guidance, help, and companionship. During a wedding, he feels bound to
proper visitation and required supervision for the slaughter of cattle. For the ten days
of mourning prior to a burial, he pays proper respect to the dead by arriving at sun-up
and staying until nightfall. During this time he often denies his own needs or desires.
By our standards, Diane is the success story representing Western independence
and autonomy. To her neighbors, Diane is worthless and useless. She is seen as
producing solely for her own good and not the good of others. Family has little
meaning to her, as she neither takes nor receives. Above all, she allows nobody to serve
her. Some say her behavior indicates she is a witch. Others fear she will use dikgabe
against them. They purposely avoid her. Even her aunt tries to avoid her.
Diane publicly fails to demonstrate the ideology behind the process of achieving,
even though she is economically productive and knowledgeable in decision-making. Her
income is used only for her welfare, which is not seen as adequacy or regarded as
laudable. Her social interactions are viewed as self-centered, contributing little to the
good of society. Although she relies on others to purchase beer, there is no harmony
between herself and the social environment. Such actions do not represent self-
sufficiency within the rules of society or cultural confines. Societys judgment of her


197
women, in particular, are striving to make decisions independently of family input, with
motherhood and employment providing excuses to do so (Suggs, 1986). Younger village
men, especially if unemployed, seldom sought household authority. The deciding factor
for the placement of control is often that of economics, independent of gender. This
modem "household elder, who is not necessarily the oldest person in the family,
controls and benefits from discourse regulations.
The one accepted exception to family control by the household elder is with
sickness. Any adult is allowed to decide the course of action for personal illness.
Others may be told, and advice sought, but the decision for action remains that of the
individual. In contrast, the ill child is under the control of the head of the household.
Jacob, although becoming old, was still the head of the household, and therefore, was
not forced to seek medical treatment. This does not always hold true when the aged
are perceived as children and lack aspects of elder authority.
Family Function
Descriptions of Batswana family function, in terms of supportive behaviors,
usually center on the positive, or the help-giving and health-promoting behaviors of the
members. (See Biesele and Howell, 1981, Suggs, 1986, Ingstad, 1989, Rosenberg, 1990.)
Equal attention should be placed on the stress-producing and problematic behaviors, as
these also influence the setting for social exchange. In the following I identify
behavioral trends providing both support and stress from the emic point of view. The
outcomes of these behaviors reflect the goodness of fu, or the degree of balance
between the needs and demands of the aged person and the family environment (Ryan
and Austin, 1989). A goodness of fit can be considered as working doors between the
aged and the family. The degree of balance in relationships also influences the balance
in exchange relationships. Not all households behaviors are mentioned, only those


256
opportunities and rewards associated with the Tswana process of achieving as a person
and as an elder. Both participation in relationships and the reactions of others have
importance. (See Table 9.3.) The finding of personhood through achieving is
conceptually possible until death. Therefore, I adapt items to include the physically frail.
The first item involves participation in meaningful events, which promotes feeling
of social contribution. Activity, such as having a small church service in the home or the
sharing of food brought home from a from a feast, opens this category to all aged.
Some of the more frail aged see meaningful participation as just being told, and then
talking about, events that they could not attend. The group is fairly equally divided
between having opportunity to participate for social contribution (57%) and not having
the opportunity (43%). The aged who express denied access to opportunities for social
contribution gave reasons for exclusion. This includes not being told in advance of
events, lack of transportation, and feelings of prejudice against the aged if they did
attend. This was true of both active and frail aged. Home-bound aged stated they were
not told of marriages and deaths, or not given an opportunity to express their thoughts.
A large majority perceive themselves as actuating self-identity through kinship
gatherings. This involves being visited and/or visiting others.. This area has import
beyond having company, as visiting elders is mandated by the law. Thus, the act of
visiting reflects respect and concern for the elder. This allotment of respect is not
commanded by power or prestige, but symbolizes the value of one individual to another.
Seventy-one percent are involved with visitations. The rest are not visited and/or have
no one to visit with.
Direct questions on the perceptions of status in the community and at home
were a dismal failure. There is no specific Setswana word for social status. People
know their place in the scheme of village life with everyone being recognized for their


284
21. Can you walk to the nearest health post/clinic? How do you get there when
ill?
22. Do you take, or should you take any medicine? Why? What
difficulties do you have in getting or taking medicine.
Now / would like to ask you about some of the activities that are part of our daily lives.
I would like to know if you can do these activities without any help at all, or if you need
some help to do them, or if you cannot do them at all.
23. What difficulties do you have with eating?
5. Can shop, prepare, and serve food.
4. If food is present, can prepare and eat own meal.
3. Must have meal prepared, serves and feeds self.
2. Must be served all foods, feeds self.
1. Must be fed.
24. Do you have any problems with your teeth? (Explain)
25. Do you have difficulty getting to the toilet on time, or with your water leaking?
5. Always uses toilet by self, no soiling.
4. Uses toilet by self, occasional loss of urine with rushing, laughing, coughing, (circle)
3. Must be taken to toilet/given pan, can control urine and bowels.
2. Urinary incontinence, bowel control.
1. Complete incontinence.
26. Do others help you with bathing and dressing?
5. Keeps self clean, and dresses by self.
4. Keeps self clean, minimal assistance with clothing, (buttons)
3. Gives self main bath, requires dressing by others.
2. Can wash part of self, main bath and dressing done by others.
1 Totally bathed and dressed by others.
27. Do you ever feel you do not look nice? Why?
28. Tell me how you can obtain water, if necessary.
5. Carries in a 5 liter bucket. (Large sized)
4. Carries in a 2 liter bucket. (Medium sized)
3. Carries in a 1 liter bucket. (Small sized)
2. Carries in a cup or uses at tap.
1. Unable to get water.
29. Do you ever go without water? Why?
30. How far can you walk?
5. Can walk any desired distance.
4. Can walk around village, up to 4 Kilometers.
3. Can walk in immediate neighborhood, less than 1 Kilometer.
2. Moves about in house/yard with assistance.
1. Crawls or cannot move about.


162
mainstay in agriculture (Kerven, 1982:240). They are the ones who plant, weed and
harvest. Men are responsible for the initial plowing, and then are traditionally free of
agricultural duties until exerting control over harvest use (Schapera, 1953:27).
The gender imbalance found with land ownership, with nearly 50% of women
owning no lands compared to 10% of men, is not startling when looked at in terms of
family authority. The eldest man in the compound can demand or command
agricultural service from the young, keeping the land under his control. The woman,
with the transfer of household responsibility, loses power over daughters and sons.
Fields are lost from non-use or transferred to family outside of the immediate
household.
Social change also enters into the picture. With the more recent growth of the
international money market, subsistence agriculture does not produce adequate and
rewarding returns (Kerven, 1982:242). The young prefer wage labor and have little
desire to either work or inherit the family lands. Older women, even if lands are
plowed, must have enough other fund resources to command continuing agricultural
assistance. As the physiological and family gerontic fund assets decrease with age, so
does the loss of fields. All those after age 90 claim the loss of land from disuse.
Residential land, like agricultural land, in owned by the populace, not the
individual. The aged of today built their original homes on land assigned by the chief.
Giving up the home means that the land and hence the building reverts back to the
government for reassignment. For this reason, even if the majority of time is spent at a
daughters or another relatives house, the aged, if at all possible, will return home for
one night a week to retain ownership of this monetary and emotional investment.
Home ownership, regardless of the type of construction, comprised one item in
the fiduciary dimension. Maximum credit was given if the aged person owned a home
in a good state of repair, as a well maintained home created less worry and fewer


140
TABLE 6.1 PERSONAL/PHYSIOLOGICAL SELF-CARE ASSETS. (N=105)
A. Eating: (Mean = 3.71)
1. Must have food served and be fed. (1.0%)
2. Must be served all foods, feeds self. (22%)
3. Must have meals prepared, serves and feeds self. (24%)
4. If food is present, can prepare and eat own meal. (11%
5. Can shop, prepare and serve food. (42%)
B. Toileting: (Mean = 4.2)
1. Complete incontinence. (5%)
2. Urinary incontinence, bowel control. (4%)
3. Must be taken to the toilet or given pan. (5%)
4. Uses toilet by self, occasional loss of urine. (32%)
5. Uses toilet by self, no soiling. (55%)
C. Grooming: (Mean = 4.0)
1. Totally bathed and dressed by others. (7%)
2. Can wash part of self: main bath and dressed by others. (2%)
3. Gives self main bath, requires dressing by others. (16%)
4. Keeps clean with minimal assistance with clothing. (30%)
5. Keeps self clean and dresses by self. (45%)
D. Carrying water: (Mean = 2.9)
1. Unable to transport cup of water any distance. (21%)
2. Carries water by the cupful. (18%)
3. Carries water by the liter. (21%)
4. Carries water in 2 liter bucket. (29%)
5. Carries water in large bucket. (11%)


98
With the formation of District Councils, tribal chiefs lost their remaining
legislative and administrative functions, sweeping away any power to regulate the
political and economic life of the tribe. The Tribal Land Acts of 1968-1973 and the
creation of national land boards withdrew chiefly power over land allocation and use
(Silitshena, 1979). These once powerful men could only hope they could preserve at
least some residual authority over formal matters of traditional law and protocol by
serving as government advisors in a upper house of Parliament known as The House of
Chiefs (Picard, 1987:179).
Within Ramotswa today, the chiefs functions are mainly concerned with non
criminal judicial matters and with serving as a vital link in communications between the
federal government and the people (Silitshena, 1979). Although diminished in autocratic
authority and power, the Malete regard the Chief Kelemogile Mokgosi and his son,
Deputy Chief Ikanena Mokgosi, as symbols of tribal identity and cultural heritage.
The kgotla continues to serve as a place of guidance and rectification of
perceived wrongs. On many mornings a family dispute, unresolved by the elder and
headman, is solemnly judged by the chief and his advisors. The kgotla remains a place
where people congregate, usually older men. Young men and women visit also, some
seeking a answers to questions, others just to talk. Private affairs are discussed in either
the old British Tax Collectors building, which today serves as chiefs official office, or in
the original kgotla building. Tucked in this buildings walls are the impala horns, still
used to call initiation classes back from the hills.
The large iron bell still notifies the people of impending important
announcements and meetings. Its ringing signifies the necessity to stop activity and
proceed immediately to the kgotla. The original kgotla building can no longer hold all
the people. Instead they almost fill the new rectangular shaped, but traditionally


241
The traditional fear of sorcery, with divisions between friends and enemies arising
from unknown, has probably always been a broken door for help and mental support
with all age groups. The need to survive with increased in rural poverty has given to
reason to magnify the fear of unknowns, separating individuals even further. Kin must
rely on kin, but even then fear may prevent help. The possible outcomes for the cousin,
if she obtained the water Khuma needed, were too great to risk.
The unification of tradition and social change has damaged additional doors.
Many relatives care but are constrained in the opportunity to provide care because of
the interweaving of continuity and change. Sepia lives with his mother on the extended
family compound. The sister lives in a nuclear family. Her master must be served
foremost. This limits time she can be away from her own home. Health is
compromised when new and traditional beliefs are superimposed on each other. Access
to the essence of being is further damaged as outsiders, in their drive to improve village
life, fail to accept the meaning and value of Tswana custom. Poverty increases the
damage to all doors.
The next chapter will investigate which aged can manipulate the broken doors
that they face. We have seen that Sego manages them fairly well. Others do not.


108
been governmentally banned. It does not take a child long to learn that disobedience
has no severe consequences. Refusal to carry water or fetch items may result in a few
yells but little more. Respect and deference for elders can be thrown aside, for parents
or guardians have little recourse. This is the one area where parents and grandparents
severely resent governmental intervention. Fear of arrest for child abuse prevents even
the mildest of spankings. Replacements for traditional discipline are absent. Concepts
of withholding privileges or using a quiet comer are unknown. Shaming the child has
little effect in todays world. The traditional action of having an older male talk to a
disobedient child is used, but only if behavior becomes a severe problem.
Growing-Up: The Agola
Adulthood begins with a process of growing-up, or agola. Agola has always
begun early in the village, and this continues in todays world. Traditionally, adulthood
began with age-set initiation. Today, completion of primary school, usually at age 14,
marks the end of childhood (Lee, 1985). The few who continue with secondary school
are in a nebulous stage, neither children or adults. They are a child in that they are a
student but an adult in their behavioral expectations of increasing responsibility.
Both teenage boys and girls are expected to wash and iron their own clothing,
shop and cook meals and tend to all their personal needs. They are seen as capable of
becoming economic contributors to the household, if opportunity arises. Therefore, it is
not long before many young adults migrate to cities or mines seeking employment.
Young women, many of whom continue to view themselves as producers, would rather
produce money than traditional crops. Unemployed young men dream of work. Those
without employment often perceive themselves as the managers or supervisors of the
household members who are dependent on their physical presence, such as younger
siblings, aging mothers or grandparents.


CHAPTER 2
THEORY
Modernization has been a bad thing for the old people. They have no
control over their children. No one seeks my advice or asks about the
past. (68 year old woman)
The old are now poor because of drought and old age. People dont
treat us well. What I want is someone who cares. Im lucky if people do
things for me and I dont have to pay them. (91 year old woman)
Some Tswana aged are efficacious in old age, retaining meaningful activities and
status. Their doors are open and work well. Others fail to meet the goals of successful
aging and lack life satisfaction. They cannot overcome the barriers of broken doors and
are unable to move forward or have others enter their lives in a meaningful way. This
chapter will explore the theoretical reasons behind the possibility of obtaining success in
aging, as an individual and as a social phenomenon. This includes the social processes
that can either open or block doors. Gerontology is presented as a specific field within
the realm of the social sciences. Selected theories on social aging are discussed. The
studys hypotheses are presented, as extracted from the presented theory.
Early Gerontological Theories
Early gerontological theories focused on the declines and losses experienced with
aging. In 1961, Cummings and Henry presented the Disengagement Theory, stating that
aging people, with an awareness of diminishing capabilities and a desire to promote self
over society in the limited time before death, reduce their amount and intensity of social
interaction. A process of disengagement takes place, beginning with the relinquishment
of given roles, such as those surrendered at retirement. This act leads to withdrawal
from other roles and activities. With increasing age and diminution of energy, social
20


196
an active and authoritative role in the management of comprehensive family well being
(Coles, 1990).
The life histories of the aged indicate women's participation in decision-making is
not a new phenomena. Jacob, like many others, found value in his wifes thoughts, with
joint decisions made regarding cattle, agriculture, employment, style of housing, and
education for children. Other aged report similar spouse interactions, with aged single
men still seeking wives, reportedly to assist them in this arena. With some families, men
nodded in agreement as women laughingly claim to be in total charge of the household,
turning to men for the automatic stamp of approval.
The turning over of direct household responsibility to adult daughters and sons is
a long standing tradition (Schapera, 1966). Adult children see this as including the
broad scope of all decision-making. Priscillas daughter, who was presented earlier,
assumes all decision-making with the formal transference of domestic duties. Alfred, as
a husband, continues having indirect power over his wifes daily activities, with neither
having control household functions.
This transference of duties may also evolve without formal assignment. Leru, as
the household bread winner, assumes the financial decision making, and extends her
power to include other areas. Only in retrospect do the aged recognize the impacts of
transference of duties. They do not foresee the congruent label of child and the
accompanying family behaviors. It is the very rare household where the aged have
supreme authority. This occurs only when a aged male is fully employed and no formal
transfer of duties has occurred between mother and daughter.
The Mosokos are actively delaying the traditional "turning-over act, saying that
Dorothy is not yet of age for this to occur. Even so, the aged couple does not have
complete authority. Some of Dorothys antagonistic behavior reflects the decision
making power struggle commonly found between parent and young adults. Young


299
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230
usual elements of friendship such as closeness, trust and openness are missing. Friends
are a "safe" person, as opposed to a enemy. The enemy is someone who is feared, as
they spread rumor, tell falsehoods and know of witches for assigning doom or death.
Overt malevolence towards enemies is to be avoided at all costs, as the enemys
retaliation will make the threatened a reality through witchcraft. Although no one had
ever personally sought out a witch, everyone was sure their enemies had personal
contact with one.
References about enemies tended to be unidirectional, with a person having
multiple enemies but seldom seeing oneself as an enemy to many. As with other
interpersonal conflicts, the naming of enemies to others is not usually done. Sometimes
the enemies of a individual can be identified, such as with the refusal of food as
demonstrated during the funeral feast. Other times, the sudden departure of a one
person with the arrival of another signals a lack of friendship.
Allegiance with friends and alienation between enemies is not a constant.
Change from friend to enemy can occur with a simple behavior, harshly spoken word, or
siding with friends enemies. More time is required for the reverse conversion, as a
feeling of safety has to be generated. The regaining of trust seldom occurs as enemies
tend to be avoided. As extended family usually provides sufficient trustees, there is no
great motivation for improving the status quo of village acquaintances.
The system of friends and enemies places limits on the extent of possible
interactions, and also upon the type of interactions, as the unknowns of who is friend or
foe to whom is not constant or bilateral. In particular, the system complicates the law
of asking and giving. In practice, this law applies only to friends and strangers, as
strangers are friends until proven otherwise. One does not ask a enemy. Also, if one
asks too often, or for too much, one is apt to become the enemy.


119
The result is a psychic distress for the aged that arises from contradictions
between self and society in the interpretation of phases of aging. It can only be
assumed that some degree of psychic distress with aging always existed, as mentally alert
aged were cut off from performing elder roles within the family. The change in
definition of the aged is not so much in kind but in direction and degree. With changes
in the definition of old, status loss occurs earlier. Disrespect for all aged is becoming
verbal and open as more and more old people are considered old-children without
status and control over others for obedience to the law.
The aged, who are past their prime in physical fitness, repeatedly expressed their
anger in being treated like a child.
Once the body begins to go, people treat you like the mind goes
also. They say I walk with my head under my feet. I know many things
and think clearly, but to them I am a child.
When I was healthy, everyone would visit. Now they say I am a
child and no one visits. I am godile but nobody cares. No one asks for
advice as I am useless. Once the body goes, people act like the brains
goes too. I am not a child!
Corresponding Change in Deference and Respect
Public expressions of old age status were of great concern to all aged, as all had
at least one incident of disrespect. Most had multiple experiences of being teased and
debased. This is one area where the ladder technique was most helpful in investigating
social change.
The aged were asked to describe the reactions of others to the old, as the
Setswana language has no special word for social status. The middle rung of the ladder
was identified as representing the reactions of others to the aged in society prior to
Independence. I asked, "How do the people of the village react to the aged today?"
Only one of the 31 in-depth participants felt the aged had moved upward. His
comment referred to self and family with overtones of disrespect on the social level.


38
/
4*
w
&
Socio-cultural
£
\
/
/
\
\
\
Figure 2.1: Theoretical Model of Access to the Good Life


76
elder." Fear of beating gave strength to any elders dominance, making disputes a rarity.
By customary norms, anger was not shown and there was no back talk.
The austerity of obedience was tempered with security within the family. The
aged claim food was shared equally; children were given to grandparents who would
otherwise be alone; and unmarried men or women shared a compound with extended
family. "We were as one, with each helping the other."
The village was the center for communal activity. All families maintained two
additional seasonal homes. When the rains began women and children moved to the
agricultural land, and men to the cattle post. Families reunited in the village during the
dry, or winter, season.
The Cattle Post
The family cattle post was an outlying area in the open veld for the grazing of
livestock. A small hut served as the temporary home. All boys received instruction and
practice in the arts of cooking, sewing, laundry and cleaning from their mothers during
the winter. They were well prepared to perform these strictly feminine chores at the
cattle posts as women were generally absent.
Grazing could change sites on tribal lands, depending on the availability of water
and quality of grass. Livestock herds consisted mainly of cattle, but could include goats,
sheep, fowls and dogs. The men and their older sons, including boys over six or seven,
tended animals. They returned to the village, with livestock, only when grass and water
disappeared with the cessation of rain.
Donkeys and horses were introduced with early colonization. The donkey
provided a new source of wealth and status for the man who amassed enough for
plowing and wagon transport. The donkey gained special significance as this was the
animal Christ rode. One man condensed the thoughts of many when he said:


229
Yesterday, I asked a passing teenager to help me with some
lifting. He did not want to assist and we began arguing. He pushed me,
and I fell down and hurt my hip. I still cannot walk well, and I am in
need of water. I havent had water since last night, How is an old
woman to wash and drink if no one brings her water?
I know the statement is one of complaint discourse directed towards the
neighbors rather than a direct request for my assistance. I offer to help her. Taking
her bucket, I make the 300 meter trip to the public tap. Khuma hobbles along beside
me, all the time repeating loudly "An English woman shouldnt have to do this for an
old lady. It is shameful that an old woman can get no water." Doors close quietly as
we pass homes.
As I leave Khumas compound, the cousin calls me into her house. She closes
the door behind us. Handing me a bowl of porridge, she says "This is for helping the
old lady. You helped her when no one else would." Stunned, I ask why no one would
assist.
Khuma has friends and she has enemies. Some of her enemies
are my friends, and some of my enemies are her friends. I know who my
friends and enemies are, but not all of hers. If I got water the friends
and enemies would get all mixed up, and then they would say bad things
about me or about her. It makes too many problems to do such things
when everyone will see and talk. It is a shame she has no children, other
than you.
That evening another neighbor, a young mother, brings me a carrot from her
garden. She explains the gift.
This is to say thank you for getting water for Khuma. It is bad to
be alone and get sick. Everyone has enemies. They know who they are
but may not know why they are an enemy. If I got water for Khuma I
could get unknown enemies who are more dangerous than the ones I can
identify. A friend can help a friend but enemies make doing so very, very
dangerous.
Everyone has friends and enemies. The term friends, in this case, refers to
people that another has interactions with on a social level. They may be neighbors,
extended family, club members, or government officials. While considered friends, the


4
which the aged trade personal/physiological, social/familial and fiduciary resources in
exchange for the services and support needed for a good life (Gubrium, 1973). It was
my belief that the elements of a good life are present in the transitional village
environment, and that those who control goods and labor wanted by others find
satisfaction. It was anticipated that aspects of tradition and modernization both
promote and hinder the process of exchange.
Little was previously known about the definition, let alone the acceptance, of the
Tswana aged as a unique population within traditional Tswana society. The commonly
held assumption that high status and power were automatic for all aged was investigated.
Central goals were to learn how definitions of old age traditionally affected application
of the eldership principle, and if supportive behaviors were withdrawn with advanced
aging. Cultural change and continuity in these social processes were seen as affecting
the present as much as the social and material changes associated with modernization.
The intent was to evaluate the assumption that the process of modernization is the sole
agent in the devaluation and degradation of old people.
The main unit of analysis was the individual. Cultural continuity and social
change set the stage for the use of their resources. Access to the good life was
hypothesized as difficult for those who are poor in resources. I found that most people
lived with broken doors. The poverty of old age superimposed upon widespread
economic deprivation, and the fact that traditional attributes of childhood with a lack of
social value were no longer limited to the extremely decrepit, were barriers too strong to
overcome.
My research includes the similarities and differences found between the aging
process in a developing country and the developed nations. Much of the theory of
social gerontology comes from the modern world. I apply Western-based theoretical
concepts to aging in a transitional peasant agricultural setting. This necessitates looking


279
Incongruity between generations, which involve definitions of a successful person,
results in old age status-anguish (Dressier, 1988). This is one of the major problems
faced by the aged of the world. (Hendricks, 1982; Tout, 1989). Both the assets, and the
process of asset use, are ineffective in overcoming this anguish, as aged are prevented in
becoming involved and participating citizen in society (Nusberg, 1988).
The Tswana aged claim they are "the living dead" and are without a place in life.
These same complaints are heard in many places around the world, including the
developed countries (Hendricks, 1982; Goldstein et al., 1983; Barker, 1990; Sokolovsky,
1990:289-291). These feelings are no different from those of the displaced disaster
victim or war refugee (Oliver-Smith, 1986; Hansen, 1990). The main difference is that
the aged are involuntarily displaced-in-place. The involuntary-displaced are
disenfranchise from the host society, and find frustration with achieving and remaining
valuable members of society (Oliver-Smith and Hansen, 1982:2-6). The difference with
the displaced aged is that their "host" is their own community. The problems are the
same.
Refugee workers attempt to reintegrate the displaced into the host society.
Group commonalties form the foundation for reintegration. Group commonalties should
also form the foundation for reintegrating the aged in their host society (Stanford and
Yee, 1991). Segments of broken doors can be mended when tradition and change are
used together for repairs, as with the Wise Elders. Traditional roles, past experience
and accumulated knowledge can make a continuing contribution in developing society,
with proper planning. (Nusberg, 1988). The strengths of the aged can assist with
meeting the needs of society, while the resources of the society assist the aged.
All societies change. The society entered at birth is never the same as the
society one exits with death, although the differences may not be perceived as great.
Old age goals do not change. Change occurs with the doors needed to meet goals. It is


298
Lauer, Robert H. 1973. Perspectives on Social Change. Allyn and Bacon, Inc., Boston,
MA
Lawton, M. Powell. 1983. Environment and Other Determinants of Well-Being in Older
People. The Gerontologist 23:4:349-357.
LeVine, Robert A 1965. Intergenerational Tensions and Extended Family Structures in
Africa. In Social Structure and The Family: Generational Relations. Ethel Shanas
and Gordon F. Streib, Eds. Prentice-Hall, Inc., Englewood Cliffs, NJ.
LeVine, Sarah and Robert A. LeVine. 1985. Age, Gender and the Demographic
Transition: The Life Course in Agarian Societies. In Gender and the Life
Course. Alice Rossi, Ed. Aldine Publishing Co., New York, NY.
Lee, Richard B. 1985. Work, Sexuality and Aging Among IKung Women. In In Her
Prime: A New View of Middle Aged Women. Judith K. Brown and Virginia
Kerns, Eds. Bergin and Garvey Publishers, Inc., South Hadley, MA.
Lieberman, Morton A and Sheldon S. Tobin. 1983. The Experience of Old Age; Stress,
Coping and Survival. Basic Books, Inc. Publishers, New York, NY.
Martel, Martin U. 1968. Age-Sex Roles in American Magazine Fiction (1890-1955). In
Middle Age and Aging, A Reader in Social Psychology. Bernice L. Neugarten,
Ed. The University of Chicago Press, Chicago, IL.
McKee, Patrick L. 1982. Philosophical Foundations of Gerontology. Human Sciences
Press, Inc., New York, NY.
Meillassoux, Claude. 1981. Maidens, Meal and Money Capitalism and the Domestic
Community. Cambridge University Press, Cambridge.
Morgan, Richard. 1986. From Drought Relief to Post-Disaster Recovery: The Case of
Botswana. Disasters 10:30-34.
Mumford, Lewis. 1987. For Older People: -Not Segregation But Intergration. In
Housing and the Elderly. Judith Ann Hancock, Ed. Center for Urban Policy
Research, New Brunswick, NJ.
Munnichs, Joep. 1976. Dependency, Interdependency and Autonomy. In Dependency or
Interdependency In Old Age. Joep Munnichs and Wim Van Den Heuvel, Eds.
Intercontinental Graphics, Amsterdam, Netherlands.
Myerhoff, Barbara. 1978. Number Our Days. Simon and Schuster Publishing. New York,
NY.
Ngcongco, L. 1982. Precolonial Migration in South-Eastern Botswana. In: Settlement In
Botswana, R. Renee Hitchcock and Mary R. Smith, Eds. Heinemann
Educational Books, Ltd., Lesotho, South Africa.
Nusberg, Charlotte. 1988. The Role of the Elderly in Development .Ageing International
(December). Pp. 9-10.


86
established on the western outskirts of town by the Roman Catholics. Chief Seboko
Mokgosi paved the way for its acceptance in the village by switching church affiliation.
At the same time, Seboko felt a need to move the tribal headquarters. A new
kgotla was built, about a ten minute walk northeast of the new Catholic mission. Two
round silos for storage of the annual tribute of grain were constructed to the side of the
traditional round court building. The royal cattle corral sat across the large open
courtyard. The corral could be used by anyone in need of a temporary livestock
enclosure. According to custom, it was also to be the burial site of the present and
future chiefs. The traditional design of the kgotla was distorted with the addition of a
new type of building, a square one with glass windows and a corrugated iron roof. The
present chief, Mr. Kelemogile Mokgosi, gives his version for the deviation.
The people were walking to Gaborones quite often to pay taxes
and do the increasing amount of government business. The elders
complained to my father that the distance was to great for the aged and
took too much time from the young. The British were approached and
they agreed to build a office in the kgotla. The British District
Magistrate collected taxes in the square building. There was no problem
in paying taxes. If a person did not have money as he could bring a
lamb or a goat and get the correct change. But, at the same time, local
tax collection signified the end of giving grains to the chief for distribution
to those in need. Our new silos were never used.
Such action was the beginning of political subordination of tribal authority, which was
occurring throughout Botswana in the 1930s (Parson, 1984:22).
Religion and Marriage
Monate and John were one of the first couples to be married at the new kgotla.
Monate, now 73 years old, sprawled herself out on a blue plaid synthetic blanket in her
courtyard to recall the time of her wedding 55 years ago.
I had finished initiation school at the church, which meant it was
time for marriage. My parents arranged a marriage with a man I had
never seen before. That was the way it was done in those days. The
church could not take that away from us. Oh, I was so scared. What if I
did not like him or if he treated me bad? Mother encouraged me to go
nicely, as my name implies. My ancestors assured me all would go well.


(LeVine and LeVine, 1985; Guillette, 1990; Biesele and Howell, 1981). Care-giving, as
well as care-receiving, is an integral part of aging in Africa.
For some years, the main models for gerontological social programs available to
developing countries have been provided by established, economically secure nations.
These established programs for the aged include guaranteed income, government
supported group housing, and long term health care facilities. While social security and
long-term care facilities may seem easy solutions to increasing problems with the
increased numbers of aged, these solutions may not be best on the national, community
or individual level. As a rule, developing nations do not have the monetary resources to
invest in such problem-solving approaches. On the community level, the transference of
such programs tends to discourage rather than encourage already existing beneficial
moral and social ties that provide support to the aged. For the individual, other salient
solutions available within the geo-political and sociocultural settings are overlooked in
favor of what is known.
I want to stress, that while the theoretical physical and social aspects of aging in
a modem society may have catholic implications, the approaches and solutions to the
aging process should not be universal. Local factors that support the aged, as shaped
by each unique sociocultural system, need to be incorporated into specific program
development. Thus, qualitative identification of local systems that influence the
socioeconomic and humanitarian aspects of aging warrant as much consideration as
qualitative evaluation of the aged as old people.
The contents consist of four parts. The first three chapters deal with the
research study itself, explaining how gerontological thought in combination with
anthropological theory and methods were used to investigate human actions and
reactions regarding the process of aging in a dynamic environment. Chapters 4 and 5
explain the evolution of the village and the determinants of who is considered old.
vi


64
migration for men seeking wage labor, although the majority of households had one
adult or aged male present. Women migrate also. The absence of daughters is the
main reason aged live alone or serve as the sole adult caring for very young children.
In Botswana only 2%-5% of the population is 60 or more years old: this estimate
varies with the source (Republic of Botswana, 1981; United Nations, 1985; Third World
Guide, 1988). The percentage of aged in this country is similar to the rest of Africa.
Aged men are almost as numerous as aged women throughout all developing countries,
contrasting with figures from developed nations. Throughout Africa, it is not until the
80 year and over age group is examined do marked differences in sex ratios occur, with
100 very aged women for every 88 men of similar age (United Nations, 1985:31). This
sex-ratio reflects extreme variation from the sex-ratios of the United States, where there
are three women for every two men at age 65, and 66% more women than men by age
75 (Hendricks and Hendricks, 1986:68). Part of the ratio discrepancies between
developed and developing nations can be accounted for through high maternal mortality
with proportionally fewer females reaching old age (United Nations, 1985:31). Other
reasons are difficult to isolate (Gage, 1991).
The total number of aged in the census areas represent 28% of the known 375
aged in Ramotswa, as reported in the national 1981 census (Republic of Botswana,
1981). Overall, there were 147 aged (41 males and 106 females) living in the 207
households, comprising 13% of the sampled population. The customary return to the
village of birth with old age has caused governments to raise questions about old people
being concentrated in the older villages and absent in cities and new towns (Hay et al.,
1985; United Nations, 1985:105). Throughout Africa, 78% of all aged live in rural areas
(United Nations, 1955:98). A concentration of aged appears to be true in Ramotswa,
giving the village a proportion of old people that is similar to that found in the United
States. The big difference between countries is that the aged must interact with


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264
Self-Help Programs
Self-help programs for the aged in developing countries are drawing interest, but
are few in number. Planned intervention usually originates with an outsiders selective
perception of need and solution (Chambers, 1983:28-31). Most programs provide for
direct employment by the teaching of new skills or establishing gardening and husbandry
programs for the active aged (Tout, 1989). These programs, while successful in
promoting income-producing opportunities, usually involve a small number of individuals,
exclude the more frail aged, and require extensive external financial and manpower
support. Many fail as models, as internal cultural strengths and inhibitors are not
considered (Chambers, 1983).
Self-help programs in Botswana have been associated with drought relief. The
government provided either income or food in exchange for labor for village
development. The aged have been active participants in these programs, with the goal
of stimulating social prestige and acceptance rather than becoming economically
independent (Hay et al., 1985; Guillette, 1990). The last government sponsored self-
help program ceased in 1989.
Defining Gerontological Problems
In gerontology, the manner in which problematical social trends are defined
becomes the basis for social policy development. In Western nations, concentration is
focused on how modern life styles have affected the previous support systems for the
aged. Gender, age, family, and income have become the important issues (Stanford and
Yee, 1991). African gerontology is no exception. The claim is that migratory
employment and breakdown of the extended family places the aged-poor, the childless
aged, the widow, and the very old in jeopardy (Osman, 1983; Tout, 1989; Ingstad et al.,
1991; Thomas, 1992). They should become the targets for policy intervention (Khasiani,
1987; Thomas, 1992).


CHAPTER 8
THE AGED AND THE COMMUNITY
The old people say we should listen. I say if there are opposing views
between two people, each should go their own way as each individual is
unique and has the right to be oneself. Let tomorrow take care of itself,
as it will not be like today or yesterday. (21 year old woman)
I have worked hard all my life to make Botswana a better country. I
worked for the government and on the lands. Now they treat me like a
dog. You know what a dog is? He gets kicked around. Lucky is the old
person who can be somebody, for no one sees or hears us. (74 year old
man)
During the past hundred years, modernization and Westernization have touched
every nook and cranny of the village. This was no accident, as new thoughts and
methods were disseminated from the top down by foreign and state governments and
carried upward by the young. (Harrison, 87:54). The aged of the present were and are
carriers of change, just as are the present day young.
Individuals are the mediators of social change, as they serve as the bearers of
culture (Social Science Research Council, 1953). Response to new ideas is set within
their perceptional framework as outlined by the past (Oliver-Smith, 1986:16).
Innovation incorporates tradition, with cultural continuity and social change existing side
by side. The old is negotiated with the new, which alters the approach to life. The
question is how much alien cultural material can a society incorporate and still preserve
basic values and patterns. As additional unfamiliarity is introduced, the new of the past
becomes the traditional and another renegotiation occurs.
One can assume that there will always be contradictions, opposition of interests,
plus real or latent conflict with negotiation (Social Science Research Council, 1953:977).
208


243
Sources of Respect and Power: Yesterday and Today
Control over the good life in traditional societies comes from the power of
knowledge (Cowgill, 1979). For the Tswana, the majority of knowledge has been, and is,
open knowledge. It is given freely for the asking. There have always been secret sacred
laws taught with rites of passage. These rites occur relatively early in adult life, placing
the aged in a position to enforce the laws. The Tswana aged have never been regarded
an distinct hoarders of traditional knowledge. Instead they were perceived as having
knowledge resulting from varied and unique life experiences. This gave them value to
others but not necessarily power.
Power and status came to the Tswana aged through social organization
emphasizing eldership (Schapera, 1955; 1966). Laws could be used advantageously.
Those who were eldest in age were the recipient of the required giving of services and
social deferments. Some laws that support elderhood still hold true to varying degrees.
Daughters continue to cook and grandchildren run errands. Other laws continue to hold
true, but the power associated with them has changed hands. Adult children, who
control family economic assets, control the use of material goods. Other laws are being
ignored as social change promotes self-serving action and wealth in the younger
generations. The aged recognize these social changes, yet seek the promises of status
and power found in the earlier gerontocratic society to which they were socialized.
African gerontological researchers tends to use the gerontocratic promises of
respect, power, and provisioning as a pivotal point for describing the trials and
tribulations experienced with aging today. (Osman, 1983; Rosenberg, 1990, Ingstad et al.,
1991). My conversations with the aged indicate that power and status are a reflection of
the finding the good life, but not the core. The core for successful aging is based on a
validation of life through continuous processes involving self, family, and society.


231
The directional flow of friendship does not have to be shared. Khuma is
threatened by the young mans asking for money for drink, and therefor, considers him
as an enemy. On the other hand, the young man sees Khuma as a grandmother. He
avoids many of the risks in the friend/enemy issue by labeling her as adaptive kin. He
could always deny any rumor or falsehood Khuma spread to others because socially
Khuma is regarded as an aged-child, incapable of accurate memories.
Within this type of setting, individuals are constantly aware of their movement in
the village. It prevents some from seeking medical assistance at the clinics, as the
crowded waiting rooms may contain an enemy. Others avoid certain shopping areas or
sections of town. Certain events seem to be immune from enemy harm, among them
thekgotla meetings, church services, and funerals. Weddings, parties, governmental
meetings and simple visitation require vigilance, and one must constantly evaluate the
consequences of action. The result is that a generalized lack of assistance given to those
who are not immediate family. (Surprisingly, there is no fear than enemy will enter an
open house unless he, or she, is an actual thief.)
The neighbors did care about Khuma but could not afford to upset the delicate
balance in relationships. It is not that they did not recognize a need or lacked
emotional involvement. The neighborhood doors shut to prevent the giving of shame
for not assisting. The fact of caring is shown through the gratitude for my assistance as
a "granddaughter." However, as the "anthropologist", I remain exempt from the areas
internal processes of friends and enemies. Why did not help come from the man who
considered Khuma his grandmother? "Carrying water is the work of women and
children."
Case 6: The Wedding Dances
Delight spread over the neighborhood this month. Ida and Mmanato Ramasus
youngest daughter is getting married. It is expected to be a grand wedding, reflecting


203
the aged who had centered nurturing on a specific child in anticipation of care
provisioning, frequently have their plans disrupted. Resentment exists on both sides.
While an emotional bond exists between the family out-of-balance and its aged,
it lacks closeness and understanding. The aged seek roles reflecting seniority and family
interdependence. Younger family members perform roles and duties with the western
epistemological stance of self-autonomy. Dorothy, like others her age, dislike the forced
subserviency and dependency stemming from the seniority principle. Decision-making,
aimed at self-good, directs many of her behaviors. At the same time, she admits a
need for having grandparents, and had concern for their well-being.
Resentment does not prevent using the aged as advisors. Grandchildren, like
Dorothy, often turn to the aged for assistance in personal matters, especially those
involving interpersonal relationships. While the research does not specifically
concentrate on grandchild/grandparent bonding, a special relationship involving closeness
on the part of the grandchild is frequently seen. A grandchild will name the
grandparent as a confidant, but the reverse never occurs. With families out-of-balance,
the aged feel very insecure with the generation gap, questioning if grandparental advice
was heeded. The degree that such insecurity is colored by generalized family tensions is
unknown, but is surely present.
The aged of these families tend to internalize family conflict. This is the group
who asked rhetorical questions such as "Did I go wrong?" and "What happened so they
do not keep the laws?" Yelling is seen as a personal deficit, rather than disagreement
between two individuals. Passive behavior is the norm when arguments occur, with the
aged concentrating on the loud words without interfering or separating themselves from
the scene. These aged also overlook their own requests when orders are ignored by
others. There is usually no second, more authoritative ordering.


204
Independence and autonomy, desired in the Western-style family, conflict with
shared functioning (Streib, 1972). Shared functioning, with family coordination of efforts
in the process of achieving, is a hallmark of the traditional culture. The present day
aged applaud individual achievements involving free thinking and self-motivation for
social success. The difficulty lies in the fact that self-direction and decision-making have
altered the interdependency occurring with shared function within the household. With
families out-of-balance, the aged definitely become far removed from their traditional
roles as gate-keepers for family decisions and actions. Family unification, if present, is
controlled and directed by the young. It is not that the young have lost knowledge of
the culture. Instead the pull of progress has distorted the push of traditionalism,
creating a schism between generations.
Extended Family Outside of the Household
Extended family outside of the household must be considered in family
functioning, as this is the unit that provides additional doors beyond the confnes of the
home. Discourse regulations may limit the depth of intimate knowledge about others,
but not the scope of caring. The use of an extended family member as a confident is
rare, the turning to kin for assistance is not. The reasons for requiring assistance need
not be explained to them.
The interdependency found with shared functioning involving extended family is
particularly notable with aged living alone. Sego, like other aged men, women, and aged
couples without immediate household members, maintains self-sufficiency by utilizing
extended family interactions. Some of these aged live alone by choice, others do not.
Many of these aged are satisfied with using extended family as informal care
providers. They have an advantage in that the selection of kin to be included in their
family function is based on congeniality and trust. All but two of the 13 without nuclear
family feel secure in terms of continuity of care if it is needed. This is, in part, because


94
Traditionally, labor was given as freely as food and other needed goods. Labor
was regimented according to age-sets, as determined by tribal/church initiation. The
group would work together to clear areas in the village. At other times, all the people
of the village would work together, either directly for the chief or for village good.
According to Senatla, the giving of labor provided reward with achieving.
We used to have to work for the chief, helping him with his fields
and village improvement. We would help him when we came home from
South Africa. Sometimes it was inconvenient but it was good as everyone
benefited. For example, about eight years after Chief Seboko built the
new kgotla, he decided it needed a fence. Every man in the village had
to cut a tree and place it on the fence. It is still a good fence. My log is
one near the end.
The free giving of organized labor conflicted with the British concept of
organized labor for material gain. The traditional labor system was abolished in 1965.
The same year it was reintroduced in a new form Food for Work. This was soon
followed with specific employment programs that assisted internal development (Prah,
1979). Volunteer labor became paid labor, and money became the reward (Campbell,
1979). Sego recognized the impact of the change.
In some ways it was good that we no longer had to give time and
energy to the chief, as we could make money instead. In other ways it
was bad, as no one really cared about the village, only the job. Today,
no one gives labor for free.
Pathways of Culture Change
The process of becoming civilized was summarized by Sego. After leading me to
the open area in front of his four-cornered house, he spread his arms in a wide circle.
See all the houses here; they are the homes of all my family. The
village was a good place years ago. In those days, the eldest male was in
charge of the home. Wives knew their place was to cook and care for
husbands and children. Children and old people both had roles and they
knew what they were. Every family was involved with plowing and raising
cattle.
The village grew in size. We had children who had children.
Change occurred, especially between the chief and new government.
Some, like myself and my nephew, who lives in that black house, stressed
education and good employment and became very modern. My sisters


226
supporting one life, or providing improved quality of life for many people? In addition,
the locally underdeveloped service-providing infrastructure, and the scope of poverty,
prohibit access to the necessary equipment and support programs for rehabilitation and
later survival. What may be prolonging life in a developed nation may be prolonging
the act of dying in other situations. What is viewed by one group as a new door is seen
by the other as a broken door.
Case 4: Elisas Teeth
I realize that I had overlooked an interview question on dental health for one of
the female participants, Elisa. Socially, Elisa is a child, as she has limited mobility and is
recognized for her nearness to ancestors. Passing by her house with Agony, a 26 year
old friend, we see the old woman in her yard with her late-middle-aged cousin. We are
invited in.
I see no harm in asking Elisa if she has problems with her teeth, as personal
health is openly discussed in detail with others. Elisa responded openly. "My teeth? I
have no problem as I am on my third set. When I was working in South Africa, the
dentist pulled all my teeth and I now have new ones."
She smiles proudly as dentures are virtually unknown in the village. Evidently,
this modem advancement has never been covered in my friends education. Agony turns
to me, saying, "No one can grow three sets of teeth. It is impossible for a person to
grow a third set of teeth, isnt it?" The old woman prevents me from explaining, as she
continues talking. "These teeth are very special. It is possible only in South Africa."
Seeing bewilderment and increasing fear on Agonys face, I try to interrupt. Elisa insists
on explaining the features of dentures. "What is special about these teeth is that I can
take them out." As the dentures gradually protrude from her mouth, Agony lets out a
tremendous scream and continues yelling as she runs out the door. She stops only when


141
Mary, a shebeen keeper, had worked hard to have a new latrine installed for
herself and customers. She felt future replacement would be impossible, in recognition
of her increasing limitations. The next-door family of six, including a physically-limited
old woman, assumed the latrine was available for their use. One day, when the old
woman walked towards the building, Mary chased her away with screams. The following
day, Mary went after the old woman with fire in her eyes, additional anger in her voice,
and a stick in her hands. The frail woman complained aloud bitterly, "There is no
place for an old woman to go." Some area residents sided with Mary, saying she had a
right to refuse outhouse use to the general public, as it would soon become full. The
other group thought it should be open to all, for most people bought beer there at one
time or another. Arguments came to a quick end a week later, when all concluded
Mary was under the spell of witchcraft. The wild yelling and stick swinging, neither of
which was acceptable behavior according to the law, was an expression of the witchs
spell over which Mary had no control. Fear overrode desire, and the neighboring family
went back to using an open area.
One might think that with the elementary living conditions, and ambient dust and
dirt, that cleanliness and grooming have little import. The opposite is true, with strict
adherence to high standards of personal cleanliness. This means clean clothes and
bodies. Poverty may limit the wardrobe, but that doe not mean dirty clothes are
acceptable. A general all-purpose granulated soap is used for everything. A limited
soap supply causes as much worry as limited food.
Grooming is always done early in the morning, including the brushing of teeth
(no toothpaste as a rule) and the combing of hair after a complete bath in a basin of
water. As age progresses, it becomes increasingly necessary for others to bring the
water, but very few (7%) rely on others to completely wash and dress them. No signs of
skin breakdown or decubiti (bed-sores) are present in the bed-bound.


61
expected that "all my children" would be sent on errands and assist with simple chores.
Matt was given all the responsibility for daily grocery shopping and carrying water. In
doing so, he found new self-confidence and new friends.
It did not take long to discover the supposed equality between races was not
quite true. Preferential treatment was always given to an "English woman," as all non
native females are labeled. I tried to discourage their efforts to elevate my being with a
concentrated awareness on my overt behaviors. I immediately sat on a goat skin instead
of looking for a chair, ate with my fingers before a fork could be found, and waited my
turn instead of going to the front of a line. Having a Tswana mother and being a
mother in my own right contributed to acceptance. Mma introduced me as her worthy
daughter, but I became known as Mrs. Matt, a title implying respect for me as a Tswana
woman.
Feedback on acceptance came through Mma when she related the village gossip.
With hands on her hips, head high in the air and a swag in her walk, she said:
this is an English woman in the village, here for her own good. She does
not see the people or their life. The village women decided you were not
like that. You see us as we are. You a person without color. We can
tell you what we want to say, not what an English woman wants to hear.
Prior to this I had found a translator and together we were administering the
pretesting. (Three other English-speakers verified accuracy in translations and
interpretation of adages.) I had noticed a marked change in answers from the first to
the third week, when the above occurred. It was now time to begin serious research on
being old in a Tswana village. I began with the village census to obtain demographics,
from which springs all further findings.
Village Demographics
The overall census involved 207 households. A household was defined as one or
more individuals functioning as a family unit. They share one or more houses on family


199
These commonalties among separate families has little influence in what Ryan
and Austin (1989) call the "goodness-of-fit" between the aged and the young. It is the
intermingling of strong points and faults of individualized family functions that determine
the balance between generations. This balance will now be discussed, using the two
family scenarios as a foundation.
The aped in balance with the family
The Kereng family exemplifies the assorted everyday problems found in Tswana
households. For Happy Sound, the rewards generated by daily behaviors of the adult
children and grandchildren outweighs stress-inducing events. She, like other aged in this
group, see her family as one that works together. Their felt-board pictures contain
actual family members performing chores necessary for household functioning. Usually
children are playing, adult daughters are cooking or cleaning, and adult sons are going to
work or tending cattle. The degree of personal decrepitude and household poverty are
not major points, as they see themselves as having a place within the unified family.
These aged feel accepted and needed. They have reasons for happiness. Some are
happy to be loved or wanted, with others happy to see the sunshine, one day at a time.
The felt-board pictures parallel their own on-going lives. These aged men and
women perceive themselves with deGnite roles and can be observed performing them.
For some, the roles are more modem than traditional, with the performance of tasks
previously assigned to the young. Others are helping children "grow-up", teaching the
laws, and providing guidance. The personal process of continual achieving is contained
in all these roles. Interdependency between household members is always highlighted.
The aged, of families in balance, describe family members as adding meaning to
their lives. When asked to explain what the young children in their picture actually do,
comments include aspects of emotional and physical caring. "He rubs my body when I
am tired." "She listens to my stories of the days of becoming civilized." "The young


35
generational exchanges increase in complexity. The aged are very apt to base decisions
on traditional knowledge without having the more formal education and informal
knowledge about Westernization, which has been obtained by the young.
Limited knowledge can also produce limited options for possible behavior. In
addition, the life situation in itself can limit options, as I pointed out earlier in regards
to extreme poverty. With limited resources, individuals cannot consider all options: a
small risk can make potential reward too threatening. Emotions, hopes, and fears
detract from the assumed rationality in decision-making.
The essence of the theoretical outcomes of aging are said to be problems with
decreasing power sources and trade resources (Dowd, 1984). The decrease in age-
related ascriptive power of the Tswana aged is well known (Schapera, 1953; Ingstad and
Saugestad, 1987; Suggs, 1987). The aged are also known to be economically poor (Tlou,
1986). This has created conflicting ground rules between generations for the essence of
trade.
Ability to participate in social exchange also decreases in other ways as old age
progresses. The number of kin and friends with whom trade occurs shrinks with death
of friends and relatives. Loss of significant others gains additional momentum in rural
Africa as emigration of the young is the norm. This situation is confounded with sensual
and ambulatory losses. These common circumstances are not deterrents in using
exchange theory as these losses can be incorporated in the dissolution of resources and a
shrinking of command and power. It is the presence and use of existing resources that
has import, especially in regard to meeting old age goals. By regarding bodily and
personal loss as lost assets for trade allows for the testing of the commonly held
assumption that such loss is a major factor in personal disintegration.
The past use of the theory in situations of famine assumes control over labor is
an asset. The presence of family and friends equates with success (Sen, 1981). I see the


CHAPTER 4
THE EVOLUTION OF THE VILLAGE
Ramotswa is better than ever before. We have schools, a tar road and
Firms to employ people. Botswana is ruling itself and the people are
happy. (73 year old woman)
It is heart-breaking to tell you all that has happened to Ramotswa. Our
culture is becoming lost with development. Our richness is replaced with
poverty. (78 year old woman)
The aged, with descriptions of their own lives, provided the organization for this
chapter on village history. It begins with the history of the Malete and their
introduction to colonization, and then explains the movement from the "days before
civilization" through "the days of becoming civilized" and ending with "being modem."
These time periods provide a framework for the discussion of social change and cultural
continuity. The goal is to examine in detail the historical and on-going sociocultural
facets that affect the meaning of life, especially for the aged. Oral histories are
compared to written history, with agreement and discrepancies discussed.
History of the Malete Prior to 1885
The eastern area of Botswana and the South African Transvaal were sparsely
populated by Sotho-Tswana groups by 1200 AD. The forerunners of the Malete tribe
lived in Transvaal, east of the Ngotwana River, which today separates South Africa from
Botswana. Tribal groups migrated, split and reformed, as droughts and population
growth occurred. Eventually, the parent groups of the seven Tswana tribal nations,
including the Malete, emerged (Tlou and Campbell, 1984: 66-67).
The Malete were first identified as a tribe of the Tswana nation near the turn of
the eighteenth century (Ngcongco, 1984:25). At this time in history, the whole of
69


269
acceptance. The one problem of adequate food supply is but a small area of needed
change in the overall setting. A small magnitude of change has a great significance
when social worth is involved (Rossie and Freeman, 1982:68). The aged, by leading
themselves within the organization, become traditional elders concerned about family
and social good. Acts of achieving become recognized. The necessary group
interactions pave the way for turning enemies into friends. The aged can see others
were living in need, without speaking of their own problems and breaking discourse
regulations. All these actions reflect traditional roles and social structure. Such actions
have a better chance of inducing further change than if the program was planned
abstractly, and administered by others (Foster, 1973:164-165).
The formation of the Wise Elders also serves to undo some of the door-sealing
effects of gerontic deprivation ratchet. Traditionally, the Tswana elder acts as the door
keeper for family and social welfare. Change has opened new exits for the family, with
a closing of the traditional doors. This program recreates value in using the traditional
door, as families need the aged for access to the desired food. Although other doors to
successful aging continues to have severe cracks, and has hinges with a tendency to work
the wrong way, a small opening exists. These minor repairs promote positive social
interactions and a reason for being. With this comes a twinkling of hope that the aged
children of Ramotswa will once again be seen as the wise elders.


124
Decreasing strength reverses the process. The process of achieving begins to
falter. Production and possession of trade items gradually decreases and possible errors
in decision-making may occur. Yet being tsofetse does not negate the need to follow
social rules, although one is increasingly separated from social function. Conceptually,
self-sufficiency progressively decreases as childhood blends into infancy. Decision-making
is not expected; knowledge of the rules may be questioned; and behaviors become those
of play.
Dependency
Logically one may ask if the decrease of self-sufficiency is not the same as an
increase in dependency. Unfortunately, the term "dependency" is used to describe a
wide variety of types and degrees of factors, ranging from the individual to international
level and reflecting anything from physical dependency and the dependency syndrome,
through the economic dependency ratio, to dependency theory.
Hansen (1990) describes dependency as a complex concept with specific
characteristics providing several layers of meaning. In its simplest form, dependency
implies a relation between any two or more factors. The characteristic of directional
flow between the two factors, which may be one or two way, is placed on the
relationship to give it additional meaning. The characteristic of need comprises an new
layer, with the term dependency reserved for those relationships in which one element
or factor needs the other to maintain its state. The content of the relationship may be
specified as well, such as substance or physical care. The final layer emerges from
characteristics of value. A value judgment is placed on the relationship, its directional
flow, need and content (Hansen, 1990).
Americans tend to view old age with a selective focus on the retention of
physical characteristics. Such traits are highly valued and susceptible to decline with
aging. In the majority of cases, the judgment value placed on the aged is negative when


118
ADULTHOOD
Figure 5.2: Applications of Indigenous Life Cycle to Contempory Life


41
action (Blau, 1973). The aged experience friction between past acceptable behavior and
emerging trends. This friction extends beyond moral conflict, as trends set new rules for
the values associated with specific resources, and changes what is regarded as an
acceptable reciprocal exchange. Cultural continuity, in contrast and in relationship to
change, must also be analyzed. On-going social customs and beliefs also affect present
reality. Such customs and beliefs can either be transferred directly to the new society or
altered to fit the new society. In either case, they affect the value of resources and the
exchange process, both in the present and in the future. My goal is to make clear the
strong interrelationship between the individual and society in establishing the good life
during old age. This includes the behaviors and values of the aged and the responses of
others towards the aged, for flow with process is not one-way.
My approach assumes the aged must have open doors for flow between
themselves and the contextual setting. Old people enter into the lives of others as well
as having others enter their lives. I do not assume the doors were originally perfect.
Large and small cracks have always existed. The process of social change can create
more damage to some and repair others. The swinging of the door is as important as
its actual condition. Resources determined how well the aged individual can use the
assorted doors.


81
ceremony. The presence of The Resident Commissioner and District Magistrate made
him stand in awe.
A second delight was the thrill of obtaining water from one of four communal
water faucets that Chief Seboko had installed. Instead of walking many kilometers, he
could get the household water in 30 minutes. These water lines were connected to
bore holes that tapped into the large underground artisan springs, which continue to
provide village water. He proudly explained that, although the water system has been
enlarged, these same taps are present today.
Sego, sitting with other aged men at the Kgotla, shared memories of hard times.
Hard times came from nature, usually in the form of drought or excessive damaging
rain.
According to custom, every household gave part of the crop to the
chief as tribute. Grains, mainly maize and sorghum, were stored in silos,
to be distributed to the hungry and poor. After the land purchases of
1926, a drought came. We had some food, but new crops were dying
from lack of rain. Our elders questioned the status of Chief Seboko, as it
appeared he was no longer a good provider for the tribe. The village was
very discouraged. Our maize was wilting in the fields, and communion
with God and our ancestors did little good.
At that time, the chiefs were believed to have powers to make
rainfall. Seboko, because he was a Christian like his father, had never
been instructed in the secrets of rainmaking. Fhologang Peba was the
one man in village who knew about rainmaking. He and another man
began the ceremony. They grew afraid and quit, because the chief was a
spectator. Seboko sent word to South Africa to have a famous rainmaker
come to the village.
Laughingly, all the men described how the famous rainmaker spent three days in
ceremony, praying and crying out for rains that did not come. They act out how they
chased him out of the village as a hoax. Then, growing serious, they talked of the days
that followed.
As soon as the rainmaker left the village, the rains began to fall.
They did not stop so our fields flooded and the maize turned black. We
call 1926 "The Year of the Black Corn." Everything was ruined. People
said trying to make rain was against the wishes of God. God acted


266
escape from the gerontic deprivation trap, which I see as the commonalty unifying the
aged. Culture and need has to be united to establish any potential program direction
and content.
Identified individual, family and social needs fall into two distinct, but
complimentary categories: that of promoting physical and mental well being, and that of
providing sources for status generation. Traditional culture dictates that the aged
needed recognized roles of contribution involving power, authority, and status.
Contemporary society suggests action that addresses the needs associated with family
poverty. I also have to consider the basic facts of life in a Botswana village. The
physical environment means any program has to be adjusted to the absence of modem
conveniences and facilities. Motorized transport is also limited.
I still have many unanswered questions as a result of my research. I have only
touched the surface of adjustment after retirement from employment in more developed
areas, and how this relates to finding the good life. The combination of positive and
negative interactions and the flow of affinities between grandparents and grandchildren
still creates confusion. How does one approach the needed education for resolving
generational conflicts in a culture without quilt or belief in self-control over life? I
wonder if the teaching of newer Western discipline techniques and the addition of
positive feed-back would restore any control and authority by adults and elders. Lastly,
can one expect the Tswana, who have reached old aged, to have an extended life
expectancy as found with African-Americans?
I do know that many of the problems the aged were experiencing were not
unique to the village (Osman, 1983; Hay et al., 1985; Ingstad, 1987; Guillette, 1990).
Everywhere, there is a strong need to enhance the filial obligations associated with the
extended family (Hampson, 1982,1985; United Nations, 1985; Tout, 1989; Guillette,
1990; Ingstad et al., 1991). In addition, the economic problems of this villages


157
recognition of the registered destitute, no attempt was made to pinpoint the individuals
level on an economic scale.
The statistical mean indicates that the average aged relied on regular
remittances. This was not reflected in reality, as only 7.6% of the aged fell into this
group. Roughly one-third (36%) shared in the use of irregular household remittances
and another third (32%) had irregular money making schemes providing money for
personal use. Even with the provision of remittances and/or irregular money making
schemes, adequate income was felt to be absent in many of these families The other
age were equally divided between the two extreme ends of the scale, having a regular
source of personal income, either through employment, rentals or pensions (12%) and
that of being a registered destitute (11%).
A registered destitute is one who lives at 10% below the poverty line and is
without family who have means to provide support. Numerous aged live below the
poverty line, but because the family has the ability to provide support no government
funds are given. It makes no difference in regulations if families do or do not provide
money (Knudsen, 1988).
What proved to be a accurate way of assessing control over money was not
discovered until quite late in the research. I mention it only because the method may
be valuable in future work. The simple question of "When was the last time you had
money in your pocket?" was so different from the usual questions asked in typical
surveys that it took people completely by surprize. They responded without thought.
Some would look in their pocket and show me what they had. Others would laugh and
say, "I found 10 thebe (5 cents) on the path last month," or hang their heads and reply,
"I havent had my own money for over a year." This question was followed with probing
into how money was obtained and how it was used. While the numbers asked these
questions was relatively small, the 19 respondents did support my growing assumption


294
Biesele, Megan and Nancy Howell. 1981. The Old People Give You Life: Aging Among
IHung Hunter Gatherers. In Other Ways of Growing Old. Pamela T. Amoss and
Stevan Harrel, Eds. Stanford University Press, Stanford, CA.
Blau, Z.S. 1973. Old Age in a Changing Society. New Viewpoints, a division of Franklin
Watts, Inc., New York, NY.
Botswana Central Statistics Office. 1974. A Social and Economic Survey of Threee Peri-
Urban Areas in Botswana. Ministry of Finance and Development Planning,
Government Printer, Gaborone.
Brathwaite, Farley. 1986. The Elderly in Barbados. Carib Research & Publications Inc.,
Bridgetown, Barbados.
Campbell, Alex C. 1971. The Rural Economy: A Sociological Perspective. Botswana
Notes and Records 13:192-194.
Campbell, A. C. 1979. The 1960s Drought in Botswana. In Proceedings of the
Symposium on Drought in Botswana. Madalon T. Hinchey, Ed. The Botswana
Society, Gaborone, Botswana.
Campbell, A. C. 1982. Notes on the Prehistoric Background to 1980. In Settlement in
Botswana. R. Renee Hitchcock and Mary R. Smith, Eds. Heinemann Educational
Books, Ltd., Lesotho, South Africa..
Central Statistical Office, MFDP. 1982.1981 Population and Housing Census, Summary
Statistics on Small Areas (for settlements of500 or more people). Government
Printer, Gaborone, Botswana.
Chambers, Robert. 1983. Rural Development: Putting the Last First. Longman Scientific
& Technical, Essex, England.
Chambers, R. and D. Feldman. 1973. National Policy for Rural Development The
Governments Decisions of the Report on Rural Development, Government
Paper No. 2 of1973. Government Printer, Gaborone, Botswana.
Chatters, Linda M. 1988. Subjective Well-Being Among Older Black Adults: Past Trends
and Current Perspectives. In The Black American Elderly. James S. Jackson, Ed.
Springer Publishing Company, New York, NY.
Clark, Margaret. 1972. Cultural Values and Dependency in Later Life. In Aging and
Modernization. Donald Cowgill and Lowell D. Thomas, Eds. Appleton-Century-
Crofts, New York, NY.
Cohen, Ronald. 1986. Traditional Social Formations. In Food in Sub-Saharan Africa. Art
Hansen and Della E. McMillan, Eds. Lynne Rienner Publishers, Inc., Boulder,
CO.
Cohen, Carl I. and Jay Sokolovsky. 1989. Old Men of the Bowery: Strategies for Survival
Among the Homeless. The Guilford Press, New York, NY.


60
questions about the world. Monate says she has lots to do. Yesterday she had helped
cook for a wedding. "But you should understand I dont do the cooking. I supervise,
which means to sit and share talk with others."
With the preliminary exchanges now over, I introduce our need for rental
housing. John is happy to accompany us to a place he knows of, the compound of Mr.
and Mrs. Mosimi. The family had built a four-room square house on their plot of
village land for an adult son who died unexpectedly. Moving in was easy, with the
transfer of three pieces of luggage from a borrowed truck, each suitcase weighing the
airlines 20 kilo limit. Household furnishings were simple: two beds, a table, six chairs,
and a one burner kerosene stove. All we had to buy were candles for light, a couple of
pots, and tableware. We carried in enough water to last until morning and made the
latrine our last stop before going to bed.
The crack of dawn brought sounds of wood being chopped, radios, and voices.
We were soon to learn there is no such thing as private space, either auricular or visual.
Mrs. Mosimi was in our kitchen at six oclock that first morning. "My master sent me to
see if our new children slept well." Thus began our incorporation into an extended
family of aged parents, two adult children and a grandchild. Family titles replaced given
names, and new role-norms for extended family corporacy with intergenerational
interdependency, replaced the American nuclear family independence.
Rra (Father) effectively, yet lovingly, demonstrated that he was the household
elder and master, befitting his 76 years. Severe arthritis kept him from walking long
distances but he remained the effective manager. Mma (Mother), born two years after
Rra, guided me through the learning of female verbal and nonverbal behavior. Above
all, I was to serve my own master (husband), show deference to Rra, and teach my son
the law. Brother was to be my guardian when my master was not home. Sister was my
equal. Her 6 year old child was also my child to discipline, teach and command. It was


Zambia
Kasans
Okavango Delta
Frqhcistown
Selebi
Philwe
Kalahari Desert
borone
Zimbabwe
Namibia
RamoUwa
batso
South Africa
Paved road
150 km
Nonpaved road
River
Botswana
Figure 1.1: Map of Botswana


90
witches, and they multiplied; some being old ladies; some being young
mothers teaching their children the secrets of witchcraft. By
Independence there were many witches of all ages. People no longer
trusted people outside of relatives. Cures for being bewitched became
very expensive, like one or two cows. We continue to live with the
problem of disease and misfortune from witches because the white man
denied it was so.
The daughter agrees that witchcraft is a daily threat that outsiders continue to overlook.
Growth of the Money-Market Economy
The primary activity of Bechuanaland administration was directed towards tax
collection to maintain a modest number of officers for law and order, while developing
Botswana as a labor reserve for South Africa (Parson, 1984:22). Opportunities for local
entrepreneurship were restricted. In 1949, there were only ten African-owned stores in
Bechuanaland as opposed to 155 European-owned ones (Parson, 1984). Small numbers
of natives entered teaching or became store assistants, clergyman, or native clerks for
the English administration. Even smaller numbers worked for themselves as
dressmakers, thatchers, or carpenters (Schapera, 1955:33). The dominant avenue for
economic relief was the road to the mines in South Africa. Low wages kept workers in
a situation of poverty. Rules forced a return home after employment. Economic needs
at home forced a return to the mines. Oscillating migratory labor became a necessity
(Parson, 1984:24-25).
John, like so many of his age-set, left for the mines soon after his marriage to
Monate. He says:
there was a recruiting officer at Ramotswa Station. I just walked up to
him. He asked which mine I wanted to go to. After I told him, he gave
me papers and a ticket to get there. It was required that you stay eleven
months, then you got a ticket to come home. After several years, I could
have stayed longer for a bonus but never did. I did this again and again.
I never thought much about it. That was the way things were done, as all
people, not just the family, now needed money.


5
at aging from two perspectives: the outsiders theoretical and experiential view of
gerontology and the Tswana view of ideals and reality. Identification and understanding
of the grass root desires and yearnings of the Tswana aged, as based on their approach
to life, is necessary. I assume that hidden behind the overt behaviors and values one
can find universal in the goals of the aged and the processes used to obtain them.
Research design purposely reflects the inclusion of possible universal components.
The Setting
Botswana, slightly larger than France, is a land-locked country in southern Africa.
(See Figure 1.1.) The Kalahari desert occupies two-thirds of the country but sand is
found everywhere. Sand is a main component of the soil in the flood plains of the
Okavango Delta to the north and the low rocky hills in the east. Only the east is
hospitable for agriculture, concentrating the population within this area. Even there one
finds environmental risks with crops and livestock. Droughts and erratic rainfall, varying
from year to year and site to site within a given area, have caused repeated withdrawal
and resettlement (Campbell, 1982:20-22).
The eastern area of Botswana and the South African Transvaal were sparsely
populated by Sotho-Tswana groups by 1200 AD (Tlou and Campbell, 1984:32).
Pastoralism and peasant agriculture provided subsistence (Campbell, 1982:20-22).
Gradually the Tswana culture emerged, encompassing various tribal groups and resulting
in the seven Tswana nations found in the country today. The Malete were first
identified as a tribal nation near the turn of the eighteenth century (Ngcongco, 1982:25).
Botswana, known as Bechuanaland, was claimed by the British in 1885. The
Protectorate served as a "labor reserve" for South Africa, providing workers for mines,
farms, and later, industry. At the turn of the twentieth century, a railway was built in
the eastern corridor to interconnect the wealth of South Africa and Rhodesia (Tlou and
Campbell, 1984:156,161). Other development was directed toward supporting the cost


3
Secondly, just as there are cultural differences in the meaning of a doors use,
there are cultural differences in interpretation of the meaning of aging. I present
contrasts between aging in Botswana and developed nations, and contrasts between
concepts of what old age should be like. This is the sort of thing that may provide
additional information for opening new approaches to cross-cultural gerontology.
In this introduction, I will present the generalized salient elements of the study.
Following this overview, I will discuss the aged and their physical and ideological setting.
The chapter ends with an interpretation of success as delineated by sociocultural values.
This information provides the foundation for understanding how the Tswana doors have
been broken, and why our own should be unlocked in the interpretation of cross-cultural
gerontology.
Overview of the Study
The study involved 105 aged (age 60 or greater) of the Malete tribe of the
Tswana ethnic group of southern Africa. The setting was an established, moderate
sized, rural village in Botswana. The village presented a typical mix of traditional and
modem thought, social rites, and technology. The investigation was done during two
research periods, each involving separate steps. The first was a two month period
during 1988, looking at society as a whole to extract specifics for gerontological research.
The second was a seven month period extending from the end of 1989 through March
1990. At this time an in-depth study of the aged was undertaken. At both times
research methodologies of formal and ethnographic interviewing, oral histories and
participant observation were used. Visual aids were added as interviewing tools with the
intensive research on the lives of 30 individuals. These tools encouraged abstract
thought, which is difficult for the Tswana to express and promoted discussion to include
that which others were hesitant to mention or considered irrelevant.
The study was based on a gerontological adoption of social exchange theory, in


PREFACE
Becoming old. It is something we all think about, including our own personal
securities and insecurities that accompany the individual process of aging. People see
themselves as being old in a setting similar to the one they know, without marked
alterations in the way the family incorporates its aged members or radical change in the
social and political environment. We have been socialized to expectations of how
individuals and groups will accept us as old people, who will provide for our needs, what
our roles will be, and the strategies we will use. These lessons provide a passageway to
a satisfactory old age, and cast a light over this final phase of life.
But what happens to the passageway when rapid social change associated with
the modernization of developing countries means that perceptions of life are topsy-turvy
and lifes expectations are remodeled? Which elements from the past can be utilized?
Which are broken or lost? Can the resulting combination of new social and materialistic
advances combine with traditional ideology to create a secure life for the aged? Or do
the old face a barricade that prevents the finding of security and happiness.
This dissertation deals with social change and cultural continuity and how these
factors block or encourage the aged of Botswana in their quest for a good life. Like all
ethnographies, a specific cultural group is involved. In this case, the people are the
Malete, a Tswana cultural group of southern Africa. The setting is an old, established
Botswana village experiencing rapid development as the country becomes modernized.
Both the "old" and the "new" are embodied in the decorum of life. The scene involves
riches for a few and poverty for many. One also finds the repeated calamities of
drought, as created by nature.
iii


50
couple was typical of many of the aged: intelligent and exhibiting the reasoning patterns
taught through culture, yet bound by limited education I found I was similarly
culturally bound with limited Tswana education. Their answers to my "Hows" and
"Whys" were elusive and confusing. I left the village, and Botswana, recognizing the
research was possible but in a very different way from the usual American approach to
gerontology.
Research Design
Social environmental theory, proposing that the aged use personal-physiological,
social-familial and fiduciary assets in a process of social exchange to provide a good life,
formed the basic constructs of research design. These basics became the foundation of
a Gerontological Assessment Form, a formal survey instrument to provide quantitative
data on the aged. The actual questions are presented in Appendix A. From this form,
specifics could be evaluated singly or grouped for comparisons.
The categorical data for the resource dimensions, such as possessions or the use
of social services, could not be equally equated in meaning or content with Western
society. Questions regarding the assets held by the aged were devised by modifying the
proposed contents, listed by Gubrium (1973), to the local environment and level of
village development. Adaptations were based on observations and statements accrued
during the initial visit. For instance, the distance to water had great meaning. The
number of indoor bathrooms did not.
Originally, the inclusion of proven life-satisfaction questions from known valid
instruments was considered for determining the good life, as this would provide
comparisons with aged in other countries. Even though such questions were previously
judged to be applicable in situations of cultural differences among Western ethnic
groups, many were found irrelevant to Tswana reality. For example, the standard
question of "How do you see yourself five years from now?" actually brought forth anger,


43
expenditure. A series of appointments with various agency heads met with
disappointment. I was informed that there was no need to study the aged, as tradition
dictated the family should take care of and support their elderly members. In other
words, as I was told, "the aged have no problems."
No matter who I talked with, the high official or the person on the street, the
custom of providing family support for aging parents was stressed. However, outside of
government buildings, individuals admitted that this was a custom often given allegiance
in abstract principle but not in reality. They pointed to social change as creating inroads
on the continuation of status and welfare of the aged. Therefore, I had no doubt that
the situation on which I based my study was present in the country.
Now was the time for self-critique for failure to obtain permission for research.
Analysis indicated my problem-centered approach for seeking approval was in error. No
agency had said the study was faulty; it just did not fit their needs. Maybe a fresh
approach was needed, something that did not threaten the concept of traditional family
support. I had used the problem-solving approach, stressing identification of social
change and areas of need. Why not have a study that reflected strengths?
With only one possibility of approval left, I applied to the Ministry of Local
Government and Lands. This Ministry administers village development programs
including the destitute funding program. This time I changed my opening format and
said, "Since the tradition of family support for the aged is working so well in this
country, I would like to investigate why, with the hope of finding strengths that could be
incorporated by other countries." Approval was immediate with a signature on the
needed formal Presidential Permission for Research application form. At the same
time, I was given the necessary written permission and letters of introduction for visits to
various villages.


70
southern Africa was affected with wide spread assassinations and conflict, known as the
Difaqane. Tribe displaced tribe and nations were divided. The Malete, under the
leadership of Chief Malete, left the Transvaal and settled in the iron-rich Tswapong
Hills, straddling the present Botswana border with South Africa. Metal craftsmanship
flourished. The trade of three iron hoes for an ox, or four for a cow, soon made the
tribe rich in cattle (Ellenberger, 1937).
Boers moved into the area during the 1830s although the Maletes first battle
with Europeans did not occur until 1852. It was then the tribe first encountered guns
and defeat. Crossing the Notwani River where Ramotswa now stands, the tribe moved
on to Ditheyane, a Bakwena tribal village to the west headed by Chief Sechele (Tlou
and Campbell, 1984:115-116). The local residence of Dr. Livingstone, and his teaching
of Christianity, was seen as added protection against further upheaval (Tlou and
Campbell, 1984:131).
Again the Malete took advantage of their trading skills, this time bartering with
early European traders in the area for guns. The price of a single barrel was four or
five head of cattle; a double barrel was eight or nine head (Ellenberger, 1937). Without
knowing herd sizes, who can say if guns were cheap or expensive? Guns were used to
protect cattle from the numerous lions of the area and to provide an easier means of
obtaining trade items during this time of peace (Tlou and Campbell, 1984:126-128).
Ivory, skins and feathers were exchanged with traders for cooking pots, matches and
cheap trinkets (Tlou and Campbell, 1984:126-128).
The stay in Ditheyane was disrupted after 10 years when Sechele, who was poor
in cattle, demanded tribute from the visiting Malete. A short distance move to
Mankgodi did not eliminate the pestering for cattle and guns by the former hosts. In
1875, Chief Mokgosi I, who had taken over from Chief Malete, took his people back to
the Tswapong Hills, and established a new village named Ramotswa. (Ellenberger, 1937).


255
from 5 to 20. While very few (4%) received the minimum, 16% scored no more than
four points above the minimum, with half the group (49%) receiving less than half the
possible points.
A correlation existed between those who received high scores for security and a
large gerontic fund (p = .009). The social/familial component had the strongest
correlation (p = .0007). In contrast to freedom from deprivation, there is no direct
economic cost to providing security. I suspect correlation is due to the availability of
assorted family members who care and love the aged parent(s) and are willing to make
social exchange a payment of past debts. Fiduciary assets are weakly correlated with
security (p = .04), hinting that material wealth and potential inheritance and do not
strongly contribute to the gaining of service and psychological support. This is
understandable as many of the fiduciary assets of the aged are generally not valued by
their adult children, and frequently adult children have more fiduciary holdings than the
aged parent. Only a hint of possible relationship between physiological assets (p = .08)
and security existed. This suggests service and support do not alter excessively with
changes in health and vigor. There was one exception in the correlations between
physiological assets and security, if individual categories are considered. Security in the
single area of delivery of rightfully due services actually decreases with increased physical
limitations (p = .002), supporting my observations that when social exchanges involving
labor cannot be made, care is not always provided. This involves the aged who are the
most in need of physical-supportive services.
Recognition of Personhood
One main reason for the Tswana wanting to grow old is to continue the "great
works" of creating a unified family whose activities perpetuate household and kin welfare
(Alverson, 1978). This last category of the good life is called personhood. Personhood
consists of the web of social relationships that provide self-identity. It reflects the


161
60 year olds having permanent loss in the 1980s. (See Table 6.5.) Four of the five
individuals presently owning any cattle are males, age 60-69. The fifth owner is a female
in her low seventies, possessing one cow that she is saving for her funeral.
Using these 73 aged to determine total livestock assets, I found 2% own five or
more cattle, with another 10% owning either 1-4 head of cattle (3 individuals) or a herd
of 15 or more goats (9 individuals). By far, the majority (63%) controlled no animals,
These people expounded on the fact of "not even having a dog or a chicken." The 21%
with a dog or a chicken or two considered these animals as an arena for in-kind trade,
with offspring or eggs exchanged for sugar or beer.
The majority of households in Botswana rely on agriculture to either provide, or
supplement, food sources (Kerven, 1982:240-242). In Ramotswa, agriculture remains a
family affair with the aged, children, and some adults participating. As land becomes
more scarce due to population growth, it is quickly reassigned if not used, as land is
insufficient for requests. Thus, the number of hectares an old person controls
determines asset strength. Control over land extends beyond possession. It implies
decision-making for production, and a say in the use of crops. Actual participation in
agriculture is not necessary for control.
The average land size, slightly over 2 hectares, is not reflective of actuality.
Forty-two percent of the aged do not control any land. Causes are varied and include
such reasons as never acquiring land because of long term migration, loss of lands from
disuse, and transfer of lands to extended family members. The remaining individuals
(58%) have control over land, usually three to four hectares, with some having larger or
smaller fields.
Those without land are mainly women, while the mode for men is to control
three or more hectares. In one sense, this is surprising as land is shared by married
couples, with land going to the widow or widower. Also, women are regarded as the


the trials and tribulations associated with research that allowed for my own growth and
understanding.
viii


58
agreed to participate in the three additional interviews involving the felt board, ladder
and ethnographic interviewing. These individuals were also frequently visited throughout
the study period to provide data on continuity and changes with time. An additional
female was added in mid-study, as one woman was eliminated due to her extensive
responsibilities in agriculture.
Analysis
The Geriatric Assessment Form was purposely designed to permit statistical
analysis, serving as a bridge between social exchange theory and data on the individual
level. Both the ordinal variables of assets held by the aged and the mix of ordinal and
nominal variables for the good life were analyzed with the STAT-VIEW statistical
program. Specific questions were asked, including means, ranges and frequencies of
variables. My questions were many. "How are age and sex related to held resources, or
assets?" "Which assets are most important to control the good life?" "Can people find
the good life without assets?" Spearman correlations, and simple and stepwise
regressions were used to determine relationships among variables. No doubt some
questions were overlooked but the data are available for more detailed analysis.
Ethnographic interviewing associated with the in-depth studies provided much
qualitative data of statistical value. Some lent itself to coding with frequency
distributions, such as household items desired. The actual numbers on the ladder rungs
had varied meanings to different individuals but could be used to show directional flow
of relationships between self and others, and self and time. The majority of
ethnographic data, like that from participant observation, was simply coded, with
repetitive events, behaviors and thoughts singled out for use in quotes and descriptions.
This method provides validity to observed family interactions and social processes
(Bernard, 1988).


167
had the present day opportunities to establish strong fiduciary assets. Prior impetus for
personal economic growth reflected desires for traditional social and familial sharing for
equality, rather than individual personal materialism. Historic loss also enters the
picture, with devaluation of assets and the actual loss of assets with calamity and
drought. My questions on cattle ownership supported the claim that cattle ownership
transfers to the younger elite who can afford purchase and grain during drought
(Amtzen, 1984a; Amtzen, 1984b).
What value do the young place on the economic assets of the aged? With better
wages and opportunities, they regard a broad assortment of furniture, which the aged do
not have, as a necessity. Radios and watches are a mandatory luxury, with gas stoves
and refrigerators quickly moving into this mandatory category. The changing emphasis
on material goods has altered the meaning of the traditional inheritance. Younger
members of society claim it is desired, but for more modern reasons. Farm land is
supplemental to employment, the household compound is a place to erect a better
house, and livestock is to add to a larger herd, if not sold with the money placed in the
bank.
A Comprehensive Look at the Gerontic fund
All aged have at least a few assets in their gerontic fund. The majority have a
fair amount, and a few are rich. All aged who are in good health retain strength at a
level that appears to surpass Americans. All Tswana aged have some sort of access to
family members. Although monetary deficits are numerous, all have a place to live.
The average score, based on weighted averages for each dimensions, was 10.1 in a total
of 15. (SD = 1.9). Although this average is relatively high, the variations between
individual funds are expansive. Of great importance is that extreme variation existed
within all age groups, except for those over 90 who are always poor in assets.


106
ADULTHOOD
Figure 5.1: The Indigenous Life Cycle


109
As in the rest of Botswana, there is a decreased emphasis on marriage as a
marker of adulthood (Ingstad and Saugestad, 1987; Suggs, 1987). In itself, marriage
today does not necessarily carry status among the young, being viewed more as an event
than a commitment. Divorce, which has always been acceptable, is more so. The only
change is that a judicial divorce proceeding has replaced the customary act of the
woman placing her blankets and clothing on her back and leaving the house. As before,
the children and all possessions become the mans property.
Producing children without marriage is the one way the woman can obtain
parental rights and, as such, is becoming more common (Ingstad and Saugestad, 1987;
Suggs, 1987). Parenthood does contain status, even though the single mother cannot
claim the respected status of married women (Ingstad and Saugestad, 1987). I feel this
is more true in regard to respect from elders, mainly the older adults and the aged who
openly protest the increase in single parenthood. Among the young, single parenthood
has become a statement of freedom from parental control, although the young mother
usually continues to reside within the family compound. There is no prejudice against
the child, as traditionally children would be produced by the single woman as insurance
in old age. Unmarried fathers are expected, but not required, to contribute to child
support. In reality, any child support becomes part of family support.
Young adulthood slowly blends into full adulthood, without ritual. Throughout
these years, be it young or middle age, the process of growing continues. Unlike the
physical maturity of childhood, growth in this stage involves learning, maturing, and
gaining from interpersonal bonds with the extended family. Middle-aged women are
freer to participate in community social affairs as her older children take over household
chores. Lessening of household responsibility is not an increase in recreational time, as
social interactions, be it informal visiting or community service, remain an integral


297
Hill, Polly. 1986. Development Economics on Trial, The Anthropological Case for a
Prosecution. Cambridge University Press, Cambridge, England.
Hitchcock, Robert K. 1989. Settlement, Seasonality and Subsistence Stress Among the
Tyua of Northern Botswana. In Coping With Seasonal Constraints. R. Huss-
shmore with J. Curry and R. Hitchcock, Eds. MASCA Research Papers Vol. 15.
The University Museum, University of Pennsylvania, PA.
Hoover, Sally L. and Jacob S. Siegel. 1986. International Demographic Trends and
Perspectives on Aging .Journal of Cross-Cultural Gerontology 1:5-30.
Hudson, Derek J. 1977. Rural Incomes in Botswana. Botswana Notes and Records
9:101-108
Ingstad, Benedicte. 1989. The Disabled and The Community in Botswana. Paper
presented at Organization for International Health Cooperation, Feb. 24-26.
Bologna.
Ingstad, Benedicts, Frank Brunn, Edwin Sandberg and Sheila Tlou. 1991. Care for the
Elderly-Care by the Elderly: The Role of Elderly Women in Changing Tswana
Society. Paper presented at American Anthropological Association. Nov. 20-24,
1991. Chicago, IL
Ingstad, Benedicte and Sidsel Saugestad. 1987. Unmarried Mothers in Changing Tswana
Society:Implications for Household Form and Viability. Forum for
Utviklingsstudier 4:3-25.
Jacobs, Jerry. 1974. Fun City: An Ethnographic Study of a Retirement Community. Holt,
Rinehard and Winston, New York, NY.
Jacome, Eduardo Garcia. 1988. Prvida: A Non-Govemmental Organization for the
Care of the Elderly in Columbia and Latin America. Paper presented at Aging,
Demography and Well-Being in Latin America. February 23-25,1988. University
of Florida, Gainesville, FL
Keith, Jennie, Christine L Fry and Charlotte Ikels. 1990. Community as Context for
Successful Aging. In The Cultural Context of Aging. Jay Sokolovsky, Ed. Bergin
& Garvey Publishers, New York, NY.
Kerven, Carol. 1982. Rural-Urban Interdependence and Agricultural Development. In
Settlement In Botswana. R. Renee Hitchcock and Mary R. Smith, Eds.
Heinemann Educational Books, Lesotho, South Africa.
Khasiani, Shanyisa A. 1987. The Role of the Family in Meeting the Social and Economic
Needs of the Aging Population in Kenya. Genus 43:1-2:103-117.
Knudsen, Thutego. 1988. Family Welfare Educators in Botswana. Can They Be More
Community Oriented? Health Research Unit, Ministry of Health, Republic of
Botswana, Gaborone, Botswana.


184
respect his wishes." Dorothys disagreement with Betty carries no weight, for she too
agrees that God (Modimo), not doctors, controls life and death.
March 11.1990
When Tobie entered our compound at sunrise I knew the message he was
carrying. The funeral would be in 10 days, a week from Saturday, giving time for the
dissemination of news and arrival of migratory family. Ignoring the increasing daytime
temperatures, I dress properly for visitation, in long sleeves, a shawl, and a kerchief on
my head.
The family had begun mourning rites by the time I arrived. Betty was secluded
in the rondaval, face down on a bed of goat skins. Male cousins had arrived before day
light for the customary guarding of the gate. A distant aunt was heating water for tea
as she carefully stepped around the quiet children. She introduced herself as Auntie,
saying that Jacob and Betty think of me as family and that makes me part of her family
also. Other women kin were beginning the brewing of bajalwa. Dorothy, reached in the
hut to remove Bettys breakfast dishes, being careful not to touch her, or her belongings,
as direct contact with the widow is forbidden. I stand in the shade of the hedge. Other
than Auntie, no one has spoken.
Later Dorothy tells her mother of visitors who have arrived. Betty requests to
see her niece and her daughter (myself). We receive Bettys solemn, vacant gaze as we
enter the rondaval. The door closes behind us, and a black cloth on the window isolates
us from the living world. Betty is laying on a goat skin. She will not sit or stand until
the time of burial.
Mourning is a time when all thoughts are centered on the dead. Only female
kin may sit with the widow. Along with touch, speech is prohibited except for barely
audible, needed commands and exchanges. Several hours of silence pass until Betty
nods, indicating we may leave and other kin may enter. Before many days pass, her


275
Many traditional patterns of behavior continue. Work and active leisure are not
divided. Visiting, talking and feasting are integral parts of lifes work. Life centers on
the present. Therefore, one is, or is not, involved with "doing." "Doing" and "not-doing"
separates achieving from idleness. It also separates the elder from the child, for without
providing the desired contributions, one is useless and has no value. The past is
ignored, making it difficult to rely on past accomplishments and experiences for
establishing usefulness or value. The future is uncertain and unpredictable, directed by
forces over which one has limited, if any, control. There is no quilt with failure, as it is
forces beyond ones control that prevents achievement. One tries to achieve with
continuous doing, but one must also accept the situation.
Those individuals perceived as not productively "doing" are shamed and teased
for acting like a child. The aged have difficulty finding ways to be productive in a
recognized manner, as economic and technological change has damaged the door of
usefulness. The aged are degraded, as contributing has more social value than being the
recipient of contribution and the rights of elderhood.
Continuity in many of the traditional laws also limits opportunities for the aged
to be of value in the household. The daughter is to cook, the son is to make repairs,
the child is to carry water. If an individual fails in duties, the duties are not assumed by
others. Often, everyone does without, unless the individual responsible arranges
otherwise. Many times the resulting deprivation comes from choice and not the physical
inability to act. Participation by the aged in household activities, because of the absence
and failure of others, is just as apt to bring shame instead of reward.
I disagree with Ingstad et al. (1991), who places the fault of lack of service on
the adult children of the aged. The claim is that because they are "the losers of modern
society," they are unwilling to serve the aged. I admit that many assume the position of
care-provider for the aged by default, but nearly all are ambitious to achieve and "want


120
I know I am respected very much because my family gives me
food and washes my clothes. I am still in charge of my house. But if I
went to look for a job now, they would laugh and chase me away. Some
old are cared for by the government and some by their children, but
some are alone and no one cares.
Those three aged who retained the status of the aged on the third rung were all
indecisive.
That could be up or down. Sometimes when I go out, people
laugh and call me a worn-out woman. Other people say, Good morning,
Mother. Then some old people are always treated nice and others are
not, even though I know of no difference between them.
The other 27 aged felt the social position had declined, with 19 of them placing the
aged on the lowest rung. The following quote are from three of these individual.
Young people liked the old people before Independence.
Civilization was not like it is today. When we were growing up, we knew
we must assist the old as we were stronger than they. In exchange, they
helped us with our problems and guided the household. Today there is
no respect for the old. I am not treated as an elder. People dont see
an elder as any different from a child. We are the same. Our children
shout at us and do what they want, regardless of what we say.
There is no respect, as in the old days. Now children do not help
the old. Before children listened to their elders and asked them for
advice. Today, when children go for a walk, all they think of are the
paths that will take them to a bar or other young.
Not many people have any manners that show the way old people
should be treated. Some shout at parents, others laugh at what their
parents say. The young dont like old people. They say the old must die,
even our own children want us to die.
Schaperas (1955) definition of the old as helpless intones a decrease of social
status along with the loss of family power. For the young, the new definition of social
old age includes this decrease of social status. The change is that open disrespect has
replaced customary deference, which traditionally indicated respect.
Much of the psychic distress the aged feel today is actually status anguish. Status
anguish is created from an inconsistency between what the aged perceive as a deserved
elderhood ranking and the publics conveyance of childhood and dislike (Lauer, 1973).


212
rates increased to 34% and would have climbed higher except that Botswana was one of
the largest per capita recipients of food-aid in Africa (Morgan, 1988). The prevalence
of severe malnutrition in children under age 5 now remains about 30%, and this may be
a gross underestimate (Hay et al., 1988; Knudsen 1988:19). Similarly high rates are
believed to present among older children and adults, as the basic cause creating hunger
in young children also causes hunger in older individuals. That cause is chronic
household poverty (Morgan, 1988).
Rural incomes are declining in real terms as inflation increases, with those in
poverty affected the hardest (Hay et al., 1985:11). Survival becomes the objective. The
need to survive forces participation in agriculture. Three/fourths of the studied
households planted with the sole purpose of obtaining food. Those not in agriculture
were mainly the aged destitute living alone or with very young children. They were also
the ones who either lost or never owned land. Very few were like Priscilla and Isaac,
who could gather enough resources for food without being rich.
If the rains were good last year, as claimed by the missionary, why are these
farmers hungry? Substantial risks in planting accompany the risks in life. Expected
production does not occur when scattered rains fail to fall on a segment of land, when
plowing is late because of community demands on tractor time, or illness and
absenteeism of sons and daughters prevents proper maintenance of the crop. An
unexpected expense means the selling of intended food grain for a monetary obligation.
For most, production is between two to five bags of grain. There is never enough to
feed the average household of 6, much less support a family, for a year (Hay et al.,
1985). Sorghum alone, even if sufficient, is not adequate nutrition. Meat, vegetables,
and milk need to be purchased to round out the diet. Food security comes only with
secure employment or regular remittances.


2
expend unnecessary work to open the door wider. At times the passerby will keep on
walking, or the occupant will stay indoors. Both know the door is broken and question
if the extra work is worth the visit. Occasionally one sees a door so damaged that it has
been sealed shut. The house has nothing to offer, and the past occupant is said to have
moved or "disappeared." No one exits and no one visits.
To Americans, the closed door provides a safe haven. To the Tswana, an open
door provides access to a safe haven. It shows that people are part of life and have a
respected place in it. The open door is more than a passageway. It is a giver-of-life in
itself. The Tswana use many doors in their movement through old age.
I use the Tswana meaning of door as an analogy for a continuing access to
opportunities to maximize potential as a person. The small cracks are those that affect
the process of "being someone." They may be disliked but are an integral part of life.
As the cracks enlarge and the door refuses to swing properly, access to personhood and
the good things in life becomes more difficult. Additional breakage creates barriers in
bringing others into ones life, and in entering the lives of others. Some obstacles are
more difficult to overcome than others, depending on the type of damage. Extra effort
is always needed to use the door. The door so damaged that it is permanently sealed
shut severs access to a full and meaningful life.
Two things will be highlighted in this study. First are the many doors that the
Tswana aged use for a rewarding life, and the fact that many no longer work properly.
Cultural continuity provides both strength and imperfections in their structure. Social
change can provide new doors. Usually, social change misshapes the old doors so that
additional effort is needed for use. Sometimes the old cracks and new imperfections
combine to form a deadlock. The door is inoperable from either side. No one can pass
through it. Both the Tswana aged and society must deal with these various damaged
and broken doors in finding access to the things they hold dear.


165
their working years, they always purchased a bed first, then a storage unit. The third
and fourth purchases were a table and straight back chair.
A leading council official, in perusal of my research instrument, proudly stated
that no one in this village was without a bed. The days of sleeping on a goat skin or
mat had gone by. That may have been true for the younger generations, but not for the
aged. Their possessions reflect the stated preference in purchases, with many unable to
obtain the basics. Nearly half (43%) sleep on the floor with a blanket, mat or goat skin
under them. If only one piece of furniture is owned, it is always a bed. With two
pieces of furniture, the second is a storage unit, usually a chest of drawers or a
wardrobe. Only when a person owned these items do they add the table. Fewer than a
third (31%) own anything above this. This does not mean assorted furniture is not is
the house. It is, for most aged do not live alone.
I was quite interested in discovering the degree that laws regulating use of
furniture were practiced. The reply to, "Do you use your daughters bed during the
day?" was always denial. I tried other methods to give permission to say "yes," and
always heard "no." Several aged, without their own house and with no or few
possessions, house-sat for migrated adult children. These homes were furnished with
beds owned by children who returned once a year. I expressed my feeling that if I knew
the owner would not be back for 11 or 12 months, I would probably use the bed if I
was sick. In all cases I was severely reprimanded for the thought. The same was true
with the use of anothers blankets, even if it was cold. One man, living in his sons
house containing easy chairs, always sat on his sleeping skin. No permission had been
given for the use of the furniture.
In each of the fiduciary categories, individuals are fairly equally distributed within
item options. Patterns of distribution between options varies with age groups, with the
older groups consistently reporting fewer fiduciary assets than the youngest one (p =


83
for total cultural conversion without regard for the impact on social and economic life
(Tlou and Campbell, 1984:140). By the end of the 1930s, Europeans in Ramotswa
claimed success in their goals although "a few relics of barbarism" remained
(Ellenberger, 1937). There was some trepidation whether the acceptance of Christianity
was a matter of course, as missionaries "labored amongst an unresponsive people"
(Schapera, 1953:58).
There is no denial on the part of the aged Malete that the ingress of Europeans
brought delightful foods, Western clothing, and money. But through the eyes of those
who lived during this period of importation, new experiences were found within the
bounds of the Tswana cultural setting. To them, it was still "the time before
civilization."
In other words, this was still a time when the traditional reigned. Traditional
here is defined as that quality of social life in which contact with the Western world did
not play a significant part of the lives of the majority of the population (Cohen,
1986:136). It does not encompass the substitution of a metal pot for a clay one, or the
covering of the entire body with cloth instead of a leather loin cloth. A traditional life is
one where basic behavior, regardless of the implements used, continues to be built on
the non-Westem organizational principles of the society.
Social unification was stressed by many. The change that did occur was a series
of adaptive responses. In part, change was to allow for continuation of the traditional
through accommodations. On the individual level, money became another trade item,
not the basic necessity for subsistence. Taxation and growing desire for luxury items
encouraged, rather than forced, labor migration of a single family member (Parson,
1984;23). Employment became new way of achieving without altering the basic structure
of the culture. The comprehensive process of achieving, embracing the concept of
gerontocracy, remained the underlying ideology.


287
The next few question are about what you eat I want you to think carefully about the
answers.
48. What did you eat for breakfast yesterday? Today?
49. For lunch yesterday? Today?
50. For supper yesterday? Today?
51. Did you have anything between breakfast and lunch, such as beer, tea, or
food?
52. What did you eat or drink between lunch and supper?
53. What did you eat or drink after supper or at bedtime?
54. How many spoons of sugar do you put in your tea?
55. How often do you drink beer? ; can of beer?
How often do you eat or drink something made with:
56. vegetables?
57. eggs?
58. meat
59. milk?
60. I will read a list of reasons people are hungry. Tell me if any are true for you.
I. No one brings me food and I am unable to prepare it myself.
2 I cannot afford to buy enough food for myself.
3 I have no one to go to the store for me.
4 It is better to let someone else eat the food. (Who)
5 I/my family was unable to grow enough food this season.
6.1 do not have firewood or matches.
7.1 want to loose weight.
8.1 cannot chew or my mouth hurts.
9. Everyone in this household is hungry.
10.1 cannot get water for cooking.
II. Do you have another reason for staying hungry. (State)
12.1 never go hungry.
61. Which is the main reason you stay hungry? (Mark one with an X)
62. How many times in the past week did you have a day without food? Why


27
should be viewed as intentional directors of their lives, accepting the fluid relationship
between people and social contexts and actively integrating role definitions with
opportunities experienced and to be had. Like other people, they seek need fulfillment
using what is available from the environment, from culture, and from others. Their
efforts involve both active and reactive responses. Individuals strive to meet ideals,
which can, and do, express a variance from social ideals and from reality. Context can
vary, as with the differing reality and ideals between the African village and American
city. The positiveness of aging, with a recognition of continuing yet varying capacities
for interactions with and within the environment, must be incorporated into theory.
Social Environmental Theory
The social sciences have provided a framework for more comprehensive
gerontological theory. Social Exchange Theory explains how individuals exchange goods
and nonmaterial resources in society in order to meet their needs. Gerontology has
adapted this theory to the special needs of the aged.
Social Exchange theory arose from Goodenoughs (1963) interpretation of culture
as emerging from shared knowledge among individuals with each person having his or
her own mental template for behavior. Behavior is interpreted as voluntary actions on
the part of the individuals, who are motivated by the returns they expect to receive from
others. The unit of analysis is always the individual who is seen as an independent, self-
directing person involved with a series of trade transactions (Cook, 1987). Each
individual negotiates for a favorable exchange. Trade is possible only when both
partners perceive a reward that is advantageous to themselves (Dowd, 1980:58).
A person must have resources in order to make trades (Blau, 1973). A social
exchange resource is any labor, service or commodity exchanged during social
interaction. Resources include tangible and abstract goods, including the use of power
and subordinate behaviors. An individual may have direct control of a resource, such


302
United Nations. 1985. The World Aging Situation: Strategies and Policies. Department
of International and Social Affairs, United Nations, New York, NY.
Wolensky, Robert P. and Kenneth C. Wolensky. 1990. Local Governments Problem with
Disaster Management: A Literature Review and Structural Analysis. Policy
Studies Review 90:4:703-725.


238
removed from the impervious knowledge and activity reserved for youth. Priscillas
dance confronted this assumption. The dance that followed discredited the thought that
the aged resemble youth in desire and thought. "It is impossible for the aged be alive
and modem."
The aged, exposed to public ridicule and degradation, sometimes bow out of
potential skirmishes. The older women, when faced with doubt of their sexuality during
the brides party, withdrew from the festivity. Other aged pretend to ignore the actions
of youth, although the hurt is deep with teasing and ridicule. Very few would purposely
face confrontation, as with Elisa in her attempt to show superiority with the dentures.
Most aged retreat.
The young do not retreat. Changes in discipline influence public, as well as
family, behaviors. The lack of deference and respect no longer results in public shame
among cohorts. The power of group-support promotes ridicule, and attempts to
disengage the aged from public activities. The aged use their age-group for solace,
instead of power. Priscilla and her cohorts effectively ended the derision caused by the
dance, but failed to win. The few opportunities to use group action are compromised
with threats of losing the whatever support they have from middle-aged adults, who lean
both right and left.
The majority of schisms are representative of those found in our society. Martin
Martel (1968:55-56) claims that with American social advancement, a shift in the
sociological prime of life from middle age to young adulthood has occurred. Life after
age 40 becomes "anti-climatic", with the aged shut out from meaningful interactions
(Martel, 1968:56). The devaluation of the aged is well known in developed society. We
might ponder if it is the social advancement that creates change in people, or does
socio-biological human behavior become more pronounced with social advancement.


181
live with hunger now. We still have one cow that we want to save for the funeral of
whoever dies first." The social worker claimed the sons were capable of supporting the
household and that the cow represented available money. She expressed anger that so
many aged must live in poverty because adult children think the government should
support the aged and so do not send remittances.
This afternoon Jacob is at his cousins house Betty and Dorothy are cleaning
for the Christmas season. The family expects tradition to hold true, with the two sons
arriving sometime during the holidays. Lack of communication does not dampen their
hopes. Betty makes it known that she wants the latrine spotless and leaves to clean it
herself. Tobie, who has been sitting by himself, is told to carry the supplies. He
continues to sit as Betty gathers rags and leaves the house.
Dorothy and I are alone in the house, except for the two babies sleeping on the
floor and Tobie, now in another room. I use this opportunity to discuss her role as the
care provider.
My grandmother is never happy with the way I clean. She is
always moving a chair slightly or rearranging the ashtray on the table.
Both my grandparents are children, unable to think or do anything for
themselves. They want me to do all the work but they will not turn the
house over to me.
Using the ladder, she rates their physical abilities as in the middle. She explains
that they can take care of their bodies but have many years. In ranking them in
comparison to the person who has money and objects to give away versus the person
with nothing, she places them near the top, yet she knows about the discontinued
pension and infrequent, irregular remittances. "They are the ones with the money.
They just wont give me any, except to buy food at the shops."
I put the ladder away. I ask how she would envision her life if she did not have
to take care of her grandparents. She pauses before saying:


271
The Aged
Diversity among the aged of Ramotswa exists, with widows, childless aged, the
physically limited, the extremely poor, and the extremely aged contrasting with the
economic elite, aged in large families, and the physically strong. All placed emphasis on
tradition, but varied in the extent that Westernization played a role in their behaviors
and thoughts. Individuals varied markedly in the strength of their fund, but no
particular group stood out as having the best or worst in the good life, either by age,
sex, family finances, or marital status.
The Tswana aged seek a good life by using the self-controlled assets in their
gerontic fund. Physical, family, social, and economic resources are used in a process of
social exchange to obtain service, care, security and recognition. The amount of held
assets varies between individuals, with some aged entering old age with very weak
dowries. Natural disasters, changing patterns of employment, limited education, and
increased demands on income prevented them from entering old age with the needed
riches. Others report having lost their assets since becoming old. Repeated drought,
death and migration of family members, new expenses, and illness tend to erode the
funds as age progresses.
Strong personal-physiological, social-familial and fiduciary resources creates power
and command, allowing the aged individual to overcome the sociocultural and geo
physical barriers to a good life. Few aged had such strong gerontic funds. The poverty
of old age, within a setting of generalize poverty, places many of the aged in a gerontic
deprivation trap. Any small loss in health, family, or savings increases their vulnerability
and decreases their already waning power over others. They must be able to use the
remaining resources effectively in order to find any life satisfaction. Demand and
compliance may provide the needed basics for life, but not satisfaction. Therefore, I see


77
the donkey is hard working animal, which devotes its life to the welfare of
people. It is not to be eaten but paid respect until a natural death. Like
the child and the dog, he must be trained and controlled through beating.
We do this because we love him.
Another loved animal was the dog. Every man owned a dog for protection and
hunting. Many of the men talked about their dogs value, including the two village men
I met the first day. The eldest of the two said:
a trained dog was the way I could get a rabbit in my pot. Spears were
for wars or when attacked by a beast. They were not allowed in hunting.
Instead, I would dig a trap in the ground or make a rope noose from
plants like the men of the Kalahari {San or Bushman} did. My dog
would chase an animal into the snare.
I never went anywhere without my dog. A snake would always go
after a dog before he went after a person. The most dangerous snakes
were the black colored ones. Dogs saved my life several times,
particularly when walking to Gaborone or Otsi.
The Lands
The "lands" were an assigned agricultural plot, usually some distance from the
village. A simple shelter was home to the family women, girls, and very young boys
during the growing season. Their men had plowed the field with oxen or donkeys in
November after the early rain had softened the ground but before the grass was green
at the cattle posts. Now it was the womens responsibility to plant, weed and harvest.
According to Mma, all ages toiled together, chatting about life and the universe.
I learned stories about baboons at the lands. Baboons were
plentiful. They would come down from the hills, and rob our crops. The
baboons are always frustrated. They want to be humans, but never
develop into people. Therefore, baboons must be treated nicely and not
thwarted, or they will use their people-like brains to become very
mischievous and bring destruction to the lands. Night was the time you
really had to be careful, as a thwarted baboon could run across the paths
and cause crops to wilt.
Agriculture was one means by which a women could develop pride. A field kept
free of weeds and birds was envied by others. Outside influences such as rain, and the
mysteries of the baboon, determined the size of the harvest. One could expect crop
failure about once in every three or four years. The fields were small, usually two to


29
it becomes possible to evaluate the individual and the aging process independently of
the degree of modernization or by standardizing the sociocultural environment.
This separation does not mean the specific social context, including the people,
social structures, and institutions, can be ignored. Historical, political and economic
features, as well as cultural values and the ongoing social construction of everyday living
experiences, are important in the process of aging. These factors can be approached
objectively in that they are external to, and partially independent of, the dynamics of the
mind (Gubrium, 1973:36).
The theory states that three overlapping dimensions of resources under the direct
control of the aged individual are necessary in order to maximize satisfaction and to
maintain the individual in a viable position as a creative agent in the exchange process
(Gubrium, 1973). The broad outline of resources cuts across all social and cultural
situations. The personal I physiological resources are of a physical, mental and
psychological nature, and are essential for integrity and active participation in the
exchange process within the environment. The social/familial dimension refers to the
people and support facilities whose contextual presences can provide positive
reinforcement for personhood. The fiduciary dimension consists of the "coin of the
realm" in a particular context. Money, goods and other barterable items, such as access
to land, are included (Gubrium, 1973).
A major assumption of Social Environmental Theory is that resources decrease
with aging (Gubrium, 1973). Maladjustment increases and social acceptance decreases
as holdings in the dimensions become increasingly limited (Lawton, 1983:665; Dowd,
1984). Resources may be used or "spent" without replacement, disappear with failing
body integrity, or may be simply lost to the environment, such as cattle death with
drought.


211
bread, she says "Take this. Ive heard of your work. Give this to the old people who
are the hungriest."
Who are the hungriest? Choices for decision-making overwhelm me. My friend,
Sego, is sometimes hungry, but he manages to get a meal at least once a day. Others
are more in need. The loaf I give Elizabeth and her nephew is shared with the
preschool great-grandchildren and quickly disappears without accompaniments or
beverage. Then I see Anah, who shares her destitute rations meant for one with her six
pre-school grandchildren. These young children spend most of the day sitting on the
ground, obeying Anna only after persistent commands. They scramble quickly to the
gate when they see Anah breaking the bread. Again, the food is gobbled immediately.
I know I will find Sepia and his 88 year old mother at home. Both have told me they
reason they sit so much is that they "cannot afford to walk." Their bread is gone in
three minutes. My last stop is at the home of Dikeledi, an 82 year old man, who lives
with his 91 year old sister. She is asleep and the way the bread is disappearing, I begin
to doubt if she will get any. Dikeledi reassures me that he will save some for her. I do
not worry about the four grandchildren at school, as they will get a large lunch of
vegetables and mealie meal (ground com). As I take my leave, Dikeledi holds me back.
People in the city with their fine houses quickly forget how people
in the village have to live. Even those with education, and thus work for
the local government, do not want to think of what it is like to be a poor
villager. Those who work see only themselves, and they have the money
to be fat. They tell me I am a useless old man. On the streets, people
call me old man in a nasty way. They think that because I am destitute
I should work. Ten years ago, the place where I worked said I was old
and worn out. They made me quit work. They took away my
independence and made me a child. Now they saywork. Who will give
work to someone as old as I am. They dont see that I tend to my sister
and the children and we have no land to plant.
Hunger is a multifaceted problem affecting young and old alike. A few families
have abundance in food resources; many have none. Prior to the 1981 drought, national
rates of malnutrition among preschool children hovered around 24%. With the drought,


51
as the Tswana refuse to predict the future. Questions regarding individual decision
making and personal independent functioning were not applicable at all, in light of the
marked norm for interdependent behaviors for household functioning.
Tswana world view does not include comparisons and ranking. One knows only
about him/herself. There are no gradations with emotions or opinion. One believes or
does not believe. One feels emotions totally or not at all. Open-ended questions with
ethnographic interviewing provided the best measurements of life satisfaction, but these
too met with limitations. Questioning about perceptions of life as it is today (rewarding,
dull, interesting, etc.?) was limited, as adjectives describing the quality of life are absent
in the Setswana vocabulary. Life is either happy or sad. Discussions of the reasons for
happiness or sadness did little to illuminate the degree of feelings, as learned thought
processes constrict perceptions to the categorical black-or-white. However, responses did
shed much light for the development of constructs for the process of achieving, including
perceptions of utility, providing direction to others and the value of self in social
interactions.
Functional utility, both in the home and community, was expounded upon by
altering the often used question of "How often does your health, and changes with age,
stand in the way of your doing the things you want to do?" When the unmodified
question was used, all respondents answered "All the time," regardless of the degree of
observable physical limitations. In the same question, aged always stated if they were
"useful" or "useless." This again reflected the dichotomy of their world view. While
reasons for the response would be easily stated, the individual could offer no variations
on a continuum, i.e., could not see oneself as having varying degrees or types of
functional value. When the initial question was changed to a request for specific
activities that could or could not be done and why this was do, data concerning health
and activity emerged.


175
you always claim illness yet you run about town and get drunk every day.
You are lazy. As a son, you are useless to Mother. How can I return to
work this afternoon with such a shameful man in my mothers
compound?
My entrance does nothing to temper the argument. The older children ignore
the scene and continue their usual pastime of idly drawing lines in the dirt. Happy
Sound, pretending deafness to the yells, indicates I should sit on the goat skin with her,
under the tree As we talk, Leru leaves for the brigade. Kaizer walks over to the
children, bending to tie their shoes. He addresses his mother with the request to visit
friends. She hesitates, and then grants permission, although pain in her voice is clear.
Turning towards me she speaks softly.
When I talk to you of disrespect, this is what I mean. The
children scream at each other. The son does not fix the roof. The
daughter acts like she directs the household. The children of today do
not follow the law. Will they follow the laws of giving food, and bathing
me, when I am an old parent?
Happy Sound smiles as she lifts the baby (now a toddler) in her arms. She looks
at him while she says:
I teach my grandchildren the laws, maybe they will keep them. I am
lucky as I have only three. With any less, they could not do all that is
expected. Any more would be too many. It is much work for the
grandmother to raise more than three grandchildren.
December 18.1989
Happy Sound and I meet on the footpath near her house. A blanket holds the
toddler on her back. Her hands hold a dozen quart sized beverage containers that she
has picked up along the road. Once used for Shake-Shake, a commercial traditional
brew, she claims she has another use for them. She will show me, if I go home with
Lined up along the side of the rondaval are numerous containers, each with a
plastic fork shading a small plant from the bright sun.
When I last visited my daughter in Gaborone, her master had
many of these forks that people had discarded near the take-away food


263
I have grave doubts if such income would be used for clothing, bedding, or traditional
health care for the aged. Yet, this potential program indicates that the government is
concerned and is trying to divide equally.
The second program under consideration is the construction of a government
supported 36 bed nursing home, to be located in the heavily populated area of
Francistown, some distance to the northeast. This will provide care to a select few aged,
to be determined by policy relating to economic and health needs. Several drawbacks
can be identified. A few aged receive benefits from a large expense. Their families
receive "something for nothing", an ambient attitude created by past drought relief
program that the government desires to combat. Equally important, this transfer of
Western ways and means of gerontological care acts in direct opposition to the desired
promotion of traditional family-centered care in the home. In addition, it is concrete
evidence to the population that separation of the aged from the rest of society is
acceptable.
The provision of care services is a growing problem in Botswana, and does need
to be addressed. A novel approach to the problem was taken by the Maru-A-Pula High
School in Gaborone, as part of their community outreach program. A housing unit of
one elongated building with several bedrooms and a kitchen area was constructed in the
middle of a small rural village. Aged who were living alone were invited to become
residents. A mix of talents and abilities in residents resulted, ranging between the very
active and alert to the care-dependent bedridden. Chores were divided according to
strengths and desires. Care was given to the bedridden. Pride in achieving replaced
fears of being alone when in need or at death. The house location allowed two-way
interaction with the village, with visits from kin and to kin. The school was proud of
their innovation. The community was proud the aged who lived there.


79
Elizabeth talked about how Chief Mokgosi I respected the church leadership,
becoming one of the first Malete to convert to Christianity and attend the new Lutheran
Church. She then talked of her familys conversion.
I remember when we first went to the church. I was still a very
young girl. There was a large room full of beautiful colored materials.
Everyone wanted this cloth, but the only way to get it was to become a
Christian. My mother joined the church, and she later had beautiful cloth
to cover her entire body.
My parents insisted I go to the church confirmation school instead
oiBoyale {female initiation training}. I was taught reading and writing.
I was baptized at that time, and given the Christian name of Elizabeth.
The girls of the school felt very different and separated from those of
Boyale {tribal initiation} so we went to the chief and asked that he make
us part of the age-regiment system. He did this by giving us a group
name.
It was easy for people to become a Christian. The Tswana have
always believed in one god, Modimo. The Christian god and the Tswana
god were really the same. With Christianity, you could pray directly to
God for important things, like rain. We continued to seek guidance and
knowledge during dreams from our Badimo {ancestors, mainly dead
parents and grandparents}. It seemed to us that, as Christians, we could
live a better life and still keep our customs. We drank tea with sugar.
This was the food of the civilized people. The church looked down on
the heathens, or people who did not join the church. The church said
they were uncivilized. Actually, we all thought and acted the same.
My parents had always used the Ngaka ya Setswana {traditional
medical doctor} and the Moprofiti {an explainer of the past and
predictor of the future, doctor of ill fortune}. The church said this was
wrong, as they were heathen witch doctors. The people of the church
gave us powerful new medicines to make us well. After that we never
used the traditional doctors, mainly because the new medicine worked so
well, not because we changed what we believed.
Senatla. Senatla, Elizabeths nephew, is 88 years old. Today, he and Elizabeth live
together with his 17 year old granddaughter and seven preschool great-grandchildren.
They reside on a small piece of land near the original kgotla. This land was given to
Senatlas father by the Chief Mokgosi I. According to custom, it was here that Senatla
built his house. It is the only remaining living quarters on the original family compound.
Senatla, meaning Industrious-Person in Setswana, was not a church member in
his youth. He spent much of his childhood away from the village. As a young boy he
went with his mother to the lands. When about six years old, he began accompanying


209
There can be no completely satisfying life to all members of a society. The aged,
operating within a framework that carries the past back further, have historically larger
perceptions of the paths that negotiation should follow.
The aged were socialized to society when sources of status were linked with
ceremony, ritual and traditional power. They introduced new status sources of the
Western kind. Employment has led to a conspicuous consumption of a modern life style
and acquisition of necessary funds to abrogate the bonds of traditional obligations
(Harrison, 1987:467). These easily adapted material forms of culture are gradually
influencing the less amenable symbolic forms.
The village young are caught in the bind of social pluralism as they mediate new
knowledge with old folklore, interpret modern beliefs in light of traditional world view,
and negotiate new ethics with customary laws. Seeking satisfaction, the adoption of the
latest Western fashions and lifestyles gives the young a symbol of supposed superiority,
which they hold with contempt over the aged (Harrison, 1987:54). The resulting fission
and fusion breaches the bond between being Tswana and being Batswana (the people of
Botswana). The paradox is that, in seeking the new national Batswana ethnicity, reliance
for direction stems from traditional Tswana tribal thought. The irony is, that with the
forces of time, todays young are very apt to be without the what they hold dear at the
present.
In much of rural Africa, the primary social struggles are not just between
traditional and new ideas, but against a social injustice found with abject poverty and
gross inequality in opportunity (Harrison, 1987:467). Social justice in not a simple
matter of the distribution of goods. It includes the provisioning of social, political,
economic and cultural rights. This chapter deals with the hope and despair arising from
the struggles of change when interfaced with the continuation of assorted traditionally
familiar meanings and symbols, and the disappearance and new acquisition of others.


147
memory for all aged (59%) parallels the western bane of aging: that of occasional
forgetfulness. This complaint occurred most frequently in the 70 and above age-groups.
The stories are familiar: that of not being able to find a certain paper, or having money
or keys in a pocket and not realizing it. This complaint increases in frequency with
older ages, but not necessarily in severity. Individuals readily recall my previous visits
and topics of conversation. The average score for those age 90 and above is like that of
others; occasional forgetting. All have accurate memories of the past. Their historical
stories are quickly confirmed by family members or other aged.
Overall, the personal/psychological bundle of the gerontic fund shows many assets
for many people. For all ages, the weighted total mean was 3.8 out of a possible 5 (SD
= .9, N = 105). This indicates that the typical aged could care for themselves and
participate in life with minimal assistance. Like aged everywhere, increased years do
take a toll, with a decrease in strength and stamina. There is a significant relationship
between personal/psychological assets and age (p = .0001). The 60-69 year old age have
weighted average scores of 4.2. This weighted average progressively drops in each ten-
year age group, although it was not until reaching 90 years or above do scores routinely
fall below 2.
Men fare no better or worse than women. For both sexes, if an individuals
scores are weak in the self-care category, they tend to be weak in social functioning,
reflecting a generalized decrease in body integrity. As a rule, it appears that major, all-
encompassing, physical limitations do not appear until quite late in life. There are
exceptions to this rule, especially for those with chronic debilitating illness.
The Social/Familial Dimension
Social/familial assets are based on the availability of interactional partners and
social structures that can be used for the creation of service and support. As explained
earlier, with meaningful others placed within the fund, they can be regarded as assets to


163
problems with environmental elements than a house needing repairs. A home, even in
poor condition, had more intrinsic value to the aged than a building provided by others.
During my initial visit to Ramotswa I met a couple of aged individuals living in
temporary housing. One was renting and the other had loan of a large tent. No such
living arrangements are present with the sampled aged.
By far, the majority (69%) live in the home they constructed earlier in life. Of
these 72 aged, 28 live in well maintained buildings and 27 live in homes needing minor
repair such as broken windows or a cracked side-pole replaced. The remaining 17 live
in homes where major repair is needed. Major repair includes the need to correct
support beams eaten away by ants, walls eroded to a degree of potential danger, or
large holes in roofing limiting protection from cold and rain.
A roof that does not leak in heavy rain is a rarity. Sound roofing is found only
on more expensive buildings, which also were the only ones with ceilings. It does not
matter if the roofs is thatch or corrugated iron, they leak. Almost everyone complains.
During rain belongings are sometimes left in place with the knowledge that they will
soon dry. At other times, it is necessary to move belongings. I, too, had to learn to
sleep in contorted positions to avoid damp places in bed and to avoid using certain
sections of the floor for storage during the rainy season. Based on the pervasiveness of
this housing trait, leaking is not included in the state of repair.
The intrinsic importance of home ownership is shown by the reluctance of the
aged to move when their housing conditions become substandard. They remain in
potentially dangerous houses, although better living conditions are available. Other
family members are usually living in the compound and many had offered to provide
housing. With the 30% who are living in another persons house, the move tended to
be to an adult childs house within the same compound. Those who move in with a


272
that the real issue with the aged is not one of age, or traditional ethnicity abutting
modernization, but one of poverty for successful social exchange.
No point exists in which the gerontic fund automatically provides a good life, or
negates its occurrence. Much depends on the openness and the swing of the doors that
are used during exchange. Some fund-poor, regardless of age, found various segments
of life satisfaction. All fund-rich reported some area of life dissatisfaction. Personhood,
or being regarded as a valuable, wanted, and respected individual was the most difficult
to obtain, regardless of resources. Most can obtain life-supportive care from others,
although some fund-poor cannot acquire basic needed services. Benevolence for
satisfying care cannot always be relied upon, although the traditions of giving goods and
assistance continues.
The diversity between individuals in finding the good life of care, security and
personhood, in conjunction with the diverse amounts of fund assets, strongly suggests
that the assorted process of social exchange is as important as the items to be traded.
These processes involve the aged, the family, and the community placed together in a
cultural context.
The Context for Exchange
Ramotswa, as a village, is full of diversity. A mix of old cultural patterns abuts,
clashes and collides with Western technology, architecture, approaches to health care,
and religions. People of all ages are puzzled as to why traditional beliefs, and behaviors,
do not blend smoothly with new thought and actions. Conflict creeps into everyday life
as new pressures overtake obedience to the traditional laws. The young are guilty and
so are the aged. Diane ignores others in her quest for the good life. Betty screams at
Dorothy. Alfred never gives Priscilla a portion of the egg money. Naturally, the
following of the basic laws, for and during social exchange, is more important to the
aged, for this is what they learned as proper and necessary for continuing life.


16
The aged Tswana have expectations and desires of things to come, but believe
that very limited control over the future is possible. Conceptually, the future is blurry at
best. "Nature" (the interrelationship between self and outside forces) determines the
future and the potential meeting of goals. It is the present, with involvement in the
process of achieving, which is the central issue, not the long range outcome or the
future. Thus, old age goals are synonymous with everyday behaviors involving self, family
and community.
One must ponder whether there is a theoretically universal uniformity regarding
the goals of all aged, or whether the cross-cultural context differs too radically to be
bridged. Differing cultural value systems and socialization processes produce varying
roles and behaviors expected of the aged (Sokolovsky, 1990). It is logical that the
positive social outcomes of these expectations produce culturally specific old age goals.
Do culturally specific variables transcend differences to produce a higher level of
uniformity? It is vital to answer this question, using data from a variety of cultures from
both developed and developing nations to produce sound theory and to plan
prophylactically for the increase in world aging.
The goals of old age in America are usually described abstractly as
independence, autonomy, good morale, health and peace of mind. To use leisure time
effectively and have sufficient guaranteed income are vital inclusions (Gubrium, 1973:ix;
Jacobs, 1974; Myerhoff, 1978). Such goals reflect the social horizons of the world in
which we live.
Presentations of goals tend to be more concrete when one looks at aging on a
universal scale. They include the desire to have and control resources acquired during
life and the guarantee of food, shelter and care. They also include features of retaining
status and respect, being useful participants in family and group affairs while
safeguarding health and energies, and the right to withdraw from life honorably when


221
The hospital is a good place, but many times the nurses do not
understand what it is like to be old. They say we are wearing out and
our disease is in our head. Sometimes the disrespect for the old worries
me more than any disease, even high blood.
Sometimes I fear the hospital. I have heard they sometimes do
things that make people die. Other times, they do things that keep
people alive when they should die. Ive heard that when people reach
the end of high blood disease {have a stroke}, the doctors can put in a
tube for food, even though the person cannot move or talk. It is good
they are alive but there is no way for them to live in village. All they can
do is stay on a blanket and be pulled along the ground. That is not the
life that God wants us to have. The meaning of life has been lost.
Sego has been listening to the women talk. He tells us of one old man who has
sick legs.
Other people and witches started the sickness. He went to the
hospital where they treated him like they wanted him to die. He went to
the traditional doctor. That doctor said he could dig a hole and put
medicine in it that would cure him. My friend didnt have a cow for
payment so he is still sick and will be sick until the curse is removed or
he dies. No one will let him live happily.
The body is the same as grass. Grass grows, goes down and dies.
It becomes green again after a rain. A person goes down without the
water of life but can become green again after death. He is a new
person in the skies. It is strange. When we want to live as elders, people
act like they want us to go down and die. When we are children and
want to die, they make us live.
For family survival, good health and food come First. Disease is approached with
just concern, as health provides the necessary buffer of labor against food insecurity
(Chambers, 1983:142,144). In Botswanas favor, emphasis on preventive medicine
promotes health, even in the poorest and most uneducated village households. Clinic
and hospital walls have multiple posters promoting cleanliness, sound infant care, good
nutrition, and safe sex. The general public strives to comply with preventive measures,
as far as economics, tradition and social behaviors allow them. The ambient social and
political settings promote compliance with maternal-child health programs. This is not
so true with other programs. Knowledge of prevention is present. The means is absent,
either in finances, material goods or beliefs.


192
his or her own good. This is a practice, not a law. Many present day aged report
accumulating items for themselves over the purchasing gifts for others. They never
mention that a law regarding income was gradually changed, even with questioning.
It seems that the present day working adults are building their own future
gerontic fund, just as their parents did. Their first purchase continues to be a bed, then
the dresser and other furniture. The middle-aged sense that if multiple blankets, radios,
watches and such are not obtained soon such items will never be obtained. Like their
parents, they do not expect such gifts in old age.
As each family varies in the degree of deprivation, so each family also varies in
the breath and depth in the maintenance of traditional laws and the incorporation of
Western thought. My purpose is to provide understanding of how these social practices
unite in family structure and functioning, and thereby influence the value and use of the
gerontic fund. Family structure is defined as at the communication network with a
family, with family function encompassing family supportive behaviors (Ryan and Austin,
1989). Each are separate and important variables in the process of social exchange as
family structure and function are the doors connecting household members with each
other.
Family Structure
The two main influential variables in structure, repeatedly referred to by the
aged, are discourse and decision-making patterns. These two factors transcend family
size and ages of the members. Patterns are similar between household, always reflecting
aspects of cultural continuity, and some degree of conflict between tradition and change.
Family discourse
The teaching of every young child begins with the cultural rules of discourse.
The majority of such regulations promote power in the elder: only the elder may show
displeasure in the face and tone of voice, one does not criticize an elder, one remains


73
improvement (Picard, 1987:98). The penalty for non-payment was severe (Tlou and
Campbell, 1984:181).
Access to money became a requirement for taxes and additional reasons. The
Malete, because of the delineation of the size of their reserve by the protectorate,
suffered from chronic agricultural and grazing land shortage (Schapera, 1953:24). In
1921, and again in 1926, Chief Seboko was forced to purchase additional lands from the
British. Every man in the tribe had to contribute five English Pounds for the land
purchases (Ellenberger, 1937). People also wanted to purchase the growing numbers of
trade goods (Picard, 1987:31).
Money became an absolute necessity, forcing at least one male household
member to become employed. Employment was available only through labor migration.
The Malete did not relish the idea of working underground in the South African mines,
but many went. Others chose farm labor in the region of Transvaal. Men, migrating for
employment, were separated from wives and children, with remittances being sent home
for the familys required expenses (Ellenberger, 1937).
At the end of the first third of the twentieth century, the English claimed success
in meeting many of their goals for "rehabilitation" and "civilization" with only "relics of
barbarism" remaining (Ellenberger, 1937). Schools had been established;, the church
had over 3000 members; and tribal customs were being discarded. To the Malete, these
were still the days before civilization.
Although changes were being made, the aged regard this time period as the days
before civilization. Some were born before the 1899 hut tax, others as late as 1929. In
either case, it was the time of their youth, the formative years when, as children, they
were socialized to their world and expectations in later life. The following is based on
their perceptions of the time period. Much of the data are supported by Ellenberger
(1937) and Schapera (1944,1955).


262
second at the Community Center. The second meeting, held nearer the other end of
the village, would allow attendance by those who could not walk across town to the
tribal headquarters. Over two hundred village aged and their families attended my
community reports of the strengths and needs of the aged.
The general findings of the study were echoed by the crowd, with specific
requests for better nutrition, treatment of hypertension and "something to make people
like us." The absence of models did not prevent their recognition of a need for a place
for old people to share common concerns and receive understanding of age-specific
needs. Throughout the presentation came pleas. "We Need Help Now!" "We dont
have years for the government to plan programs, for by then we will be dead." The
present determination and past cooperation of the aged suggested the time was ripe for
immediate intervention.
Existing Plans and Projects in Botswana
Botswana has recognized there is a need to institute programs for the aged. This
is a welcome reversal attitude since the beginning of this research in 1988. The desire is
to create policy that avoids the pitfalls of past public support programs. These
programs, usually associated with drought, promote the public concept that it is the duty
of the government to provide economic support during adversity. Anger arises as not all
individuals receive equally (Guillette, 1990).
The most wide-sweeping program under consideration is tax relief for households
containing aged. This will certainly relieve some of the economic burden of support. I
cannot help but wonder if the aged member will directly benefit, other than receiving
their share of purchased food. My research has shown the use of money is controlled by
the person receiving it. Expenditures for family nutrition, and for the education of
young children, take priority. Remaining funds, in this case in the hands of the tax
payer, are usually used for personal goods, either from necessity or the desire for luxury.


132
plow so I use the land for my one goat, two dogs, three cats and four
chickens.
With a wink in his eye, he continues. "Oh, Priscilla, I will be late coming home
today. I have some thebe {coins} so will stop and buy some bajalwa {traditional beer}
on the way home." After Alfreds departure, Priscilla frowns and says:
ever since we lost our two cows in the recent drought, he likes to drink.
He buy beer with money from the chicken eggs. Its awful, the way he
drinks. Its not every day but sometimes he takes too much. When he
comes home, he accuses me of romancing with other men. Now, who
would want a worn out woman like me? I hate beer so much I will not
brew bajalwa, and that is the only way I can see to make my own money.
People laugh because Alfred uses the field as a cattle post but it gives
him something to do. In some ways I am lucky too. I have lots of
clothes from the better days when Master was working, a daughter at
home, and a nice house.
Maria and Martha
This morning I find Maria sitting on the ledge of her rondaval. Martha, her 63
year old daughter, has given her a basin of water for bathing. There is no
embarrassment or covering of the body. Maria starts the feminine chit-chat.
Do you remember I when told you about the varoom of cars?
Those were the good days, when I could walk and do things. Now my
bones ache. I only sit, too old to even pull grass from the yard. Even
Martha is becoming old. Our husbands died long ago. There was no one
to plow, so my land went idle. I think someone else uses it now.
Martha supplements the statement with:
her nephews family plows her land and we get some grain from them.
Food is a heavy problem. It is so expensive, especially milk. Without
milk, we do not have breakfast as black tea makes Mma sick. Sometimes
my brothers and sisters send us money and sometimes I help a neighbor
make bajalwa, but money is always dear.
Martha walks into the house to get her mothers clothes from a cardboard box.
Each have a bed and several blankets. Otherwise the house is bare except for a box of
laundry soap. While she is gone Maria tells me about the comfort and care she gets
from her daughter.
I can talk to her, especially about my sadness from everyone
dying. Three people died in my family last year, including the one


258
role in the schema. The more recent association of economic success with social status
is recognized by all. In addition, the aged emphasize a social ranking based on the
traditional social structure of society. Structurally, age in itself determines the level of
status in any verbal exchange. All aged should be given respect, independently of other
markers of status. For data in this area, I took advantage of the two concepts involved
with respect that are presented repeatedly by the aged: that of being "listened to" and of
being "useful."
The concept of being "listened to" involves obedience to orders, respect for
advice, and subservience to the elders authority. Almost two/thirds (61%) of the aged
said they were not "listened to" by the family, indicating that the aged not only lack
family respect but also power. Being useful implies making valid contributions for the
welfare of other. Sweeping to avoid idleness is not being useful. Sweeping in respect of
the needs of others is useful. Over half (57%) see themselves as having no use-value to
the family or society.
The final question concerns the view one has towards ones present life. Life is
described as either happy or unhappy. Happiness comes from on-going recognition, by
self and others, of achieving and of doing. Happiness is present, or it is not. Various
events could make one cheerful or depressed at the time, but the aged insist on
separating specific events from the overall feeling. No gradations of contentedness with
life is possible. Two-fifths (44%) of the aged were happy, with the remaining 56%
seeing life as unhappy.
Sex and age have no bearing on perceptions of personhood. Full personhood is
obtained by 14%, with another 16% finding personhood in all but one area. Nearly
one/third of the total group find no aspect of personhood (13%), or only one segment
(18%). All aspects of the gerontic fund are related to the finding of personhood,
although the social/familial dimension had the lowest correlation (p = .03). Again, this


259
is not what one would expect in a setting where family is regarded as insurance for a
good life in old age. Does one have to buy happiness, respect, and power with
physiological and fiduciary holdings?
The Gerontic Fund and the Good Life
All aspects of the gerontic fund, including the total score, strongly correlated with
the total good life score. All aged with strong holdings found a relatively good life. The
fiduciary dimension had the strongest correlation (p = .0002). Everyone who scored in
the top quarter of the good life also are in the top quarter for fiduciary assets, although
a few with minimal economic assets are able find various aspects representing success.
Strong correlations are also found with the social/familial dimension (p = .0006) and
physiological/personal dimension (p = .0008). The majority, with social/familial holdings
in the upper quarter, tend to find the good life, while very few of those in the lower
quarter can succeed. The same is true with physiological/personal assets. Age and sex
played no role, yet the very old are the group that stand out as fund-poor age group.
One must keep in mind that there are numerous individuals in the other age groups that
are equally fund-poor.
The diversity in life satisfaction cannot be accounted for in terms of gerontic
funds alone. There is no statistical threshold in held assets below which a good life
becomes impossible. A few of fund-poor, of any age, are able to exceed the midpoint in
life satisfaction, although they never reach top scores. In additions, some aged, with a
relatively strong fund, are unable to find any aspect of the good life.. Some old people
with extremely limited assets may be skilled in the substitution of one asset for another
to obtain what is desired. The fund-rich may not know how to use their fund effectively.
I think other factors are also involved.
It seems likely that a larger household size, with its increased availability of
benevolence, would increase life satisfaction. This concept is false (p. = .42). Neither


172
admit that all families are inherently different. I have added the dimension of time by
presenting the families as seen in different months over the research periods.
The Kerengs
September 15.1988
Mogolokwane insists I use the English translation of her name, Happy Sound, as
she likes to "feel modern." During her 69 years she has learned assorted English
phrases. Idioms are liberally scattered throughout her speech. "Okey-dokey" is her
favorite.
Widowhood came soon after the birth of her last child, Leru, 37 years ago.
Agriculture was her way of life for many years. After widowhood, she relied on a large
goat herd for income. Today, the goats are gone and Happy Sound no longer goes to
the land. Time has not diminished her love of plants and beauty. She enjoys tending
flowers scattered about her grass free courtyard. A simple, two room, "four-cornered"
house and a traditional rondaval sit in the courtyard. Her daughter, Leru, lives in the
more modem home. Leru, now divorced, is a typist for the local carpentry brigade.
Her three children, ages 14,10 and 11 months, sleep with Happy Sound in the rondaval.
A married daughter lives in Gaborone. Another son lives in northern Botswana.
Happy Sound is washing the breakfast teacups in a pail of water. My arrival
signals a good opportunity to divert herself from physical work and proceed with the
equally important work of teaching me about old people. Taking the baby with us, we
sit in her house to escape the cold, spring winds. The small fire adds little heat.
Obviously chilly, Happy Sound pulls her own thin blanket around her, ignoring the
warmer blankets on her 14 year old grandsons bed.
"Okey-dokey, lets begin. Young people do not ask about being old, so you must
leam and tell others." She is explaining her many kin when the 14 year old grandson


72
killed many cattle and the remaining large native animals in the surrounding areas (Tlou
and Campbell, 1984:126-128).
The Malete, like other Tswana at the beginning of colonization, were a self-
subsistent agricultural and pastoral people. Agricultural practice was, and still is, limited
more by resource availability, mainly water, than lack of creativity. Planting was mixed
and eclectic, with sorghum, maize, beans, melons, pumpkins, and sweet cane (Schapera,
1967:16). Animal husbandry was valued equally with arable agriculture, representing an
investment-portfolio with practical and symbolic implications (Alverson, 1978:10).
The Tswana based class divisions on kinship lines and rank within class on herd
size (Campbell, 1971). Each person knew their status, and could move up in rank but
not class. Wealth differences were outwardly minimized through the process of mafisa,
the custom of loaning cattle and small livestock. The holders of such loan cattle
benefited from the milk and the offspring, and could use the cattle in plowing. In turn,
they were expected to provide service and political support (Schapera, 1953:28).
The peasant economy and lack of formally recognized religion were considered
by the colonialists and missionaries as the "the heart of paganism." Christianity based on
a money market economy was regarded as a necessity (Ellenberger, 1937; Thou and
Campbell, 1984:183). Thus, early development was aimed at eliminating the traditional
economic and religious practices in order to promote economic gain for the British.
Lutherans built the first church in Ramotswa at the turn of the century. In the 1930s,
the village was described as mostly Christianized. The claim was that the heathen
practices for mystical control over the universe were rapidly disappearing, and that the
people were becoming "civilized" (Ellenberger, 1937).
Economic change was instituted in 1899, with the imposition of a monetary hut
tax. An additional "native tax" was placed on each hut in 1919. These taxes were used
to pay British administrative costs, with extra money going to the Native Fund for village


92
The present aged refute these writings. "It has always been the duty of children
to provide for parents. As a lad, I lived very meagerly in Kimberly, sending most of my
money home." "We always respected our parents, and the aged were well cared for."
"Money was always sent by working sons. Their wives, or the remaining siblings, gave
good care to aging parents." "I myself took good care of my mother, giving her gifts of
food and clothes, as well as sending money."
According to the aged, there have always been old people in the village, some
living a very, very long time. Old people never lived alone during this time period.
They shared their compound with married daughters and/or daughters-in-law, as very few
women were not married by the age of twenty. Elizabeth says:
the old people were always respected in the old days and never went
hungry. When I married, our place was with my parents, as that was the
law. When my husband left, he sent me money. I used it to pay the
costs of his parents, my parents, and our children. If we did not take
care of our parents, our children would not take care of us as we grew
older!
It is impossible to determine the true historical situation at this time. One
cannot deny the effects of time and social change on recall. The "Good Old Days"
tendency is real, even in Botswana. No doubt, some parents were not supported
monetarily by their absentee children. Their numbers cannot be told. Life histories
repeatedly report that the husband sent his income to the wife. This money, under her
control, had varying degrees of adequacy for household expenses. The claim is that the
aged were supported economically, and given food and care equal to that of the rest of
the family..
The ageds claim of past economic support is very different from the picture
painted by Schapera (1953,1966). There are two possible reasons for a
misinterpretation by this English anthropologist. Western monetary economic principles,
when applied to traditional subsistence systems, can easily overshadow the true picture.


201
member, interweaving everyones individualized, and diversified, version of the roles of
modernization and tradition. Conflict was resolved near the fulcrum between the
extremes, with family behaviors predominantly centered on buffering hardships in life.
In Happy Sounds eyes, she still has someone to cook for her. She also has
grandchildren to serve her, although they are in school much of the day. Happy Sound
is like other aged living in families-in-balance. She is able to place traditionalism in a
modem setting and accept or resolve the conflicts between being an elder in her own
eyes and frequently that of being a child in the eyes of her family.
The aged out of balance with the family
Being out-of-balance, or in a state of disparity is said to exist when the aged and
the immediate family have multiple unresolved problems based on differences in the
approach to life. Like all aged, the aged of these families stress cultural continuity, and
the obedience of traditional laws in family life. The adult children are no more
progressive in thought, or behaviors, than others. Young children accept, and refuse,
commands with equal frequency as those in families in balance. Unlike the family in
balance, these families are unable to mitigate differences.
Disparity was found in all types of households, large and small, rich and poor,
educated and uneducated. It is at a minimum when the central care provider is also
aged, as with Martha caring for Maria, and greatest when adult granddaughters are the
central care providers. The numbers of households with disruptive disparity appears to
be large. Of the 33 aged who created their ideal house, 70% either completely refuse to
put in any family members (8), put in only one young grandchild (6) or one specific
adult (8). In all these cases, multiple kin are residing in the household. The reasons
given reflect the extent of tension arising from conflict. "If you have more than one, it
just causes too many problems." "I want only my oldest daughter here, not the others.
The others should get their own house, as they disobey the laws." "One person would


123
At the same time, society incorporates the concept of adequacy in self-
sufficiency. The physically well, middle income, full-time housewife who insists on others
performing all household chores would not be considered self-sufficient, as performance
of these duties is incorporated into the social definition of her responsibilities. For the
Tswana, the receiving of service is a right of motherhood and has no bearing on self-
sufficiency. Instead, socially defined adequacy is based on the ability to trade abstract
organizational abilities for control over, not performance of, household activities.
The final step in self-sufficiency is social recognition that the previous steps are
being followed in a laudable manner. The most laudable are performing behaviors that
meet ideal social standards. Social acceptance is greater for the working father than the
father who is supporting his family with unemployment benefits, although both follow
the rules of conduct and legal laws. For the Tswana housewife, participation in cooking
rituals, such as conversation with the cook, brings greater recognition than non
participation. Cooperation and sharing self with others are lauded social traits in
demonstrating self-sufficiency.
When superimposing this concept of self-sufficiency on the Tswana life cycle, it is
easy to see that in infancy and early childhood no self-sufficiency is expected in any
form. Everything is given. As childhood progresses, elementary resources of energy and
agility emerge. The child is told how to use these resources. Socialization for the rules
with an awareness of social sanctions occurs. In early adulthood the acquisition of trade
items and the opportunity to experiment with decision making occurs. The individual is
expected to follow the rules and will feel increased positive feedback in social responses,
with errors expected as a part of learning. Progression through the life cycle brings
increasing self-sufficiency, with an interactive blend of self, family and social welfare.
This process continues through elderhood. Total application, with understanding, comes
with becoming completely grown-up.


84
As a pragmatic group, the Tswana willingly gave up the external trappings of life
and substituted sturdier and more time-efficient imported technology. These material
objects, such as the metal plow, were incorporated into peasant farming with the
retention of underlying structure and function. The same was true with the new water
sources and extension of lands. Participation in the introduced activities of school and
church was to "learn the Europeans ways in the hope of making life easier," not to
make their life like his. The principles of eldership continued, with obedience to the
laws perpetuating family interdependency and community solidarity.
On the structural-functional level, the traditional and new were intertwined. As
in the case of conflicts for initiation into adulthood between the church and tribe,
accommodation with unification of the two types of schooling under one name allowed
for the continuation of horizontal social and political village interactions. Peers
remained unified and continued to function under the rigid seniority principles.
The concept of English taxes was mitigated by the concept of tribute. Colonial
government was a parallel to chieftainship, with both playing an active role in vertical
social and political relations (Parson, 1984:17,23). The giving of allegiance to
authoritative leaders and displaying decorous behavior towards social elders was
unquestioned.
The English government and the Lutheran church both claimed to have
influenced individual and tribal decisions to discontinue "heathen practices" (Schapera,
1970; Tlou and Campbell, 1984:134). The cessation of rain-making was frequently
attributed to outside intervention (Ellenberger, 1937; Schapera, 1970). The disastrous
results of the last Malete rainmaking ceremonies were probably quite influential in the
decline of ceremonies to alter supernatural events. Other heathen customs were
reported to "have disappeared because of conversion", such as placing a stone in a tree
on arrival to the village and the purifying of the Army before war (Ellenberger, 1937).


188
other funerals. More than one hundred kin begin the walk back to the Mosoko
compound for the funeral feast. Only the fast of foot manage to find a ride from the
few trucks parked nearby. The young women squeeze together to let Auntie sit on the
tailgate, but there is no room for her aged brother.
Dorothy and Auntie are silently sharing Bettys pain within the hut when I arrive
back at the compound. Guests are seeking a place to sit on the still damp mud. The
scent permeates the air. I sit with the women. The funeral feast begins. Holding out
my palm, a niece uses her fingers to give me a scoop of sump. Gradually all the food is
distributed, and is consumed slowly with the fingers. An occasional guest will shake her
head in refusal of a serving from one woman, an enemy, but willingly take the same
food from another. No liquids are served.
Some of the guests begin to leave. I move towards the cooking area. Dorothys
father joins me, in order to introduce himself.
There was much work to do in Johannesburg. I couldnt leave
until the middle of last night. I was afraid I would not get here in time
for the burial, but I made it to the church. Mother saw me come in and
I know she was happy. I knew Father was sick and would die soon. I
wanted to visit him but had no time. Instead I gave $600 Pula {300 U.
S. Dollars} to the burial fund. That is much money and I can be proud
of what I did.
I have not yet talked with mother. Mother will stay in the hut
today. She was fed the feast foods by Dorothy. It will probably be a
couple of days before she can sit up and go outside, as she is very weak.
Another difficult time for her will be in winter. She will have to take
Fathers belongings out of the house, and share them with the family.
Long ago father designated certain things to certain people, the rest will
be given away by Mother. The taking out of clothes is a very sad time,
as Betty will picture Jacob and cry bitterly. She will wear a black dress
for a year and should stay in the compound a month or more. I have
black necklaces for the children to wear to demonstrate their mourning.
I will wear a black cloth on my right arm to show I lost a father. The
next year will be a very dangerous time for us and those who talk to us.
We must warn others with the wearing of black.


218
entity ail day. He had found the doors for community acceptance broken and sealed.
Only his sister and mother care.
The one way Sepia can maintain any degree of integrity as an old person is to
uphold any traditional laws he can. The use of the make-shift bed, over the use of his
brothers, was his way of demonstrating the importance of law in finding personhood.
My touching the brothers blanket was a threat to his ability to maintain the law in his
own home. Like many aged, he finds pride in maintaining laws, even though it means
deprivation of needed comfort.
Many relatives care about their aged, and love them as parent. However, ability
to give care and express love must be considered in relation to social rules and the
boundaries of the present. Body massage and breast holding (between kin-related
females) or genital grabbing (between kin-related males) is often a more meaningful
expression of love than direct service. Direct service is influenced by law. Since Sepia
and his mother live in one household and the sister lives in another, the sister must put
her master and children first. The aged and others say this is proper, a part of the
traditional law. The assistance given to Sepia by his sister fulfills his and village
expectations, although they may appear inadequate or lacking by Western standards.
Sepias story raises various thoughts considering the outsiders when they work in
one cultural system and come from another. I will use the social process of sharing as
an illustrative example. The custom of asking and giving for equality applies to kin and
non-kin. Anyone who has signs of wealth is frequently asked for money, as they have
more than the asker. Many an outsider visualizes the system as begging, or as being
dependent on aid, or as another sign of laziness. Outsiders frequently fail to see the
inherent social value of the system.
Chambers (1983:13-16) states that outsiders praise their own belief system and
degrade the equally valid local system. I found the same was frequently true with the


265
The identification of trends stressing the specific vulnerability of particular aged
tends to override that which is generic and common to all older persons of the society.
The present cultural and psycho-social forces that could be used to promote well-being
are forgotten in both the definition of risk and the approach for planning.
The real issue with the aged is not one of age, living conditions, or traditional
ethnicity, but one of poverty for social exchange. The deprivation trap (Chambers,
1983:111-114), which pulls a household below any possible threshold for economic
recovery, also exists on the individual level. Aged individuals are the poorest of the
poor: economically, socially and physically. The same interlocking clusters of poverty,
physical weakness, social isolation, powerless and vulnerability exist for the aged. Many
of these clusters are age-related, and intensify as age advances. Each economic, social
and physical loss become a ratchet, increasing old age isolation, powerlessness and
vulnerability. This always makes any doors to the good life harder and harder to
manage.
The aged of Ramotswa, many of whom lack numerous gerontic fund assets, can
no longer manage the present-day doors to care, support or acceptance with the process
of exchange. The impacts of the resulting gerontic-deprivation trap are shown
repeatedly in family function and communications, and in social interactions. The loss of
rights is a by-product of the resulting social detachment (Chambers, 1983:115).
Vulnerability is great, for the end-product is the loss of personhood. No one hears
them. Their activities are play. It does not matter if the aged maintain the customs of
ceremony or sweep from idleness. They are the valueless, and frequently the unwanted,
burdens of society. This is why all the aged cry out for help.
Aged Children Become Wise Elders
I feel this call for help should not go unanswered. I am aware that standard
gerontological approaches of service delivery, or monetary assistance, will not promote


18
Frequent visits to kin about the village are interspersed with paying respects to bereaved
families and attending social and political functions.
These norms may clash with introduced ideas of individualism, self-reasoning and
self-concern that are promoting new values concerning property, less division of labor
between sexes, and more emphasis on the economic expressions of social status.
Following new norms, without piety to traditional responsibilities, often negates social
success in this rural area, especially when judged by the aged. To the young, economic
success is often equated with social success.
For the aged, the good life is not the same as the meaning of success to a
younger adult. With the aged, success is not based on conquering poverty or having a
better life than before. Success is determined by the temper of current family affairs
and being included in acts of on-going family and social solidarity. Status comes from
continual striving, in opposition to previous achievements or past events. Involvement in
the process is more important than the ownership and control over multiple possessions.
Material goods reflect economic achievements as a working adult, not success in old age,
and are not considered in successful aging. Deprivation should be absent, but plenty is
not a requisite. An old person finds more joy in living in the hut built as a young adult
than in moving into a more modern square house provided by son or daughter.
The successful aged person also contributes to the economic welfare of the
family, either directly with money-making schemes or indirectly with agriculture and/or
household management, including child care. This old person also has ultimate authority
within the household, even if major responsibilities have been delegated to others.
A caring family is an overt marker of success: a daughter to tend house and
prepare sufficient food, a grandchild to sleep with and to fetch a cup of water, and a
son to do heavy chores or repairs. The old man should have a wife although a husband
is not necessary to a womans success. The more intensive and comprehensive family


278
finding partners willing to make trade that provides benefit to the aged is equally
necessary in both settings, as the geo-physical and sociocultural barriers to the good life
are rigid and unforgiving.
The assumption that assets decrease with age appears to be true. The aging
process can account for physical losses. Other loss occurs without reference to age, such
as that caused by calamity over which the aged have no control. Ambient economic
inflation and changing values also takes a toll.
The universal goals of a guarantee for food, shelter and care while remaining
respected, useful and healthy participants in society until the advantages of death
outweigh the hardships of life (Chapter 1) hold true for the Tswana. The behaviors
involved with goal attainment differs from the West. Fulfillment of basic biological
needs should come from others, as a right of age. Respect comes from being served
and usefulness comes from serving others. Independence, self-sufficiency and autonomy
are important, but lay below the surface in the process of achieving. Contribution, as
determined by Tswana culture, involves group good rather than promoting oneself.
Death is an honorable step forward in the circle of life, not to be feared or
unnecessarily delayed.
There are many indications that the Tswana aged resemble aged of the
developing world, in that, as a group, they are the poorest of the poor; economically,
socially and emotionally (Goldstein et al., 1983; Diouf, 1985; Brathwaite, 1986; Tout,
1989). Others tend to regard them as a human liability to the family and society
(Hendricks, 1982). Isolation, powerlessness, and a loss of rights is a by-product of the
resulting social detachment (Chambers, 1983:115). For the aged-poor, wherever they
may be, the psycho-social implications of poverty are greater than the monetary
implications, as the end-product is the loss of personhood (Streib, 1990). No one hears
them. They are the valueless, and frequently the unwanted burdens of society.


CHAPTER 1
INTRODUCTION
One must always "be doing" as the process of achieving gives meaning to
life. (79 year old man)
How can I do things? The door to my house is broken and my son does
not obey the laws to fix it. To toss aside the Tswana laws is to toss aside
the value of living. (60 year old woman)
This is a study of broken doors. A door controls a passageway. It has two
functions. It lets people into our homes and lives, and keeps the unwanted and
undesirable out. We tend to think of the door to our house as something that should
be closed and locked. We use it selectively at our own discretion, opening it to others
when sound reasoning and knowledge provide good reason.
In the Botswana village of Ramotswa the door to the Tswana home is usually
ajar. The open door is a signal that one is home, and the passerby is welcome into their
homes and life. It also means that the person inside wants to be included in the large
and small events happening outside.
There were no true doors when the present day aged were younger. A stick
placed across the entrance indicated no one was at home to provide company or assist
you if you were in need (Schapera, 1944; 1953). Actual doors were introduced during
British colonization (Schapera, 1953:26). In contemporary rural Botswana, all homes
have wooden doors. Many are imperfect. The people say it is because of "nature,"
external to the way the door has been used. The people overlook small cracks, as these
are considered normal. Other doors have larger gaps and deficient hinges. They do not
swing properly. This is more serious as the occupant and visitor have to pause and
1


210
The purpose is to enumerate aspects of a functioning, evolving society as it is at this
point in time, and as it effects the aged. The contradictions and ambivalence found with
change will also be addressed as I inquire into the interpretation of social justice and the
criteria for status and success.
Social Interactions
In this section I present primary material by cases, or situationally united
descriptions of events, conversations and behaviors experienced in the village. As with
the family, I stress the setting as an arena for social exchange. My observations reflect
my role at the moment of the event, which could be an anthropologist, a
"granddaughter", or a member of the community. A discussion follows each scenario.
When my feelings are put in, it is mainly to emphasize the differences between my
society and the Tswana, and between myself and others. They are not to be interpreted
as a judgmental statement.
Case 1: The Hungry People are the Lazy People
I am taking a short cut through the church yard, when one of the European
missionaries asks how my work is proceeding. In response to my reply that I see much
hunger, she boils out:
there should be no hunger in the village this year! The rains were good,
and the drought is over. It is the fault of the people if they do not have
food! They are too lazy to plant. They want us to give them everything.
Last year the destitute in the village of Otsi were given blankets. Do you
know what they did? They sold them to get money for bottled beer.
I had heard similar comments before, from assorted expatriates and government
workers and knew refutation would fall on deaf ears. I continue walking. A few
minutes later I stop at an food shop that is off my usual beaten path. I asked the
owner, a middle aged village woman, if she has any tomatoes or fruit. "There is only
cabbage, that is all Ive had all week. I always have cabbage as it is cheap. Many
people are hungry in this village as food is so expensive." Wrapping up four loaves of


88
changed. Once, everyone had enough to eat. If crops failed, others gave
you food. The law was carried out. If a person was in need for
something, he could ask another and it was given. No tally was kept as
someday the person who gave would be in need and he would be given.
We always gave if we had what was needed. Unfortunately, it became so
that all you could ask for was a little bit of sugar or salt. Now people
ask for money. If I have money, I give part of it.
Once it was good to live in a round house. The children would
gather dung and bring water and I would always be mixing mud to keep
the house looking nice. In the late 1950s the chief gave permission for
people to make four-cornered {square} houses. My children were then
old enough to have their own house. They wanted the modern house
with the tin roof, even though it is very noisy when it rains and is hot as
the wind doesnt flow.
It wasnt just my children who wanted to be modern. I did too.
My mother used to make clay pots. I preferred the iron pots so I did not
learn how to make the clay ones. Now I wish I had clay pots for beer.
Beer was always cool to the tongue in clay pots in contrast to the metal
ones. There were many things my mother did not teach me as she also
preferred the modern new substitutes. Sometimes I must go without, as I
know nothing about wild foods or working with leather. The one thing
elders made sure I learned were the laws about what is right and wrong.
They would beat me, even when I was a young adult, if I disobeyed the
laws.
An older relative, passing by the house, stops to see what we are talking about.
She reconfirms the multiple beatings. "People seldom did wrong as they knew the
punishment of pain and shame. The entire village would know that you did wrong."
Wanting to know more, I ask if they ever purposefully went against parents wishes.
The answer was a loud "NO!" Just for fun I tell them about one of my childhood
escapades. Happy Sound lives up to her name and laughs loudly. Not to be outdone,
she says:
we never went against elders but we had ways of having fun. My mother
wanted me to go to school every day. There were days I did not want to
go so I pretended to have a stomachache. She would take good care of
me in the morning and then I would play. She thought I was a sick child
as I did this many times.
The women giggle. It is the type of laugh that says, "you are right but I will not
say so." One woman becomes more bold, not wanting Happy Sound to become the only
star.


164
more distant relative, almost always another aged individual or grandchild, usually have
to change compounds.
The number of aged who had left the village to live with migrated children is
unknown. Several aged are resisting pressures to do so, as they prefer living under
poorer and more limited conditions to the breaking of the custom of living ones last
days in the village of birth. The concept is similar to the Western desire of aging-in-
place, resisting forced moves to new areas with the loss of friends and home.
I heard accounts of dissatisfaction from two who had made such moves and had
returned. Others told stories of unhappiness, never satisfaction, regarding friends who
experienced moves to the city. A similar dissatisfaction existed with those who had
moved to a new location within the village. A change of residence was not truly
accepted by the aged until external causes, such as "my house finally fell down,"
provided a valid reason. Even then, moving was something to be endured. For these
Tswana, aging-in-place means living in ones own original home on family land. Second
best is living in the village of birth.
Possessions owned by the aged have great meaning to them, making ownership
of furniture an important asset. The meaning of possessions is compounded by the laws
regulating the use of others belongings. According to law, a table in the home can be
used by all, as it was a necessity for meal preparation and open storage. Chairs are
under the control of the owner, to be used with approval. Chairs are not an extremely
high priority item as sitting on the ground, or a small wooden and leather stool, is often
preferred for meals, conversations, and watching the sights go by. Other basic furniture
is to be used by the owner only. This included beds, dressers, chest of drawers, and
wardrobes. Personal goods, including blankets, radios, silverware, flashlights and other
material objects, commonly shared in Western homes, are also restricted to owner-use.
The regulations influence the purchase of items by individuals. The old state that during


96
the universe without stimulating guilt or shame (Alverson, 1978). Diligent adherence to
tribal laws for harmonious solidarity continued to be rewarded with social approval and
respect (Schapera, 1953:61).
The claim is that labor migration and economic change weakened parental
control. Schapera (1953:50) stated that young people no longer looked to their parent
for guidance, and tended to act more upon their own responsibility. Comments from
the present aged indicate that they did not feel they were independent of elder
authority. They say the efficacy of beatings and traditional sanctions continued to
promote wide observance of social gerontocratic standards.
I believe that the present aged contributed to the power-shifts within households
as the employed, not the aged, began to control expenses and payments. Yet,
household family cohesiveness remained all important as the money market economy
increased in intensity. Power-shifts were unrecognized by the younger income-producers.
They did recognize that modern did not always mean better, as pointed out by Happy
Sound. Economic deprivation mandated changes in the laws, such as restrictions on
giving, and increased limitation on the use and exchange of items.
The structural environment created during this time was one of
underdevelopment (Parson, 1984:33). Ramotswa was but part of an entire country of
poverty. The mechanism was the new economic system. The outcomes of labor reserve
economy were the creation of new social strata and new economic and political
relationships. The subsistence system became a Peasantariat, a term embracing the
persistence of a peasant type of agriculture and society with the proletariat attribute of
working for wages (Parson, 1984:123). Professional and educational elites emerged in
the transformed and redefined tribal and village hierarchy (Parson, 1984:34).
The structural shift of political power from the chief to colonial government was
accompanied by a functional shift. In the publics eye, colonial governmental


137
agent in exchange. This area is evaluated in terms of abilities to perform daily activities
that, in turn, allow for culturally laudable interaction with family and society. This
differs from the usual Western style of functional assessment of the elderly, which
focuses on impairments in relation to approaches for needed services to retain autonomy
and independence. It is the "doing" and "being" in the achieving process, rather than
remaining "autonomous" and "independent," that give social value to this category. No
attempt is made to evaluate personality.
General descriptions of the aged frequently include the demarcation of the
degree of decrepitude. These terms, such as active, frail and decrepit, can become
pseudo labels, borrowed from the outside world, unless one knows how the terms are
negotiated within the culture where they are applied. In Ramotswa, the person
receiving large amounts of service from others is not necessarily frail. They are normal-
old, receiving service as a right. Decrepitude, which implies a need for 24-hour
supervision and care, is seen by the Tswana as a term applied only to the dying. Aged
requiring major assistance are not seen as needing the continuous presence of others.
Not infrequently, they are the only household member at home as these people are
always seen as being capable of doing at least one or more things, such as feeding
themselves or conversing.
The large discrepancy between my outsiders interpretation and the cultural
interpretation resulted in much error when I attempted to place individuals in these
categories. When the terms "frail" and "decrepit" are used in these writings, it is to
indicate limited and extremely limited physical status, according to Western standards.
For the research, I used the three generalized categories only to select representative
individuals with varying health levels for in-depth studies .
A more valid measurement of the personal/physiological assets was grounded in
terms of abilities to perform activities of daily living (ADLs). Activity is evaluated


274
Goodness is expressed through achieving in the present. The products of achieving are
to be shared. The major change is that sharing by the youth is directed more by choice
than moral obligation, with the direction of flow going to other young. They believe the
goodness of mankind is exemplified by being strong, active and achieving for self
advancement. The aged believe the goodness of mankind is shown through the wisdom
of experience and achieving for the good of others. This goodness grows in strength as
one grows in eldership.
The aged have always been divided into groups: social elders, those becoming
limited, and the decrepit aged or children who will soon become ancestors influencing
life from the sky. Before modernization became as pronounced, the aged child was one
nearing death and had a relative short time of managing the broken doors, which I
believe have always accompanied extreme old age. These traditionally broken doors
prevented access to continuing achievement, as the decrepit aged were expected to think
and react like a child. Limitations on acts of "doing" limited opportunity for meaningful
exchange and increased their disengagement from the family and society. These same
divisions of the aging process continued, except that the timing has been advanced. The
periods of elderhood and "becoming old" have been shortened with a premature
assignment of childhood.
Traditional metaphysical beliefs continue to influence behavior. Achieving is
thwarted by outside forces, separate from the individual. Potential unwanted ill forces
arising from enemies and sorcery continue to be avoided by limiting interpersonal
relationships. Kin are regarded as safer than non-kin, but even then, not all can be
trusted. Social change compounds the situation. The result is that people are leery of
social exchange with those not considered safe. The imperfections in the doors leading
assistance have enlarged, preventing entrance or exit for both the aged and others.


136
under study. For the Tswana, the importance of functional interdependency within the
extended family and the value placed on social interchange during the continuous
process of achieving are vital parts of the three dimensions. The instrument developed
to measure the fund, Appendix A, is situationally dependent for the group under study.
It would need to be modified for use with other groups, especially non-African.
In this research, the tested gerontic fund consisted of twenty separate items,
reflecting each of the three dimensions. Each item had a score range of 1 to 5, with 1
indicating no held assets and 5 representing a high degree of possession. Each
dimension was scored by adding item scores within it. As unequal numbers of items
were in each dimension, the individuals sub-totals were then weighted by dividing this
score by the number of answered questions. This provided a statistical equality between
dimensions. The individuals total fund was determined by dividing the total of the
three weighted sub-totals by three, resulting in a number between 1 to 15, with 15 being
the maximum possible score.
The rest of this chapter explains what specifics constitute a gerontic fund for the
aged Tswana. Incorporated with this are comments that exemplify the relationship
between desired fund and lifestyle. The findings reflect the size of the fund at the time
of research. No attempt is made to document changes in the individuals assets with
time. Variations in holdings, within and between age groups, are common. No person
is the same, with some controlling large total funds and others striving to maintain the
smallest, while others are rich in one dimension and poor in another. Diversity is so
great that I avoid presenting a picture of the average old person. Instead, I emphasize
the meaning of asset ownership, the modes, and the extremes.
The Personal/Phvsiological Dimension
The Personal-physiological dimension of the gerontic fund involves the levels of
physical and mental health that merge to allow for the use of self as a participating


CHAPTER 11
CONCLUSIONS
You should write a book and tell the world how we, the old people, must
live. Tell them the people are good but the way people act is not good.
(78 year old woman)
No one allows us to live happily. It is the society that makes us live like
this. I think my family cares but it is hard for them to show it. (67 year
old man)
I began my field experience with thoughts on the contextual value of gerontic
fund. As I became more and more involved with the villagers, I realized the fund could
not be isolated from the setting and the way of life. Everyone, the young and old alike,
attempted to manipulate identical doors; defined by the early culture, constructed on
tradition and modified with time. Gradually, doors became kaleidoscopic, with each
person placing their own values, knowledge, and goals on door selection and use.
The aged also built their approach to life on their culture, experienced life
according to tradition, and modified life with time. Only when the past, and its doors to
the aging process, is comprehensively interwoven with the present can one evaluate the
diversity of the aged. The past also provides commonalties among all aged. The shared
commonalties are as important as the diversity (Standford and Yee, 1991). This chapter
concentrates on the commonalties and diversity among the individual Tswana aged,
between the aged and the village population, and between the Tswana aged and other
aged in the world.
270


25
delegated to function at the periphery of society (Cowgill, 1979). The aged are thus
placed in an isolated position at the undesirable end of the life cycle.
Contrasting with Disengagement and Activity Theories, Modernization Theory, as
applied to gerontology, totally attributes failure of the aged to reach goals on social
change. The value of Modernization Theory is that it has stimulated scholars to
construct an historical record of aging in various settings as modernization progressed.
The theory has a problem however, as it implies that an historical before/after
bifurcation occurs with the onset of industrialization and modernization. The "before"
carries the uncritically accepted assumption that the aged were inevitably treated as
honored members of the family and community as long as they lived. The "after"
stresses that, with the introduction of social change, the aged have no valued functions
at all. At this new point in time, it is the ageds responsibility to reorient attitudes and
activities to mesh with their unwelcome place in society (Cowgill and Holmes, 1972).
(The antithetical position of assuming that a changing societys goal should be to
facilitate social efficiency and personal adjustment of aged members is just as naive.)
The assumption that all old people always held a high position in society is
quickly becoming recognized as myth. The aged, whether contributing members or living
liabilities, were not treated the same in all societies prior to the introduction of
modernization. Glascock and Feinman (1984), in a Human Relation Area Files cross-
cultural survey, found practices of non-supportive and death hastening behaviors directed
towards the aged in 84% of traditional societies.
In many places of the modern world the aged are regarded as lacking ability to
direct their lives and become known as the undesirable (Hendricks, 1982). They are cast
out of main-stream society (Cowgill and Holmes, 1972). In many ways the situation of
the aged is similar to that of other dislocated people. Instead of physical displacement
from war or natural disaster, the aged are uprooted-in-place. Displaced refugees are no


166
.006). The average weighted score for the 60 to 69 age groups was 3, the exact middle
of the possible score, with 11% scoring in the above average range. The 70 to 79 year
olds and the 80 to 89 year olds are just about as apt to score in the mid-range as to
score slightly below. It was the over 90 age groups that had minimal economic
resources, with a mean of 2. The minimums of the oldest people were equal to the
mnimums found in other age groups, although their mximums were consistently and
considerably lower. The oldest also had the least variation among scores. No one was
totally without any contents in the fiduciary dimension, always having at least a little in
two or more of the categories, usually support from irregular remittances, limited
material goods or owned housing. (As one person commented, those who were without
fiduciary resources were probably dead.)
Overall, the fiduciary dimension is the weakest of the three dimensions. The
average weighted score for the fiduciary dimension is 2.78, or roughly a full numeral
below the other fund dimensions (3.75 and 3.57). Is the evaluation of economic
resources too harsh for the ambient setting of poverty? This I doubt. The casual
observer can notice that the fancy housing in town is usually owned by younger adults.
My initial errors in interviewing for livestock ownership pointed out that cattle is owned
by sons, not aged parents. In my survey of household belongings, I find younger
members own more than aged parents (p = .0001). Almost never did I enter a home
that did not contain at least one bed, several chairs and a wardrobe controlled by adult
children. They are also the ones to have radios, watches, paraffin stoves, and easy
chairs. They also have direct control over household remittances. Such facts strongly
support the assumption that an additional "poverty-of-old-age" exists within the
generalized setting of poverty.
The poverty-of-old-age, although always present, became more pronounced as
age groups progress. I believe history plays a role, in that the majority of aged never


66
interviewed. Many of the men unavailable for interviews were reported to be in their
eighties. Absent women varied greatly in age, ranging from 60 to mid-eighties. Since
absentee ages could not be verified, they are excluded from age-related statistics, but
their reported ages, when averaged, were similar to the interviewed group.
All reported aged men, tended to fall between the aged of 70 and 85. The age
mode for the interviewed men fell in the 80-89 year old group. None were over the age
of 88. For women, the mode was in the 60-69 age group. See were over the age of 90,
a proportion similar to a group studied by Ingstad, Brunn, Sandberg and Tlou (1991).
Figure 3.3 demonstrates the found age grouping and sex. The noted age-grouping
imbalance between sexes supports the concept that men remain employed as long as
possible.
Despite the age group imbalance, average ages of the old were similar. The
average age of the 82 interviewed females was 74.49. The average age of the
interviewed males was similar at 75.82 (N = 23). Fifty-nine percent of the males were
75 or older, compared to 44% of the females (although no males were past 88 years). I
use the age of 75 in this instance to make comparisons to the United States aged. In
developed countries, the young-old (below 75) outnumber the old-old (75 and above)
two to one, including the African-American population (Hendricks and Hendricks, 1986:
41,380). In Ramotswa is a situation where nearly half (47%) of the aged are
demographically classified as old-old.
Employment may affect the village demographics in regards to the ages of the
aged population, but there is good reason to think there are other causes. Usually
mortality curves are thought of as bath-tub shaped, with a falling of death rates after the
neonatal period and a rise with senescence. In sub-Sahara Africa, a different pattern
occurs. Life expectancy is lower, ranging in the 50 year old bracket for most countries
(United Nations, 1955:101). In Botswana, the 1980-1985 life expectancy is 50.8,


277
It is true that many generational gaps exist, with the young feeling independent
of the aged. At the same time, the many of the principles of eldership are recognized
by many of the adult children and grandchildren. Much of the present conflict stems
from differences between the aged and others as to when the label of childhood is
assigned. Childhood negates parts of the seniority principles. There is no need to give
respect or ascribed status to old-children, for they have never been so rewarded. Old-
children continue to be separated from household function and community recognition.
The cracks in this original main door for establishing and maintaining elderhood have
been enlarged to an extent where even the younger, active-old are prevented access to
the good life.
This original broken door, now almost sealed, exists in tandem with the other
doors to achieving and social participation, which have also been damaged. The
interweaving of cultural continuity and social change present a situation where it is
difficult to use ones assets for effective social exchange for either group or individual
good. Care and service is available in the village. Assets alone do not provide access to
freedom from physiological deprivation, security in be served, and recognition as a
person. The individual aged must know how to use the broken doors, and find people
willing to help in keeping them open. Some aged can manage the various doors, using
choice, knowledge and assets. Very few can manage all.
A Comparison to World Aging
The Environmental Exchange Theory used in this research was developed in
America and designed for primarily for use in developed nations (Gubrium, 1973). The
three dimensions of resources necessary for successful aging have been shown to be
equally important in Botswana as in America, although some of the actual components
do not apply cross-culturally. The correlation between assets and the good life does not
account for as much variability as the theory suggests. I believe that the process of


93
Old age support emerging from the congruent peasant economy, with kin providing
subsistence and care, could occur independently of the newly superimposed Western
economic system. Secondly the Tswana, like the neighboring San, use complaint during
discourse as a means of control over others (Rosenberg, 1990). Increasing household
poverty may have prevented the aged of this era from receiving as much as they wanted.
Complaint is a means of insuring fulfillment of as many wants as possible, and not to be
taken at face value.
The Money Market Economy and Social Life
Social change resulting from a money market economy came with startling
rapidity in all Tswana villages during the 1950s (Picard, 1987:118). People did not want
to appear antiquated. Traditional crafts of pottery, leather work, woodwork and
basketry were tossed aside as purchased items carried more social value (Campbell,
1971). Cash was needed on a daily basis to purchase the necessities and services of
modem living, including school fees, clothing, chairs, beds, and metal roofing for square
houses (Thema, 1972; Parson, 1984:25). At the same time, a permanent change in diet
occurred. Tea and coffee replaced milk as the preferred beverages. White bread was
now regarded as a necessity. Milk, meat and agricultural produce became items for sale,
as items that could be sold were no longer given away (Thema, 1972).
Cattle, once used to emphasize important kin relationships and events and thus
cementing position in society, became a direct economic asset. Variations in herd size
reflected household economic status, with cattle purchased and sold as desired
(Campbell, 1972). A survey of this area in the early 1950s showed 11% of the
households had no cattle; 18% owned fewer than 10 head each; 19% had 10-20 cattle;
and 40% owned 20-50. Chiefs and a few others had as many as 100-500 cattle each
(Schapera, 1953:23).


169
assuming the young-old individual has sufficient assets, as this class of aged showed the
most variation. Some relatively young aged have very few assets to provide access to the
good life. At the same time, increasing age brings the warning that assets are
diminishing (p = .0001). The correlation can be explained by losses in the
personal/physiological dimension, smaller loss in the fiduciary dimension, and even
smaller loss in the social/familial dimension.
The decreased assets with age is, in part, a reflection of the rapid growth of the
money market economy, the corresponding social change, and the environmental
disasters that have occurred in recent years. In addition, the decreased funds found with
the oldest of the old stimulates that thought that once loss occurs it cannot be the
rejuvenated, at least to the previous level. This is very true with cattle ownership.
Neither can loss be compensated for with increase in other areas. This assumption
should hold true with both the young and old aged, although it cannot be proved with
facts as no such measurement over time occurred. It is strongly supported with
comments from all aged reflecting that they have less now than before. The comments
of 73 year old Monate are representative: "Since getting old, I do not have the strength
of before, the family or the things that money can buy." Toto, at 94, says, "Now that I
am very old, I have nothing left; no family other than a grandchild, no money, and no
{physical} body that I can be proud of."
Daily life-style does have a strong influence on what is regarded as valuable
within the gerontic fund. Life style, as a composition of individuals, families, social
structures, functions, and values, also influences how the fund can be used. The
following section takes a broad look at the family setting in which the aged function and
the condition of the doors for social exchange.


176
store. When I told him I wanted to make money by selling plants but
that the seedlings needed shade, he gave them to me. Last month,
before Leru cooked vegetables, I saved the seeds. Look, the tomatoes,
green peppers, and squash have come up. I am waiting for the orange
seeds to sprout but it has been a long time. This month I sell the plants
for 25 thebe. Next month, when the rains are good, they will cost 50
thebe {25 U. S. cents}.
Kaizer joins us, with a pail of water in tow. Gently, he and Happy Sound water the
plants. I promise to return.
February 3,1990
I hurry to visit Happy Sound before the older children arrive from school. She
sits under the tree, nibbling peanuts from a small cellophane bag. The toddler is
sleeping on a goat skin near by. Kaizer walks towards his mother to make his wishes
known: "Make me tea, now!" Happy Sounds moves to the fire, eating the nuts as she
talks. "Kaizer is master of the house. If Leru were here, she would be making the tea.
The women of this house have pride in serving the master." When she sees the children
coming, the bag of nuts is quickly hidden in her apron.
I bought these nuts with 25 thebe from the plants. I gave some
nuts to Kaizer, because he is master. He does not get extra food at night
by being the master. Sometimes I give some of my dinner to the
children if they are very hungry. If they see my nuts, they will want some.
These nuts are mine and I do not have to give them to others.
The children are in the house. Happy Sounds drops the now empty peanut bag
on the ground to pour the tea. Kaizer takes the cup without a word of thanks and
enters the square house. As I leave, Happy Sound says "I have a good family. I help
them and they help me."
March 6, 1991
Today is Happy Sounds turn to build her preferred house on the felt board.
The entire family comes over to watch. Past experiences has taught me that vast family
discussions occur on what is the ideal, with the authority of the aged overruling the
childrens preferences during this pretend situation. It is also a time when the parent


32
provides strength to limited resources. The ideal is to have both sufficient resources and
more power than others. (Any power held by the aged should be seen as relative to the
power held by other age strata.) When either power or resources is absent, command
may or may not be possible, as either resources or power alone may control one
particular exchange but not another.
Older people in industrialized societies have minimal age-related power to
control daily social interaction (Gubrium, 1973). They are required to rely on the
dimensions of their resources. The debasing of exchange commodities is recognized by
both parties. When resources are insufficient, and power is absent, the social exchange
process ceases and is replaced with demand (Gubrium, 1973).
Demand is the claim that services and goods are due without anything given in
exchange. There is no ability to enforce this claim. Others are requested to give and
have nothing given in return. Fulfillment of a demand depends on the relationship
between the donor and the receiver. The aged use compliance to improve relationships.
Complying to the wishes or family and friends is the only means for survival (Dowd,
1984).
Compliance does not stimulate resource regeneration. Any remaining power
disappears (Dowd, 1975:590-593). Aged in this situation obtain what they need by
relying on others, as beneficence has replaced reciprocity (Dowd, 1984). Support,
through beneficence, is always available unless generalized prejudice against the aged
becomes great. Then social breakdown occurs (Dowd, 1984). At this point, no aged are
successful in social transactions. Old people are regarded as incompetent and unwanted
trade partners, and undeserving of beneficence. Once individuals are labeled as socially
aged, they experience this negative feed-back, which reinforces their social exclusion
(Hendricks and Hendricks, 1986:106). With social breakdown, the position of an aged


48
of themselves, opening "closed windows" (a phrase used in psychology for bringing
hidden thoughts to the surface). The aged men and women I encountered in the village
included the pain of remembering with responses to my questions. Yet, they insisted
they be included in the study when I returned. Is contribution to theoretical growth,
sharing new knowledge with professionals, and sending publications to the National
Library of Botswana enough to justify the stirring of thoughts and emotions with the
congruent raising and dashing of hopes of the populations studied? Thus began my
strong commitment to share findings and thoughts, in both verbal and written forms, on
the local level which included government, tribal administration, and the people
themselves.
It was also during these early visits that I began discovering my own suppressed
thoughts regarding cultural differences. These windows needed to be opened in order to
be successful in my goals. Even though behavior may have been correct in American
society, it did not mean it was correct in Botswana.
During an early visit, I was seeking the health clinic. Everyone I approached
with a simple request of directions said, "I do not know." In complete frustration I
settled myself on a roadside rock to recuperate. Two old men ambled along and we
exchanged names and asked of family and health. Afterwards, we began talking in
depth. They talked of past and present living conditions. When I asked, "May I visit
your house?" Both old men doubled over in laughter, pointing to each other and to me.
Their broken English and my limited Setswana was insufficient to clarify what was so
hilarious. A bystander finally explained that my selection of words intoned I was
offering myself as a prostitute! Luckily, the faux pas was taken humorously.
When I finally chanced to ask the men where the clinic was located, they pointed
to the building directly behind me. My earlier attempts to gain directions failed because
of another social faux pas. Thus, I learned my first lesson in the Laws: always begin any


104
obligations of providing physical care. Interestingly, the majority of both sexes planned
to return to their village of origin with advanced age and felt sure their own children
would provide them with comprehensive, traditional care.
No mention was made of family or social roles of the aged. Their activities were
viewed as play as the aged did not produce income. Old people were called usurpers
and parasites, taking but giving nothing in return. Only with questioning did it emerge
that parents provided child care to the adult childs children or maintained the village
home to which the urbanite returned.
Old Age as Defined by the Rural Sample
The four villages, including Ramotswa, were scattered in each of the four
directions from Gaborone. Distances varied from 20 to 100 kilometers. All were of
Tswana culture but of different tribes. Uniformity existed among villagers in their
responses. All participants had at least one aged relative, usually several, living in the
village.
Rural respondents stressed filial piety as a right of aging. Stress was on physical
assistance. The needed economic assistance blended with household needs.
Remittances were for household use, not for elder support.
In defining the aged, individuals divided old people into two categories: the
active-old, or elders, and children. The active-old were not colleagues, as in the city, but
elders. Childcare for women and political participation for men were identified roles for
elders. Active aged who cared for grandchildren or participated in kgotla activities were
not abstractly regarded as children,, although many young and middle-aged adults were
quick to identify reasons why they should be classified as such. Chronological age had
no bearing on the life cycle phase, although everyone was well aware of who was
chronologically the oldest with seniority rights in a given situation.


9
below the PDL (Hudson, 1977). Since that time, rural poverty has been increasing, with
estimates of households in absolute poverty always ranging well over 50% (Knudsen,
1988:17). The number of impoverished households reflects the fact that the richest 10%
of Botswana families control 42% of national household income. That leaves 58% of
household income to support 90% of the people (Botswana Central Statistics Office,
1974)
Rural household income, whatever the amount, must be considered in
conjunction with dependency ratios, or the numbers of economically dependent. The
1981 national census for Ramotswa reported a population of 13,000 with 2,845 actively
employed (Republic of Botswana, 1981). Thus, 20% of the village population supports
the others. The employed include those in agriculture, but most are migratory workers
who claim Ramotswa as their home. The majority of households are without a locally
employed adult. Remittances from migratory family members are a must in order to
survive. Many grow the majority of their own food to ease expenditures. (These
individuals are included in the employed if any food is sold.) Even this attempt at
survival does not always guarantee food, as rainfall is erratic and drought is common.
Deprivation is rampant, according to the outsiders view. There is a lack of food
and lack of what we consider necessities for normal living. To the Tswana, the situation
is reality. It is real not to be sated after a meal, to be cold in the winter and to have
the roof leak during rain. Life could be better, just as yours and mine. Like us, they
strive to improve life, only their expectations are placed in village reality.
Members of the household work to maintain a status quo, as gain is improbable
and loss pushes people further into poverty. Families who wants to survive cannot
afford to be lazy or stupid, for then they will lose what they have (Chambers, 1983:107).
Chambers (1983) calls this situation a "deprivation trap." Poverty unites with a loss of
power to increase physical weakness, vulnerability, and isolation that cannot be


144
TABLE 6.2 PERSONAL/PHYSIOLOGICAL SOCIAL FUNCTIONING ASSETS
(N=105).
A. Participation in Family Activities: (Mean = 3.1)
1. Does not attend or participate in any area. (19%)
2. Attends events, no participation in events. (18%)
3. Able to do minor tasks. (17%)
4. Able to participate except for heavy tasks. (25%)
5. Full participation. (31%)
B. Walking: (Mean = 3.8)
1. Can not move about or crawls. (3%)
2. Moves about house/yard with assistance. (19%)
3. Can walk in immediate neighborhood, less than one kilometer. (17%)
4. Can walk around village, up to 4 Kilometers. (25%)
5. Can walk any distance. (31%)
C. Hearing: (Mean = 4.2)
1. Totally deaf or unable to recognize sounds. (3%)
2. Understands loud voice with difficulty. (11%)
3. Can understand raised voice without difficultly. (6%)
4. Understands normal voice with occasional misinterpretation. (14%)
5. Hears well. (66%)
D. Vision: (Mean = 3.7)
1. Totally blind. (9%)
2. Minimal vision, can see color or shapes. (8%)
3. Can see to perform basic functions. (19%)
4. Can either recognize people walking past the house or fine handwork. (26%)
5. Can do both, recognize people and do fine hand work (38%)
E. Memory: (Mean = 4.2)
1. Does not remember any of the past or present (1%)
2. Very forgetful, remembers only main events of past. (1%)
3. Tends to forget present events, aware of past. (10%)
4. Minimal difficulty with recent recall, (forgets where put item) (59%)
5. Remembers well, knows where things are placed. (29%)


250
dry area or feelings of safety (14%). Only 4% lack all protective factors. Safety is the
least mention item on the scale, but is a major concern for those who feel being
bumped or pushed when walking on the footpaths is a real possibility.
Freedom from deprivation is the segment of the good life where the aged scored
the highest, with a mean of 14.93 out of 20. Scores of 17 to 20 are held by 40%. Only
4% score below half of the possible points. Sex and age have no bearing on the these
scores. The total gerontic fund correlates strongly with the amount of deprivation (p =
0.0001). Both the physiological and fiduciary dimensions had a strong influence (p =
0.0004 and p = 0.0001, respectively). Those who controlled money-producing assets in
conjunction with good health were the least deprived. These findings suggest that as
health and finances decrease the amount of deprivation increases. This is not what one
would expect in a society where care of the frail and economically dependent aged is
mandated by social mores. In fact, the social/familial assets correlated very little with
freedom from deprivation (p = 0.046). Two reasons come to mind: the situation of
generalized family poverty creates deprivation for everyone, and secondly, that with the
wage-earner making decisions for expenditures and labor and the coinciding
displacement of the aged from family affairs, physiological need fulfillment is
overlooked. I believe both play a role.
Security in Being Served as an Old Person
Security in being treated as an old person concerns the receiving the customary
rights of service as an elder, regardless of the degree of decrepitude. This category
involves both subjective and objective data. The reader should keep in mind that the
concept of security is under evaluation, not the actual care delivery. Responses do not
always reflect what may be reality. Several aged are sure a migratory daughter would
quit a job to return home, and provide continuous care, if chronic illness occurred. My


288
63 I have a list of places people go. Tell me if you went there regularly before, if you
go there now, and how often.
Visited in past Visits in present Frequency
Community Center
Kgotla
School PTA
Church
District Council
Clubs/Organizations
Library
Bank
Other
64. How do you usually get to other places in the village?
5. Walks by self.
4. Walks with others.
3. Dependent on ride, usually gets one if wanted.
2. Dependent on ride, may be unable to get one.
1. Never attends because of distance/transportation.
65. How do you get transportation when you need a ride?
5. Access to bus and a car in the household.
4. Access to bus and a car in the village.
3. Access to bus and a car outside of the village.
2. Access to bus only.
1. No access to transportation.
66. What difficulties/problems would you have if you wanted to ride a
bus?
5. No problems, walks to bus stop and gets on by self.
4. Rides bus, walks to bus stop, needs help getting on.
3. Can walk to bus stop, cannot get on.
2. Does not ride bus because of a lack of money.
1. Can not get to bus stop or ride because of health limitations.
I have two more stories. Here is the first one. Mr. and Mr. Jonas were born in 1920.
Their roof started to leak a couple of weeks age. Their son, John, said he would fix it.
They were expecting John next Saturday. He sent word to tell them he did not know if
he could make it because a friend at work was getting married.
67. What do you think John should do?
68.Using your knowledge of how people behave, what do think really happened?
Mrs. Ntseane is a very old woman who has been unable to walk outside for a long time.
She lives with her daughter. One morning Mrs. Ntseane does not get out of bed and
her daughter sees that she is very sick with a fever.
69. What do you think the daughter should do?
70. What do you think families actually do when an old person is dying?


99
thatched and painted meeting-hall. Directly next door is the district Police Station with
uniformed officers.
The royal corral, once shared with villagers for the overnight pasturing of cattle,
stands empty. The royal burial ground is still used for the chief and his immediate
family. The grounds around the cemetery and kgotla building are kept clean by those
judged guilty of crime. They work during daylight, advertising their shame to the village.
Not far from the kgotla, about a five minute westerly walk, is the new village
library, a modem building with numerous books. The community center with a large
meeting hall is anther 10 minute walk to the northwest. Various groups, such as the
Village Development Committee, Red Cross, and burial societies, meet here during the
day. Frequently large parties are held here by different organizations on Friday or
Saturday nights.
The main shopping area is a ten minute walk south of the community center.
The post office, a small bank, grocers, general produce stores and one of the two public
pay phones line the dirt road. Bus service, northwest to Gaborone and southwest to
Lobotse, originates here. There are no signs or benches; one just knows where to stand,
which bus to ride and when it leaves. Early in the morning smartly dressed men and
women going to work in the capital or Lobotse fill the bus. Later in the day the riders
are mothers with babies and the aged, going shopping, visiting or to work the lands.
The first bus stop, directly south of the village center, is at the Ba-Malete
Lutheran Hospital. This medical center offers out-patient and in-patient care,
pharmaceutical service, plus surgical and maternity facilities. The physicians are
European with Tswana nurses serving as translators. It is also here that the bus begins
its journey on the one paved village road.
Traveling west, the bus passes several government housing units with electricity
and running water, small grocers, the Catholic church with its elementary school and


206
it is meaningful. Sitting replaces touch. Silence replaces crying. It is a time to respect
the need to be alone instead of showing respect with group involvement.
Betty, during her confinement, had continuous comfort in knowing that kin were
performing the necessary rituals of greeting and guarding. Alone in thought she was
never alone in the compound. To the Tswana, this was the way it should be.
Confidence in Dorothy handling the situation was never questioned, as there was always
someone, like Auntie, to encourage and indirectly direct.
The division of labor continues to depend on age, sex, and kin relationship
during crisis with death. It is never questioned that work may not be done, or
responsibilities never carried out. When a person becomes tired, another will take over
without being asked. All kin contribute monetarily, physically and emotionally. This is
the time when tradition reigns, with respect given to eldership and the principles of
seniority followed. With the passing of the crisis, life returns as before.
The parting remarks of Sego after the funeral seem all so true. Why do fine,
old-people fail to find emotional peace and security? Why is it that adult children give,
or do not give, to their parents when they are in need? Adult children do have more
money and material possessions than the aged, in spite of the generalized poverty.
Many times they also fail to recognize the needs of the aged extend beyond that physical
hunger.
Many aged function in a setting of hunger for continuity in customs, meaningful
roles, and family interdependency. Some aged seek to fulfil their hunger in a liberalized
territory where known rules for social exchange no longer apply. Others seek the
traditional approach. The irony is that the segments of continuing traditional law also
prevents fulfillment. Many adult children do care but are prevented from succeeding in
helping through custom and poverty.


FINDING THE GOOD UFE IN THE FAMILY AND SOCIETY:
THE TSWANA AGED OF BOTSWANA
By
ELIZABETH A GUILLETTE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1992


148
be utilized to obtain goals through use, investment or trade. The value of this approach
lies in the scientific ability to analyze the good life in terms of the social/familial assets,
which provides a basis for success or failure in regards to fund applications. The death
of a person is regarded as a lost, non-renewable asset while the birth of a grandchildren
or great grandchildren rejuvenates this aspect of the fund. Trade can occur with
individuals either within or outside of the fund.
This dimension includes assets of adult children, related children of school age
and below, extended family, continuing contact with social agencies and access to
motorized transportation. (See Table 6.3.) These assets, in reality, provide access to the
good life, as care and satisfaction cannot be considered an automatic outcome.
Measurement of size and density of ones social networks do not quantify the types of
interactions. Outcomes of support and satisfaction depends on the adequacy of the
relationships via the exchange process (Ryan and Austin, 1989). This is a slightly
different approach than the more common concept of regarding children as automatic
providers. For some readers, this may mean a switch of emphasis in the meaning of
children. Also, the fund does not consider family functioning, in itself. Family
functioning is considered later, as a setting for gerontic fund use.
The social/familial components vary in two distinct ways from developed nations.
First, I have placed a stronger emphasis on grandchildren as an asset. Grandchildren
from migratory workers are sent to live with aging parents to learn village customs and
provide care in the later years. Numerous aged report having the child since infancy,
with the expectation of having a care provider as the child matured. It is common to
find infants and/or preschoolers under the direct care of grandparents. Very young
grandchildren are viewed as an asset, in that "they create a reason to live." School age
grandchildren are desired as they signify the availability of provisioning for old age


107
frequently, singing and cooing as they stay together in one section of the house. Visitors
are welcome to see this "Gift from God," who requires total care.
Childhood is entered as the infant learns to walk and obey simple commands.
Overall, childhood is regarded as a carefree time for play. It is a rare child who owns a
toy of any kind. A long piece of string is shared among girls for intricate games of
jumping rope. A well worn soccer ball is the center of attention for older boys. When
these two items are lacking and there is no chore to perform or school to attend,
children sit or walk idly through the village. Imaginative play and play with adults have
little place in this society.
The child is considered to be incapable of spontaneously recognizing important
facets in life. Learning is through rote memorization, in school and at home. Facts are
not to be altered and hence there is little encouragement to apply them to different
situations. Reasoning is called cleverness, appreciated only if not disruptive. It, too, is
not encouraged as a child is not expected to act independently of others. A child is to
do as told. Activity is assigned through command. The form of command is "Go buy
bread!" with never a please or thank you. Overt signs of appreciation for assistance are
not part of the childs upbringing.
Outwardly there is an appearance of harshness and rigidity in childhood.
Actually, it is a teaching that "all things come to those who wait." The minimal
pressures for self-direction and decision-making reflect a learning of necessary concepts
for gerontocracy (Rosenmayr, 1989). The goal is to instill respect for elders, with
obedience seen as a necessity for household and social functioning. Beneath the
directiveness is a security that, if true difficulty arises, there will be someone to help, be
it a parent, older sibling or neighbor.
Ideally, the child must learn obedience to the law before becoming an adult.
The child-beating used to control the behavior of earlier generations of children has



PAGE 1

TIIB CLAfvfP LOADER OF Escherichia coli DNA POLYMERASE Ill: KINETICS OF THE ATP DEPENDENT STEPS IN THE SLIDING-CLAfvfP LOADING REACTION By CHRISTOPHER R WILLIAMS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2003

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' Copyright 2003 by Christopher R Williams I

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, I This document is dedicated to my parents family and Heather Runyan.

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' ACKNOWLEDGMENTS Foremost I would like to thank my advisor Linda Bloom Ph D ., for outstanding guidance and providing an exceptional working environment in a state-of-the-art research laboratory. I thank my committee at UF Drs. Daniel L Purich Arthur S Edison, J. Bert Flanagen and Alfred S Lewin for their assistance not only in guiding my project but in guiding me to become more wise and perceptive as a scientist I must also acknowledge my committee at Arizona State University Drs Neal Woodbury Yuri L Lyubchenko and Kenneth J Hoober for their assistance in the early stages of this dissertation project I acknowledge Manju Hingorani Ph D for helpful discussions at the Keystone Symposia meetings and protein preparation Mike O Donnell Ph D for the generous gifts of clamp loaders, Martin Webb Ph D ., Myron F Goodman Ph D ., and Jeffery Bertram for the gift of the phosphate binding protein plasmid :, and help in characterization and use ofMDCC-PBP and Petr Kuzmic Ph D ., for contribution of the cu s tom version of DynaFit and assistance with kinetic modeling I would like to thank my following colleague s for their friend s hip and invaluable discussions regarding this project and s cience in general: Brandon Ason, Ryan Shaw John C Lopez Gabriel Montano Ph D Gregory Uyeda Jose C lemente Ph D ., Joyce Feller Ph D Finally I would like to thank my parents for their unwavering support of my endevours and for purchasing my fir s t microscope and chemi s try set s years ago 1V

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,I TABLE op CONTENTS Page I ACKN"O 'WLEDGMENTS IV I LIST OF TABLES .. .. . .. ........... .... . . ... ...... ...... .. ..... ... .... .... ... .................... .......... .. .............. x I LIST OF FIGURES . .......... .................... .... .. ........... .. .............. .... .. ....................... .. .. ............. .. Xl ABS1RACT ....... ........ ............... .......... .. ....... : ....... . ................. .......... .. .. .. .... ....... ...... xiv CHAPTER 1 INTRODUCTION ST A TEMENT OF PROBLEM .... .. .......... ... ..... ...... . ..... . .. .. .. . 1 The Processive Escherichia coli DNA Polymerase III Holoenzyme ............. ............. 1 DNA Polymerase III Holoenzyme ... .. . .. .. . .. ........................ ................. . ........... 1 The p Sliding Clamp .... .. ... .................... ............. .... ... ............... . .... ................ 2 The DnaX Clamp Loader Stoichiometry and Organization of Subunits ............ 3 Clamp Loader Subunit Functions ..... . .. .. . ......... ........... .. .. .. .... ........ .............. 4 The Mechanism of P Clamp Loading .......... .. . ................ ....... ..... . .. .......... .... .. 5 Importance of the E. coli Model Replication System . ............. . .. .. ..... ......... ..... 7 The General Problem Under Study and Research Questions Addressed .... . .. ..... ..... . 8 Conformational Dynamics of the Clamp Loading Machine .. . ...... ........... ... .... 10 Enhancement of the Clamp Loading Machine by its Clamp .... ............. ...... .. ... 13 The x and 'I' Subunits are Required for Optimum Activity of the Clamp Loader14 Application of the Analyses of the Clamp Loading Machine to Other Complex Molecular Machines ....... ............. . .. ........... ............ ........ ...... ....................... 17 Novel Hybrid Devices Based on the Clamp Loader and Sliding Clamp . .......... 19 '' Off-the-Wall ' Example of the Clamp and Clamp Loader in a Novel Device ... 20 Design of Research Project .. ....... .. . ... ............ ............ .. .. .. ...... . .................... ....... 21 2 LIIBRA. T'URE REVIEW .................... .. ...... ..... .................................. ....... .............. 24 DNA Replication in Esch e ri c hia coli ............. ....... .......... ......... ........... ...... .......... 24 DNA Polymerase ill Holoenzyme ............ ........................................ .. .................. .. 25 The t Subunit is the Coordinator of Pol ill Holoenzyme Function and Processivity .. ............ ... ......... ...... ......... ...................................... .. ............... 29 Structure of the Sliding Clamp Processivity Protein . ....................... . ............ 31 The DnaX Clamp Loading Machine .. . ..... ......................... .. .... ... . .. .. .. .................. 35 The AAA + Superf amil y of Motor Proteins ..................................... ........ . .. ..... 40 V

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X-ray Crystal Structure of the Clamp Loading Machine .............. ..................... 45 Structure of the Nucleotide Binding Site and the Proposed Confo1mational Change of the y Subunit ...... .. . ................. .. ................................ ......... ......... 48 X-Ray Crystal Structure of the o Subunit Bound to a~ Monomer and the Mechanism for Opening the Sliding Clamp ................. .................. ....... .. ... 52 DNA structural requirements for clamp loading by r complex .. ..... .. ... ....... . 56 Mechanism of the p Clamp Loading Reaction Cycle by y Complex ... ............... .... 60 Mutations of the~ Clamp and r Complex o and y Subunits : Effects on the Clamp Loading Mechanism ............................. .................... ....... . .............. .. 65 The Clamp Loading Machine Within Polymerase III Holoenzyme ............. .... . 68 Clamps and Clamp Loaders of Bacteriophage Eukaryotic, and Archaeal Organisms ....... .. .... ........... ............ ............................ ...... : ......... ......... 71 Bacteriophage T 4 Clamp and Clamp loader .................. ............................ .. ...... 73 Eukaryotic PCNA C lamp and Replication Factor-C Clamp Loader . ..... ..... ..... 75 Archaeal PCNA Clamp and Replication Factor-C Clamp Loader ... ..... .. .. ..... 80 3 MATERIALS AND METHODS ..... ........ . ... .. ........ . ..... .. ..... ...... .. ..... ..... .......... 84 Proteins Reagents and Oligonucleotide Substrates ... ......... .......... ... .. .................... 84 DNA Polymerase III Proteins .... ... ....... .. .. ............................ ..... ............ .. ....... 84 Reagents .......... . .......... .... . .. ...... .. .......... ................... .... ... ... ............... .......... 85 Oligonucleotide Substrates ...................... .. . ............................................... ........ 85 Purification of Esc h e ri c hia co li Phosphate Binding Protein . .. ...................... .. ... 86 Labeling of Phosphate Binding Protein with MDCC .... ............ .. ............. . .. . .. 89 Characterization of MDCC-Labeled Phosphate Binding Protein ...... .. ......... . .. ......... 90 Removal of Free-Inorganic Phosphate ( P i) Contamination with the '' Pi-mop '' .. 91 Concentration and Efficiency of Labeling of MDCC-PBP .. ............. : ............ .. 91 Characterization of the fluorescence-molar response ofMDCC-PBP to Pi Active Site Titration of MDCC-PBP ......................................... ... .. . ....... ........ 93 Fluorescence Anisotropy Binding Assays .................. .. ............. . .... .................... .... 94 Calculation of Ani s otropy .............. ...... .. ... ..... ............... ................... ....... .... 94 Steady-state Measurement of Clamp Loader RhX-DNA Binding Kinetics .... 95 Pre-Steady-State Measurement of C lamp Loader RhX-DNA Binding Kineti cs ..... .. .......... .......... ........... ............. ...... ....................... ............ ..... .... 99 Fluorescence-based MDCC-PBP ATP Hydrolysis (ATP ase ) Assay .. .......... .. .... . 101 Steady-State Kinetics of ATP hydrolysis .... .... ........ .. .. ..... ..... .. .. .. .. ... ........... 101 Pre-Steady-State Kinetics of ATP Hydrolysi s ................... .. ............. ............. 104 Computer Modeling of ATP Hydrolysis Kinetic Data ...................... ............... 109 Correlated Pre-Steady-State MDCC-PBP ATPase Assays and Fluorescence Anisotropy Bin ding Assays ..... ..... .................. .. . .. . .............. ....................... 110 Stopped-Flow Dead Time Dete1rr1inations ........................................ ................. .... 112 Determination of the Dead Time for the Biologic SFM-4 Stopped-Flow .... .. .. 112 Determination of the Applied Photophysics SX 18MV Stopped-Flow Reaction Analyzer Sequentialand Single-mix Dead Times .... .. ............... . 114 Vl

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I I 4 ATP-DEPEND E NT CONFORMATIONAL CHANGE IN THE CLAMP LOADERl 18 In.troduction ........ ... .... .. .... .... .. .......... . .. .... .. .... ............ ................. . ...... ..... .. .. .. .... 118 Steady-State Characterization of y Complex ATP Hydrolysis DNA Binding and Clamp Loading Activities ..... ... .... .. .............. ... .... ......... ......... .. .... .......... .. ........ 120 Enhancement of Steady-State A.TP Hydrolysis Kinetics of y Complex by~ clamp ... . ....... ... ..... ....... ........................ ... ................ ................ .... . ..... ...... 120 Steady-State DNA Binding and Clamp Loading Activities of y Complex ...... 122 Pre-Steady State Kin~tics of DN~-Dependent ATP Hydrolysis by y Complex ...... 127 Pre-Steady-State MDCC PBP ATP as~ Assays for y Complex in the Absence and Presence of~ Clamp ........................ ......... ........ ..... ... ...................... .... 127 Pre-Steady-State MDCC-PBP A TPase As s ays at Different Concentrations of y Complex in the Presence of~ ............. . .......... ................ .. ......... .... ........... 129 A TPyS-Chase of Pre-Steady-State ATP Hydrolysi s Activity by y Complex ... 131 Pre-Steady State Kinetics of p Clamp Lo~ding by 'Y Complex Initiated at Different Steps of the Reaction Cycle ... ... ...................... .. ..... . ................... ..... .. 135 Kinetics of Clamp Loading when 'Y Complex is Equilibrated with ATP .... .. .. 135 Kinetics of CJamp Loading when y Complex is Equilibrated with ATP and p 137 Kinetics of Clamp Loading when 'Y Complex is Added Directly to a Solution of ATP p and DNA ................... ... ... ..... .. ....... ......... .. .. .. ...... ............ .... 141 Kinetics of ATP Hydrolysis During Clamp Loading when 'Y Complex is Equilibrated with ATP ........... . ............................. ... ... .. .. .... ................. .. ........ ... 14 2 Kinetics of ATP Hydrolysis when 'Y Complex is not E quilibrated with ATP ..... ... .. 144 Kinetics of Fonnation of the Two Populations of 'Y Complex . .... ........ . .... ............ 146 Computer Modeling of ATP Hydrolysis Reaction Kinetics .. ................................... 149 Discussion ... . .............. ........... .... .... ..... . . ... .. . ...... ....................... ........ .. ........ 153 5 CHARACTERIZATION OF THE MINIMAL CLAMP LOADER COMPLEX AND COMP ARI SON TO GAMMA COw>LEX .. ........ .......... ..... .................... ... 163 Introduction ......... ...... ... ........ ... ....................... .. .................................... . ..... .... 163 "( 3 00 is the Minimal Clamp Loader Complex Which Can Bind DNA .................... 165 Analysis of DNA Binding Activity of the Individual Subunits or Sub-complexes of the Clamp Loader . .................... ................ .................... 165 Analysis of the DNA Binding Activity of y 3 00 Minimal Complex in the Absence and Presence of .................. . .. . ... . .... .. .................. .............. . .. 16 7 Comparison of~ Clamp Binding Affinity of the Minimal Complex and 'Y Complex 169 E quilibrium ~pyr ene Binding Activity of the Minimal Complex and 'Y Complex 169 Apparent Dissociation Constant for ATP Binding the Minimal Complex or y Complex ....... ....... . ..... .... ............. ............ ... ............. ............ .. ................. 171 Kinetics of ATP Hydrolysis by the Minimal Complex Measured Using the MDCCPBP A TPase Assay ............................. .... ... ....... . ... ........ .. .... .. ... ........ ... ..... . . 173 Steady-State ATP Hydrolysis Kinetics of y 3 oo Minimal Complex in the Absence and Presence of~ Clamp ........ ........................ .................. .. ... .. ... 173 Pre-Steady-State Kinetics of ATP Hydrolysis by y 3 00 Minimal Complex in the Ab s ence and Presence of~ Clamp ...................... .................................... 176 Vll

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Kinetic s of ATP Hydrolysis when the Minimal C omplex is not equilibrated wiili ATP ....... ....................... .. .................. ......................................... .. ............... .. .. .. .... 180 ATPyS-Chase of Pre-Steady-State ATP Hydrolysis Activity by the y 3 00 M i nimal Complex ... .. ....................... ........ .. ............................................. '. ... 183 ATPyS Chase of Steady-State ATP H y drolysis Activity by the y 3 oo Minimal Complex or y Complex ................................ ............. ................ .. . ............ .. 186 C lamp Loading Activity of the Minimal Complex is More Sensitive to ADP than y Complex ............................................................................. ........... ... .. 188 Pre-Steady-State Kinetics of Clamp Loading by the Minimal Complex Initiated at Different Steps of the Reaction Cycle .......... ........ ......... ................. .......... .. . . 190 Kinetics of Clamp Loading when the Minimal Complex is Equilibrated with ATP and ............. ....... .................... ...... ... ...... ................ .............. ..... .. 191 K i netics of Clamp Loading when the Minimal C omplex is Equilibrated with ATP .................. ............... ................. .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 194 Kinetics of Clamp Loading When the Minimal Complex is Mixed Directly with a Solution of ATP p and DNA ..... .... .................. .... .... . ........... .. ....... 195 Direct Real Time Correlation of the Minimal Complex DNA Binding and ATP Hydrolysis Kinetics in the Presence and Absence of P Clamp ...... ..... ..... ... ...... 196 Discussion ..... .. ..... . ........................ .. ........ .. .. .. .. ............... ....... ....... ... ... ..... .. ... .... 200 Understanding y Complex Kinetics by Characterization of and Comparison with y 3 oo Minimal Complex ... . ................ .. .............................. ..... .......... 200 1 3 00 is the Minimal Complex with DNA Binding Ability and Binds ATP and with Affmity Similar to y Complex ..... .................................... ..... .......... 202 Pre-Steady-State ATP Hydrolysis and DNA Binding Kinetics: Analyses of the Active and Inactive Clamp Loader States .... .. ............. ..................... .. .... ... 202 The x, and 'V Subunits Missing from the Minimal Complex, May Facilitate the Conformational Dynamics of y Complex ............. ...... ... .. . .... ...... .. .... ........ 210 Clamp Enhance s the Switch from Inactive to Active Clamp Loader Populations ....... ..... .......... ...... ........ ....................... ......... .. . ... .......... . ... 211 Experiments when the Minimal C omplex was not E quilibrated with ATP Reveal Slower Confor1national Change Kinetics than y Complex .. .. .......... 213 The Nature ofNucleotide Binding to the Minimal Complex and 'Y Complex .. 214 6 CONCLUSIONS AND RE C OMMENDATIONS ....... .................... .. ............ ..... . 221 Introduction . ...................................... .. .. . ............ ........ ....... ............... ................ 221 Steady-State Kinetics of the Clamp Loader in the Absence or Presence of~ .......... 224 Kinetics of ATP-Dependent Conforrnational Changes within the Clamp Loader . 226 A Possible Mechanism for~ Clamp Enhancement of C lamp Loader Activity ........ 233 The Missing x, and \I.I Subunits are Responsible for the Kinetic Differences Between the Minimal Complex and y Complex ........... ............................... 236 The x and 'V Subunit s are E co li Clamp Loader AAA + Adaptor Proteins . ..... 237 Programmatic Recommendations .................................................. ............ .......... .. 239 Pre-Steady-State Kinetics of p Clamp Binding ... ........ ....... ..... . .. ............. .... 239 Fluorescence Lifetime Measurement ofMDCC-PBP Titrated with Inorganic Phosphate .......................... .. . ....... ....... .. ............... ..... ............................. ..... 241 V111

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Further Investigation of ATP Binding and Nucleotide E xchange by the Clamp L oader .................................................................................... ..... ........... ...... 2 4 2 Analysis of Clamp Loader Confo1mational Dynamics by Circular Dichroism Spectroscopy .. .................................. ................................................ .... ....... 244 Identification of the Putati v e C lamp Loader DNA Binding Surface ................ 245 The xand 'I' AAA + adaptor hypothesis ............ ................................................ 2 46 APP E NDIX I COMPUTER MOD E LING OF E XP E RIMENTAL KINE T I C DATA ................. ..... .. 2 48 DynaFit Script F or Fitting Shown in Figure 4-9 ... .................................................. 2 48 DynaFit Script For Fitting in Shown in F igure 4-1 O C ............................. ............... 2 4 9 DynaFit Script For Fitting in Shown in Figure 5' 6 ...................... : ..................... ..... 2 51 Simulation Mechanisms for E quilibration steps: Kin T ekSim Program ... ........ ...... 2 52 DynaFit Output Indices ................. . ............................................................... .. ... .. 255 F itting for y Complex Data in Figure 4-9 . ...................... .... ................ .......... 2 5 5 Fitting for y Complex Data in Figure 4-1 OC ................................ .. ..... ............ 25 8 Fitting for Minimal Complex Data in Figure 5-6 ........... ............. .. . ................. 260 Fitting for Minimal Complex Data For Estimation of Conformational Rate Constants From a Single Data set ..... ................ .. ......................... ................ 262 Alternate Dynafit Model with a '' Branch' Step at After Hydrolysis of Second ATP265 Alternate Dynafit Model Applied toy Complex in Figure 4-9 ....... : .. ............ 2 65 Alternate Dynafit Model Applied to y Complex in Figure 4-1 OC . ................ . 267 Alternate Dynafit Model Applied to the Minimal Complex in Figure 5-7 ....... 269 Alternate Dynafit Model Applied to y Complex in a DNA Binding Assay in the Absence of p Clamp ................... . ..... . . . ............ ...... .... .. ............................ 271 LIST OF REFERENCES .......................... .. .................. ... ................... . ....... ..... ... .. .. .. 27 4 BIOGRAPIIlCAL SKET C H ................. ...... . .. ................................ ........ .. . ..... ... ........ 287 lX

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' I LIST OF TABLES t Table page 2-1 Clamps and clamp loader s through evolution ........ .. ........................... ... .. ........ .. ... 72 3-1 Assay and protein buffers ....... ... .. ...................... ......... ........... ..................... ....... 85 3-2 TG-plus media contents .............................. ............... .. ........... .. ...... ... ... .. ... ........ . . 87 4-1 Steady-state ATP hydrolysi s kinetics of y complex in the absence and presence of ~ a .... ...... ............ ..... .. ................................ ................ ....... .. ... . .. .. 121 5-1 Steady-state ATP hydrolysis kinetic parameters for the minimal complex and y complex in the absence and presence of ~ a .. ....... .. ................. .. . ... . . ..... .. . .. .... 17 4 5-2. Steady-state A TPyS-chase assay results : comparison of the minimal complex and y complex in the presence or absence of~ ............ . .. .... .... . . . ....... .. ......... ...... 187 X

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I ' LIST OF FIGURES I Figure page 1-1 Schematic of the clamp loading ~eaction for processive DNA synthesis ... ............. 6 2-1 The crystal structure of~ sliding clamp ....... ..................... .......... ........... .. .............. 3 2 2-2 The crystal structure of a AAA + motor protein . : ..................... ....... ......... .............. 4 3 2-3 The crystal structure of the ry 3 oo clamp loader .. ... .. .......... .................... .. .......... .. .. . 46 2 4 The crystal structure of the o-~ 1 complex ...... .... ..................... .......... ..................... 53 2 5 A schematic cartoon of the basic steps in the clamp loading reaction and initiation complex formation . ........ .............. . ........... ...... .. ............ ....... . . .. ............ . .... 62 2 6 Architecture of the polymerase III holoenzyme at the replication fork organized by the DnaX clamp loading machine ... ............. ..... ............................. . : .................. 69 3-1 P i titration analysis ofMDCC-PBP . .............. .. ....... .. ..... ........... .... ... .. .......... .. .. . 93 3-2 A plot of observed reaction decay rates as a function of NBS concentration ......... 113 3-3 Fluorescent decay amplitudes plotted as a function of experimentally observed decay rate constants to dete11nine dead time of the SFM-4 stopped flow .. .. .. .. 114 3-4. Sequential-mix reaction fluorescent decay amplitudes plotted as a function of experimentally observed decay rate constants to determine dead time of the SX 18MV stoppedflow .. . ...................... ........ . .. .................. .. .................. .... 116 3-5 Single-mix reaction fluorescent decay amplitudes plotted as a function of experimentally observed decay rate constants to detennine dead time of the SX 18MV stopped-flow ............. ........................... ............................................. 117 4-1 Steady-state DNA binding and clamp loading activities of 'Y complex ................... 123 4-2 Steady state kinetics of clamp loading as a function of ATP concentration ........... 125 4-3 Kinetics of ATP hydrolysis by y complex in the presence and absence of .. . ..... 1 2 8 4-4 Quantification of the number of ATP molecules hydrolyzed by 'Y complex in the fir s t turnover of~ clamp loading ................. .............. ........................................ 130 Xl

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4-5 1 Pre-steadys tate kinetic s of ATP hydrolysis by y complex in the presence and absence of the~ clamp in assay s with and without an ATPyS chase .............. ... 132 I 4-6 Kinetic s of clamp loading measured in reactions that were initiated at different stages of the loading cycle ................... ..... ................. ...................................... 13 8 4-7 Kinetics of ATP hydrolysis during the clamp loading reaction when y complex is equilibrated with ATP ............................................ .. ....... ................................. .. 14 3 4 ~ 8 Kinetics of ATP hydrolysis in reactions initiated by the addition of y complex to ATP and DNA ........ ...... .................... .. ............................................................. .. 14 5 4-9 Kinetics of ATP hydrolysis when y complex is incubated with ATP for a defined period of time prior to addition ofDNA ................ ...................... .......... .. ........ 147 410 Kinetic modeling of ATP hydrolysis reactions ........... ... .... . ........ ......... .......... .. 150 5-1. Change in steady-state anisotropy for RhXs s DNA in the presence of individual subunits s ub-complexe s, and y complex with or without ATP ............... .. .......... 166 5-2 Steady-state binding of the minimal complex or y complex with RhX-pt DNA with and without~ ........ ........ .. ....................................................... .. . ............ ........... 168 5-3 Steady state anisotropy binding activity of 'Y complex or the minimal complex with ~ pyrene in the presence or absence of ATP ....................................... ........ .. ......... 170 5-4 Minimal complex or y complex binding ~p yr en e as a function of ATP concentrationl 72 5-5 Kinetics of ATP hydrolysis by the minimal complex or y complex in the presence and absence of .. ............................................... .... ..... ................ .................... .. 178 5-6 Kinetics of ATP hydrolysis of the minimal complex or y complex directly mixed with pt DNA and ATP ................................ .... ....................... ...................... .. . 181 5-7 Pres teady-state kinetic s of ATP hydrolysis by the minimal complex when chased with non-hydrolyzable A TPyS ............ ... .. ............................ .......... . ............... 184 5-8 Effect of increasing ADP concentration on the steady-state clamp loading reaction for the minimal complex and 'Y complex ......... .......... .......................... 189 5-9 Pre-steady-state kinetic s of the clamp loading reaction initiated at different steps 192 5-10 Direct correlation of the kinetics of DNA binding and ATP hydrolysis by the y 3 oo minimal complex in the absence and presence of~ clamp ........................ 198 5-11 Kinetic modeling of ATP hydroly s is reactions ................... .... .. . ..... .. ............... .. 203 6-1 Kinetic modeling of ATP hydrolysis reactions ........................................... ........... 229 Xll

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I I A-1 KinTekSim simulation of 'Y complex, no equilibration with ATP ................. . ...... 253 A-2 KinTek.Sim simulation of the minimal complex equilibrated with ATP ............... 254 A-3 KinTekSim simulation of the minimal complex, no equilibration with ATP ........ 255 A-4 DynaFit Fitting of the minimal complex ( Figure 5-6 ), no equilibration time with I ATP ............... ................................................. ..... ........................... ........... .... . 262 A-5 DynaFit fitting of the minimal complex ( Figure 5-7B black trace ), Single experimental data set 1000 ms equilibration with ATP . .. ......... ....................... 265 A-6 Figure 4-9 data for 'Y complex fit to alternate model with a '' branch '' step after hydrolysis of two ATPs ...... ........................................ ...................................... 267 A-7 Figure 4 lOC data for 'Y complex fit to altern ate model with a '' branch '' step after hydro I ysis of two A TPs ....... ..................................................................... ......... 269 A-8 Figure 57 black trace data for the minimal complex fit to alternate model with a '' branch '' step after hydro I ysis of two ATPs ... ... ........ ...... .. .... . .... ......... ... ..... 271 A-9 Example of the alternate model applied to DNA binding ( anisotropy ) data for a reaction of y complex and Rhx.-pt DNA ...................................... ............ .......... 2 73 Xlll

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulftllment of the Requirements for the Degree of Doctor of Philosophy THE CLAMP LOADER OF Esc h e ri c hia co li DNA POLYMERASE Ill : KINETICS OF THE ATP-DEPENDENT STEPS IN THE SLIDINGCLAMP LOADING REACTION By Christopher R Williams December 2003 Chair : Linda B Bloom Major Department : Biochemistry and Molecular Biology DNA polymerase III holoenzyme the principal enzyme responsible for E. coli chromosomal replication, synthesizes stretches of DNA thousands of nucleotides long at a rate approaching 750 nucleotides s 1 without dissociation. A ring-shaped DNA sliding clamp '' ~ '' topologically links the polymerase to DNA A clamp loader '' y complex '', assembles~ on DNA in an ATP-dependent reaction This dissertation project was undertaken for investigation of the mechanism of~ clamp loading by the clamp loader for processive replication ATP binding and h y drolysi s activities of the clamp loader promote conformational changes modulating its binding affinity for the clamp and DNA Using fluorescence-based steady-state and real-time stopped-flow methods the kinetics of these dynamic conformational changes were measured. Pre-steady-state ATP hydroly sis assays perfor1ned in the absence or presence of~ re s ulted in biphasic or monophasic kinetics respectively Biphasic kinetics suggested that the clamp loader equilibrated with ATP, exists in a mixture of two dominant species. Addition of~ X lV I

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I converted this mixture into a single activated population, effectively increasing its concentration These experiments in addition to adenosine 5 -0( 3-thiotriphosphate ) ( ATPyS ) -chase assays showed that activated y complex hydrolyzed one ATP per sub11oit at a rate faster than ATP dissociation The hypothesis that y complex exists in multiple species with ATP was confrrrned by measuring pre-steady-state clamp loading kinetics in DNA-binding assay s. When y comple x was equilibrated with ATP a mixture of two species fo11ned which when mixed with DNA and P exhibited biphasic DNA binding kinetics When equilibrated with ATP. and~ rapid monophasic DNA binding kinetics resulted. Direct mixing of y complex with DNA~ and ATP displayed slow monophasic kinetics limited by the conformational change rate The rate of the conformational change s separating the two dominant species was determined by investigating their evolution in equilibration time with ATP Computer modeling of these experimental data revealed a conformational change rate of ~ 4 5 s1 Comparison of the kinetics of a '' minimal ' clamp loader complex missing 'X and 'V subunits revealed that these subunits facilitate the conformational changes in y complex required for modulation of~ and DNA binding affinities during the clamp loading reaction xv

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t CHAPTER I ' INTRODUCTION STATEMENT OF PROBLEM The Processive Escherichia coli DNA Polymerase ill Holoenzyme DNA polymerase ill holoenzyme (pol III holoenzyme) is the principal enzyme involved in replication of the Esc h e ri c hia co li chromosome ( Kornberg and Baker 1992) The pol III holoenzyme copies the parent chromosome with surprising speed and processivity moving along the DNA at a maximum velocity reaching approximately 750 nucleotides per second, without release from the template at distances of over thousands of nucleotides Polymerase III holoenzyme must possess these functional characteristics to complete rapid replication of the chromosome for cell division Rapid and processive DNA synthesis is required for all forms of DNA metabolism and genome maintenance including DNA repair and recombination, and is evolutionarily conserved in all branches of life underscoring the significance of advancing the detailed understanding of the proteins providing these functional mechanisms DNA Polymerase m Holoenzyme Pol III holoenzyme is a multi-protein complex consisting of ten distinct subunits ( a, s 0 ~ t y o o', x and \JI) A core of the holoenzyme consisting of the a-5 to 3 polymerase s-3' to 5 exonuclease and 0 subunits is paired-up by the 't 2 protein-dimer ( McHenry 1982 ; McHenry and Crow 1979 ). The -c 2 -dimerized pol III core termed pol III ', was originally purified from cells and isolated from pol III holoenzyme The replication activities of pol III core and pol III were dete11nined to be distributive in kinetic and product s ize determination assay s, whereas holoenzyme could synthesize 1 I

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, 2 stretches of DNA with high processivity (Fay et al ., 1981 ~ LaDuca et al ., 1983) Clearly these were incomplete forms of pol III holoenzyme and were missing important factors necessary for complete and rapid replication of the chromosome Another form of pol ill termed pol III*, contained all of the subunits of pol III holoenzyme except one the subt1nit (Fay et al 1982) Addition of this subunit was all that was required to convert the distributive pol ill* enzyme into a highly processive enzyme with DNA synthesis activity matching pol ill holoenzyme The subt1nit is I initially associated with the replication for k DNA at sites called preinitiation complexes where polymerase III holoenzyme assembles for DNA synthesis Several early biochemical studies revealed th at a complex consisting of the t ; y,o,o x and~ subunits within pol III holoenzyme comprised a complex responsible for ATP-dependent placement of the '3 subunit at primed sites on DNA for forrr1ation of the preinitiation complexes and were required for conferring processivity in replication (Maki et al 1988; Maki and Kornberg, 1988) The p Sliding Clamp Solution of the X-ray crystal structure of the~ subunit revealed that it was a ringshaped dimer of crescent-shaped protomers (Kong et al ., 1992 ). was termed a ''DNA sliding-clamp'' due to its ability to topologically link pol III holoenzyme to template DNA in such a way that holoenzyme remains tightly associated with DNA, yet has significant freedom of movement on the DNA The P clamp has an inner pore lin ed with a-helices that act akin to '' skates, '' traversing the clefts of the major and minor grooves in the DNA backbone as the clamp slides along This inner pore has a diameter large enough to encircle duplex DNA as well as hybrid RNA-DNA duplex structure found at

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3 the sites of preinitiation complex fo1mation A combination of hydrogen bonding hydrophobic and ionic interactions at the dimer interfaces strengthen and hold together I the f) clamp subunits p binds directly to the core of pol ID holoenzyme through the a subunit (Stukenberg et al 1991 ) The f) clamp has a dissociation constant for dimerization in the range of ~ 6 0 x 10 1 1 molar and is stable enough to remain on circular DNA for ~ 100 minutes ( Yao et al ., 1996) The extraordinary stability of the circular P dimer in solution and on the circular chromosome stresses the need for some mechanism to open p for its loading onto and disassembly from DNA The DnaX Clamp Loader Stoichiometry and Organization of Subunits The other processivity proteins within pol ill holoenzyme (t y,6,6' X and 'If) form a complex that perfortns the duty of opening and loading onto DNA for formation of preinitiation complexes on primed DNA at the leading and lagging strands at the replication fork (Kelman and O'Donnell 1995) These proteins foxm the DnaX complex ''clamp loader ," with the subunit stoichiometry [(DnaX) 3, 6 1 6 1 X. 1 '1'1], in whic~ each subunit executes a unique function (Pritchard et al ., 2000 ). The dnaX gene forms both t and 'Y subunits by a translational frameshift that fo11ns a stop codon defining the 'Y subunit C-terminus approximately two-thirds the length of the complete mRNA (Flower and McHenry, 1990 ~ Tsuchihashi and Kornberg 1990 ). Therefore, y and t subunits are identica1 in the first two-thirds of their sequence and structure As a consequence the terminal domain oft acts akin to they sublinit and the llnique C-te1minal domain oft has a specialized function in coordinating the holoenzyme at the replication fork (Dallmann et al ., 2000 ). Due to the requirement of the isubunit for dimerization of the pol III core the DnaX complex associated with pol III holoenzyme most likely has a I

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4 stoichiometry of t 2 Y1 (Pritchard et al 2000 ) E ach of the other subunits ( 8 8 X and 'V ) is present in a single cop y in thi s clamp loader ( Onru s t et al ., 1995 ). For i n vitr o reconstitution of the DnaX clamp loader the x, and \j/ subunits bind the y subunit through 'll'Y interaction ( Glover and McHenry 2000 ) and greatly increase the affinity for the 88 I subunit s for the complex ( Ol s on et al 1995 ) The structure of the E. co l i clamp loader recently also revealed existence of a trimer of y (DnaX ) subunit s ( J eruzalmi et al ., 2001a ) Clamp Loader Subunit Functions The x s ubunit interacts with s ingles tranded DNA binding protein ( SSB ) at the replication fork (Glover and McHenry 1998) This x,-SSB interaction is thought to be involved in a primase-to-polymera s e switch prior to fo1mation of preinitiation complexes ( Yuzhakov et al ., 1999 ) The 'I' subunit ba s no known function other than for1ning a structural bridging contact for x to the clamp loader through \j/ interaction with they subunit ( Glover and McHenry 2 000 ; Xiao et al 1993b ) The function of the x-SSB interaction i s well characterized however there i s no known function for the x and 'V subunits directly in loading the clamp in DNA It i s the 8 s ubunit that bind s to and alone ha s the ability to open the clamp ( Jeruzalmi et al ., 2 001b ; Leu e t al ., 2 000 ) The s urface o f 8 s ubunit which bind s to~ i s concealed by the 8 subunit when the clamp loader is in an '' inactive '' state (Jeruzalmi et al ., 2 001a ). T h e -candy s ubun i ts tran s duc e the energy from ATP binding and hydrol y si s into mechanical work within this clamp loading machine to load the clamp on DNA (Bertram et al ., 2 000 ; Onrust et al ., 1991 ).

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5 All of the subunits of the clamp loader except x, and 'II are members of a diverse s uperfamily of AAA + ( ATPase s Associated with a variety of cellular Activities ) molecular motors (Neuwald et al ., 1999 ). This AAA + superfamily contains conserved I sequences and structures for nucleotide binding and hydrolysis that drive confo1mational change s in these motor proteins In the DnaX clamp loader ATP binding and hydrolysis by the -r ( i e ., through the N-tenninal y-region ) and y s ubunits promote confo11national dynamics that modulate the binding affinity for the p clamp as well as DNA for the loading reaction The 8 and o have been identified as AAA + superfamily members based on sequence and structural homology of o ', and structural homology in the case of o however neither has the ability to bind or hydrolyze ATP, although they are believed to actively participate in the conf or111ational dynamics of the clamp loader for the loading reaction (Jeruzalmi et al 2001a ; Podobnik et al ., 2003 ). The Mechanism of p Clamp Loading F igure 1-1 depicts a simplified schematic of the clamp loading mechanism ( under stud y in this dissertation ) for processive DNA synthesis by polymerase III Initially ATP binds to the clamp loader s-randy subunits Binding of up to three ATP molecules causes conformational changes in the-randy subunits and therefore changes the overall s tructure of the clamp loader Although the exact nature of the s e ATP-dependent confo1mational changes is not yet known there are two major consequences following the confor rnational changes The clamp interaction s urface of the o subunit becomes exposed, and a DNA binding surface on the clamp loader either forms or becomes exposed Interaction of the o subunit with p has been biochemically and structurally characterized showing that o induces a conformational change in the p subunit that t

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6 A. B. PClamp Clamp Loader Complex 5 '' -====----------3'_.. Primed-Template DNA D. 5,:::::' 3 +ATP conformational changes +dNTPs C. s--3 I ;;.,,,E. Polymerase ill Core ADP + Pi 5''-===:::::::::== 3 Figure 1-1 Schematic of the~ clamp loading reaction for processive DNA synthesis A ) clamp and the DnaX clamp loader are present with a primed-template DNA s ubstrate B) Binding of ATP increases the affinity of the clamp loader for both and DNA by conformational change s producing exposure of the o subunit-~ interaction surface and formation of a putative DNA binding surface Interaction of o with induces conformational changes in the clamp that open it at a single interface C ) Primed-template DNA triggers ATP hydrolysis and rapid dissociation of the clamp loader from the loaded clamp D ) DNA Polymerase then binds the same surface of~ that was previously occupied by the clamp loader E ) Incorporation of deoxyribonucleotide triphosphates ( dNTPs) by polymerase then proceeds without polymerase dissociation from the template triggers opening at a s ingle dimer interface ( Jeruzalmi et al ., 2001b ; Turner et al ., 1999 ). The nature of the putative DNA binding surface on the ATP-bound clamp loader remains unknown However it is clear that ATP binding is required for the clamp loader to bind DNA and further that the clamp loader preferentially places at the 3 -end of the primer on a replication-proficient template ( Ason et al 2003 ) B i nding primed-template DNA triggers the clamp loader to hydrolyze ATP ATP hydrolysis causes release of the clamp

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' 7 loader from ~' which then tightly closes on DNA when the o subunit-interaction is removed The discharged clamp loader is then in some inactive state at this point in the cycle, and most likely must undergo some as yet undefined conformational changes in order to continue loading clamp s. Immediate removal of the clamp loader from the loaded clamp is essential since the clamp loader and the polymerase a subunit share the same binding surface on the clamp Once bound to its clamp polymerase can processively replicate thousands of nucleotides without further dissociation from the I template Importance of theE. coli Model Replication System In E. coli, the clamp loader and~ clamp are utilized in DNA replication from initiation at the origin to DNA partitioning of parent and daughter chromosomes upon termination (Katayama, 2001; Levine and Marians 1998 ). Aside from its interaction with pol III core clamp has been found to interact with all five E coli DNA polymerases, as well as several other partner proteins in DNA metabolism such as the MutS UvrB, and DNA ligase (O'Donnell and Lopez de Saro 2001 ; Tang et al ., 1999) The clamp and DnaX clamp loading machine subunits are functionally conserved across evolution to all branches of life suggesting a fundamentally similar mechanism for processivity in all DNA metabolism (Ellison and Stillman, 2001) Increasing structural analyses also reveal that the composition of these clamps and clamp loading machines from s uch organisms as diverse a s a bacteriophage and a human are remarkably similar and that all clamp loaders utilize AAA + motor proteins (Davey et al ., 2002) Using E. co li as a model replication system has been outstandingly valuable for the dete1mination of how complex multi-protein machines interact and work together in a well-regulated efficient manner for DNA replication It is important to dete11nine how the individual

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8 protein subunits of these complex biological machines carry out their own functions and comm unicate with each other for completion of their tasks. Additionally, understanding of these mechanisms will allow for a more complete comprehension of the important DNA metabolic tasks in which they are involved The ~neral Problem Under Study and Research Questions Addressed The general problem under study in this project is the many remaining unknown and questionable aspects of the mechanisms by the clamp loader for clamp loading and the results and interpretations of other investigations Confo1mational changes in the clamp loader are a requirement for modulation of clamp loading However, the kinetics of clamp loader conformational changes are unknown Different nucleotide-dependent clamp loader confo11national species have been identified in proteolytic digestion experiments but how their abundance and dynamics control the loading reaction cycle is not understood mechanistically How do the nucleotide-dependent conformational dynamics within the clamp loader subunits drive this machine for clamp loading? Chapter 4 of this dissertation details an investigation that addresses this question. The studies describe the existence of distinct confor1national species of the clamp loader and the kinetics of their activity in the clamp loading mechanism Chapter 4 further addresses the questions concerning how and ATP affect the kinetics of clamp loader confo11national changes An additional key question asked in chapter 4 is that of the nature of nucleotide binding to the clamp loader and whether there is interdependency between ATP binding and the conformational dynamics Computer modeling was applied to the experimental data for analysis of these important questions Sliding clamps of all organisms studied to date are known to enhance the activity of their respective clamp loader How the clamp affects they complex clamp loader for I

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I 9 promotion of its own loading onto DNA was tested in this research project Although it is understood that p increases the ATP hydrolysis energetics of the clamp loader, it remains unknown what the kinetic mechanism of this enhancement may be By comparison of y complex with the y 3 oo minimal clamp loader in chapter 5 along with experiments presented in chapter 4 this dissertation addresses how p clamp affects the specificity and stability of nucleotide binding by the clamp loader and therefore the kinetics of the confo11national dynamics during the clamp loading reaction for its I enhancement In comparison to the AAA + clamp loading machines of other organisms outlined in chapter 2, the E. coli clamp loader contains two additional subunits (x, and w). It is known that x, and 'V assist in the assembly of other subunits in the clamp loader, and the x subunit has a role in primase-to-polymerase switching at the replication for~ but do they serve any direct function in the clamp loading mechanism by y complex? Chapter 5 of this work outlines a detailed characterization of the y 3 00 minimal clamp loader This minimal complex is missing the x and 'V subunits and is used to assess their roles in the clamp loading mechanism by direct experimental comparison to y complex. Are the x and~, subunits 'AAA + adaptor '' proteins of y complex? Do they provide an example of a novel adaptor function through their effect on the conformational dynamics of the clamp loader? Although not originally hypothesized the findings presented here along with fmdings presented elsewhere suggest that they are Finally what can the details learned here about the E. coli clamp loading machine tell us about other molecular machines driving and regulating complex and diverse cellular tasks in other organisms? This machine is essentially a molecular switch that

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10 catalyzes a fundamental reaction in all fo11ns of life Can the knowledge gained through study pf this clamp loading machine be used for development of novel biological or I hybrid molecular machines? I Conformational Dynamics of the Clamp Loading Machine Solution of the X-ray crystal structures of a minimal y 3 00 clamp loader complex and a C-tenninal truncated y subunit has recently given information revealing some of the structure-function relationships of the DnaX clamp loading machine (Jeruzalmi et al ., 2001a ; Podobnik et al ., 2003) The subcomplex of ( y 3 0 1 0 1) fotms a minimal complex with clamp loading activity similar to the complete DnaX clamp loader (Onrust et al ., 1991) Each of the y 3 o, and o subunits is a AAA + superfamily member, and is composed of three structural domains that form ''C-shaped '' molecules The N-terrninal domains-I and II encompass the conserved nucleotide binding site (in y subunits only) and the third C-ter1ninal domain-III forms an oligomerization region for this heteropentameric complex Domain-II generally fo1ms a mobile '' hinge '' between domains-I and III ATP binding and hydrolysis by they-AAA + motor subunits directly trigger confor111ational changes in these subunits Although the o and o' subunits are AAA + proteins they do not have the ability to bind and hydrolyze ATP, but most likely do undergo conformational changes caused by y subunit conformational movements The structure of the y 3 oo' clamp loader complex showed that the complex was arranged as a heteropentameric ring through the C-terminal oligomerization domain, and that there was extensive contact between each of the five subunits extending '' down '' in asymmetric orientation from the oligomerization domain The nucleotide binding sites were found in the interfaces between the o -1 1, 1 1 -1 2, and r2 y 3 subunits. At least one and possibly two of these interfacial nucleotide binding sites i s thought to be constitutively open whereas

PAGE 26

I 11 the third is deeply buried in an interface This lead to a model describing a sequential series of individual r-subunit confo1mational changes whereupon ATP binding to an open interface caused a conformational change opening the adjacent interface and so on (Jeruzalmi et al., 200 la) where the status of ATP binding to the nucleotide binding sites is communicated between the subunit,s of the clamp loader modulating its affinity for clamp and DNA There are several limitations to the mechanistic inferences made in light of these structures. The clamp loader structure was deter 1nined in the absence of nucleotide Therefore the observed subunit interactions most likely do not represent the functional complex, as it would appear in the cell and the overall conformation was thought to be largely due to a crystal-packing artifact The truncated-r subunit structure was detem1ined in the presence of non-hydrolyzable adenosine 5 -0-(3-thiotriphosphate) (ATP-yS), and crystallized as a tetramer (not as it would appear in a functional complex) The structure contained electron density consistent with A TP-yS molecules bound to two protomers, ADP bound to a third, and no nucleotide in the fourth A TP-yS is sufficient for both clamp and DNA binding by the clamp loader but does not confer clamp loading activity in biochemical assays (Bloom et al ., 1996) Therefore the A TP-yS-bound 'Y subunit structure revealed important features of the conformational change in this motor subunit. However the 'Y subunit was truncated and a substantial portion is missing from its C-terminus Only the conserved AAA + nucleotide binding domains-I and II were present This means that the different conformations observed in each of these structures may be considerably different than the actual structure in solution, or within the cell The experiments presented in this dissertation do not address structure specifically however

PAGE 27

12 these in vitr o studies do directly address the kinetic s of the confor1national dynamics of the cl~mp loader in solution conditions during real-time clamp loading reactions There are significant nucleotide-dependent conformational cha nges in the clamp loader that must be made during the clamp loading reaction for intersubunit communication modulating~ clamp and DNA binding Currently only proteolytic protection assay s (Hingorani and O'Donnell 1998 ) have provided biochemical proof of the nucleotide-dependent conformational change s, however there is no kinetic detail known regarding the nature of these confo1 rnational changes during the clamp loading reaction It has been previously hypothesized that the rate-limiting step of the clamp loading mechanism occurs in the clamp loader away from DNA (Bloom et al., 1996), and could be ADP-release or additional confor1national changes that '' reset '' the clamp loader for resuming the clamp loading cycle The results of this dissertation address the kinetics of the nucleotide-dependent confor1national dynamics of the clamp loader and add detail to what is known about the c lamp loading mechanism initially asking, how do ATP-dependent confonnational dynamics drive and regulate this molecular machine for clamp loading ? The kinetics of nucleotide binding and hydrolysis by the clamp loader DNA binding and~ clamp loading have been determined using several biochemical analyses in this project Together these explorations have lead to a hypothesis based on conformational dynamic s for the internal workings of the clamp loading machine Here it is proposed that the clamp loader exists dominantly in two distinct confo1mational states that are either 'activated '' or ' inactive' for c1amp and DNA binding when in equilibrium with ATP This project has addressed the kinetics of the ATP-dependent

PAGE 28

' 13 confo11national changes separating the two clamp loader species in the clamp loading mechanism and additionally shows that there is complexity in the confo1mational dynamic s between these two major states This added complexity probably arises due to the proposed asymmetry in th e three nucleotide binding sites, and the possibility of having differential affiniti~s for ATP within these sites causing a complex mixture of conformational intermediates within the clamp loader El ucidation of the nature of the additional mixture of confo11national states will require further study but overall this I project reveals kinetically higher populations of two major states modulating the function of the clamp loader in the clamp loading mechanism. Enhancement of the Clamp Loading Machine by its Clamp It has been known for some time that the~ clamp enhances but does not trigger, the ATP hydrolysis activity of the clamp loader in the presence of DNA (Onrust et al 1991) This feature of a sliding clamp enhancing the DNA-dependent ATP hydrolysis activity of its clamp loader is a general characteristic in bacteriophage (Pietroni et al ., 2001) eukaryotic (Yoder and Burgers, 1991 ), and archaeal organisms (Oyama et al ., 2001) ; thus it is a fundamental attribute in all clamp loading mechanisms However the mechanism of how a clamp enhances the activity of its clamp loader has remained a mystery Part of the originally proposed research project was to dete1mine how the E co li~ clamp affected the kinetics of ATP hydrolysis by the clamp loader This dissertation addresses the mechanism of~ clamp enhancement of clamp loader DNA dependent ATP hydrolysis and reveals several important findings interrelated with nucleotide binding and the confo1111ational dynamics of the clamp loader It comes as no surprise that the mechanism of~ clamp enhancement of ATP hydrolysis activity is related to a conversion of the equilibrium between the dynamic confo1mational states of

PAGE 29

14 the clamp loader It is hypothesized that the clamp converts the conformational equilil;>rium of the clamp loader with ATP into a completely '' active '' population (i e ., a I complex of ATP-bound clamp loader and~) An extension of this hypothesis is that the~ clamp affects the affinity and specificity of the clamp loader for ATP effectively 'trapping '' the nucleotide within th e clamp loader promoting f or1nation of the active bound complex poised for loading onto DNA Selective binding and trapping the active clamp loader conformation may also stabilize a putative DNA binding surface on the clamp loader as well An increase in the apparent concentration of this active complex poised for clamp loading would provide a direct mechanism for the increase in ATP hydrolysis turnover rate observed in kinetic assays Due to the considerable evolutionary conservation of clamp loader function across all branches of life (Davey et al ., 2002) the hypothesized mechanism of clamp enhancement of clamp loader activity pre se nted in this dissertation could also apply to clamp loading for processive DNA synthesis in all other organisms as well The x and 'I' Subunits are Required for Optimum Activity of the Clamp Loader The 'X and 'V proteins do not bind DNA do not bind or h ydrolyze ATP, and are dispensable for clamp loading activity At the replication fork in vivo, the 'X subunit is involved in an important interaction with SSB and a primase-to-polymerase hand-off through association with the clamp loader but not directly involved in preinitiation complex formation by the clamp loader A subcomplex of the clamp loader (y 3 00') of which the crystal structure was determined does not contain the 'X and 'V subunits yet maintains nearly the same ATP hydrolysis and clamp loading activity as the clamp loader with 'X and 'V ( Onrust et al ., 1991) Although the structure of the x-'V dipeptide is kno~ it is still a mystery where it binds to the clamp loader For these rea sons the y 3 oo clamp

PAGE 30

' 15 loader has been called the minimal clamp loader complex and in some cases it has been called the clamp loader itself This dissertation encompasses a detailed biochemical characterization of the ( y 3 00 ) minimal clamp loader, and comparison of its activities to the '' y complex' clamp loader This work shows that there are in fact several mechanistic distinctions between the minimal clamp loader and y complex clamp loader These differences have generally revealed that the minimal complex is slightly hindered in its nucleotide binding and I hydrolysis properties and hence clamp loading activity Therefore the X, and \JI subunits are required for optimum clamp loading activity conceivably by strengthening the intersubunit communication within the clamp loading machine Previous studies revealed that the x and \JI subunits did in fact stabilize the clamp loader by increasing the affmity of-randy subunits for oo ( Olson et al ., 1995 ). One of the most obvious deficiencies in minimal complex function is the loss of the ability to stabilize ATP binding in an active complex with clamp This is hypothesized to be the result of possibly slower conformational changes in the minimal complex, and perhaps ''looser '' confo11national dynamic s in the minimal complex leading to more conformational complexity ( i e ., more inactive inte1mediate conformational states ) between the major two states hypothesized for the clamp loader This research show s that the population of inactive confo11nations of the minimal complex is several-fold greater than the population of active conformation in equilibrium with ATP clamp does sustain the ability to enhance the ATP hydrolysis activity of the minimal complex However the results of kinetic analysis of ATP hydrolysis activity reveal that this enhancement is less than that of y complex and that must do more '' work '' to convert the large population of inactive

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16 confo11national states of the minimal complex into the trapped active confo1mation. To date there is no other steadys tate and time-re s olved biochemical analysis comparing y complex to the minimal complex in such a comprehensive manner as that presented in ' thi s di ss ertation, and the result s have allowed a much more clear appreciation for the clamp loading mechani s m catalyzed by the DnaX clamp loader The clamp loader i s a AAA + protein containing machine The x, and \JI subunits are structurally unrelated and much smaller than the AAA + subunits of the clamp loader Since the x, and 'V s ubunits seem to be uniqu e to the E. coli clamp loader and most likely other gram-negative bacteria ( Xiao et al ., 1993a ; Xiao et al ., 1993b ), they provide some unique functions in these prokaryotic clamp loaders compared to those of other organisms They pre s ent a foim of s ubstrate s pecificity to the clamp loader for the SSB coated lagging strand at the replication fork for assistance in preinitiation complex fo1 mation for Okazaki fragment synthesis The s e characteri s tics of the x, and 'I' subunits associate them with a new and growing li s t of AAA + adaptor proteins. AAA + adaptor I proteins provide a s imple and effective way for modulation of AAA + machine function givin g the machine better control over s ub s trate s pecificity and rapid redirection of its s pecific activity ( Dou g an et al ., 2 00 2). Alon g with their function at the replication fork, the x, and 'V subunit s are proposed here to provide increa s ed s tructural s tability and facilitated conformational chan g e s for inter s ubunit communication within the AAA + clamp loadin g machine It i s thu s hypothe s i z ed that th ey are in fact AAA + adaptor protein s and reveal a novel adaptor function for thi s newly defined s ub s et of proteins

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I 17 Application of the Analyses of the Clamp Loading Machine to Other Complex Molecular Machines Like the clamp loader there are many other enzyme complexes that perform complicated biological tasks such as assembly and disassembly of new protein-protein complexes protein-cofactor complexes and protein-DNA complexes Orchestration of I protein-protein and protein-nucleic acid interactions is likely to utilize converging mechanisms to drive the diverse functions of these multi-protein enzyme complexes The intersubunit confo11national communication observeq structurally and functionally by the clamp loaders drive several essential jobs within the large replication complexes in all forms of life The clamp loader catalyzes assembly of a new protein-nucleic acid complex without making or breaking any covalent bonds of either component The energy for this assembly is derived from within the clamp loader by ATP-dependent confo1mational changes that essentially switch the clamp loader between 'on '' and '' off' states. Understanding of subunit recognition and conformational communication driving I similar switching mechanisms in molecular machines is a complicated task in science, but explorations such as that presented in this dissertation for the 'Y complex clamp loader could help accelerate other investigations Not only for ATPand GTP-utilizing energase enzymes but also for transmembrane transport machinery cellular cargo-transport motors, chaperone and protein modulating complexes, proteases, etc The investigations outlined in this dissertation are a highly focused and detailed in vitro examination of the mechanism of a molecular machine s mechanism of switching between active and inactive states for assembly of a protein-DNA complex at the expense of energy from ATP binding and hydro]ysis The details learned here about protein conformational changes and communication of these changes within the machine could

PAGE 33

18 be applied to other solution-based structure-function studies for a broad range of proteins A key result of this and prior work shows that the info11nation for conformational changes in these proteins is stored in the structure of the proteins themselves akin to the storage of protein folding info1mation encoded within secondary and tertiary peptide structure (Fersht and Shakhnovic~ 1998 ). For example the~ clamp is not simply an unintelligent ring-shaped protein dimer but contains structural infonnation for release of a spring like trigger for opening of one of its sturdy interfaces (Ellison and Stillman 2001 ) In the clamp loading machines nucleotide-dependent intersubunit communication induces the extraction of intramolecular-stored information for subunit conformational changes that, in turn can induce changes in partner proteins Molecular motors and switching mechanisms are common in many energase enzymes other than the AAA + proteins that make up the clamp loader under study here For example several molecular motors move along the cellular architecture of microtubule and actin cytoskeletal networks (Mehta et al ., 1999 ; Vale 2003) These motors power transport of organelles and other cargo throughout the cell drive chromosomal segregation in cellular division during mitosis, give cells the capability of motility and power the contraction of muscle fibers that give animals the extraordinary strength needed for a diverse range of movement and let their hearts beat for entire lifetimes ( Vale and Milligan 2000 ). The machines powering these processes are not much unlike the clamp loading machine detailed in this dissertation They each generally contain several domains including nucleotide binding site s, protein-protein and protein nucleic acid binding regions hinge-like domains, multisubunit oligomerization domains and regulatory subunit or cofactor interfaces (Vale and Milligan, 2000) The

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' 19 conformational movements made by several of these nucleotide dependent energases drive their functions give them processive activity and allow them to generate molecular power that is matched on a relative scale only by man s largest macromolecular machines (Baker and Bell 1998 ; Ellison and Stillman 200 I ). Several molecular switches also act I through conformational dynamics allowing protein-protein and protein-nucleic acid interactions in signal transduction cascades (Franco et al ., 2003 ; Phillips et al ., 2003 ), chromatin structural modification (Hakimi et al ., 2002) transcriptional and translational ' regulation ( Mazumder et al ., 2003 ), and ion channels driving action potentials for neuronal signaling The ras-GTPase activating protein provides an example of protein protein contact directly related to the subtinit interfaces within the clamp loader A conserved '' arginine-fmger, residue is positioned between two proteins for catalysis of nucleotide hydrolysis inducing conformational changes that switch the activity status of the protein ( Ahmadian et al ., 1997) Nearly all cellular processes are driven or regulated in some way by phosphorylation / dephosphorylation mechanisms, but the activities of energase molecular motors and switches ( Purich 2001) also appear to have great importance in maintaining cellular function in all organisms. Therefore the significance of investigating the inner workings of a molecular machine such as the E c oli clamp loader extends far beyond the understanding of the mechanisms of DNA metabolism Novel Hybrid Devices Based on the Clamp Loader and Sliding Clamp U nderstanding of the mechanism of the clamp loader molecular machine ; structurally and functionally how protein subunits come together and communicate with each other based on nucleotide binding status can be exploited for development of novel molecular hybrid devices In even simple terms the clamp loader is a molecular switch, activated by ATP binding and inactivated by DNA-dependent hydrolysis of ATP The

PAGE 35

20 inactive and active states of this molecular switch are driven by confo1n1ational changes and fi.ther affected by binding a partner protein ( the clamp). Essentially, the reaction I mechanism under study in this di sse rtation is a molecular switch that entails placement or I removal of (protein) rings on ( nucleic acid ) rod s or hoop s. T his activity produces a topo]ogical connection for another machine (polymerase) to the rod or hoop structure, essentially increasing its concentration at the preferred catalytic site ( template DNA) The clamp loader switching mechanism could be utilized in other systems that require regulated activation/deactivation or in sys tems s uch as memory or communications device s that require a simple ( binary ) on/off signal. By modification of the protein properties of the clamp loader subunits it is feasible that this energase machine / switch could be custom-tailored for guided a sse mbly of proteins ( or other materials ) other than the clamp, onto specific substrates in order to control the putative complex concentration, and activate or deactivate it specifically through NTP binding and hydrolysis ' Off-the-Wall'' Example of the Clamp and Clamp Loader in a Novel Device There is a need for understanding and improvement of industrial catalysis systems (Co ontz et al 2003) Briefl y, for homogenous catalyst s, where the molecular catalyst and reactant are di s persed in the same phase there exists a seemingly simple problem of se paration of the reactants and catalysts from product s The development of het e rogeneou s cataly s t s have provided a so lution by direct separation of catalyst and reactant in different phases but in mo s t cases do not provide the surface area for cost efficient devices One could envision use of a cla1np loading machine to place ( and remove ) a ring-shaped clamp either with the ability to bind a specific catalyst, or containing the catalyst itself onto the reactant This proces s would allow a substantial I

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I 21 increase the heterogeneous catalytic surface area on an insoluble nano-sized rodor hoop shaped reactant. Perhaps the products would easily be separated in solution or gas phases These devices would be '' smart'' devices where the catalytic components contain the infor1nation needed for their assembly and disassembly for disposal or recycling For example, a spring-loaded ~lamp that can be placed and removed at will by a specialized machine with easily regulated activity Improvement of industrial catalysts is only a single (imaginative) example of how the functional knowledge about sliding clamps and I clamp loading machines could be used for design of a novel hybrid device With imagination and lots of money these and other perhaps more significant biologically functional devices could be developed Design of Research Project This research project was developed on two fluorescence-based experimental methodologies for investigation of the kinetics of DNA dependent ATP hydrolysis and clamp loading activities of they complex clamp loader and the y306 minimal clamp loader complex. The two experimental methods utilized were an anisotropy binding assay and an E. coli phosphate binding protein-based MDCC-PBP ATP hydrolysis assay The anisotropy binding assay was employed for investigation of the dynamics of the interactions between the clamp loader and fluorescent-labeled DNA or fluorescent labeled clamp Studies of the DNA dependent ATP hydrolysis kinetics of the clamp loaders were perfo11ned with the MDCC-PBP ATPase assay Both methodologies allowed for steady-state and time-resolved measurements of clamp loading kinetics For the anisotropy binding assay depolarization of the emission from a fluorescent probe reports on the rotational dynamics of the fluorescent-labeled species (Lakowicz,

PAGE 37

22 1999). By monitoring changes in anisotropy one can measure binding dynamics and obserye intermediates that arise in real time during a given reaction This enables elucidation of the kinetics of discrete steps involving interactions with the labeled species in a reacti'on mechanism To study binding kinetics of the clamp loader with clamp as well as the binding kinetics of the clamp loader with DNA in the presence and absence of p clamp on steady-state and pre-steady-state time scales, the fluorescence depolarization of X-rhodamine-labeled DNA ( RhX-DNA ) or pyrene-labeled P clamp (Ppyrene) was measured ATP hydrolysis kinetics were measured with the MDCC-PBP A TPase assay This assay utilizes a site-specific mutant of E. co l i phosphate binding protein (phoS gene product ) that allows covalent attachment of an environmentally sensitive fluorescent probe (N-[2-(l-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide, '' MDCC '') near the phosphate binding cleft (Brune et al ., 1994 ). Binding of inorganic phosphate (Pi) product ( i e. from ATP hydrolysis by the clamp loader ) to the labeled-phosphate binding protein is both rapid and tight and produces a substantial increase in fluorescence intensity readily measurable in a fluorimeter or stopped-flow real-time detection system Computer modeling was perfo1med u si ng two different programs for simulation and fitting of experimental data for investigation of the clamp loader conf onnational change mechanism The KinTekSim program (Barshop et al ., 1983 ; Dang and Frieden 1997 ) was used to te s t model reaction mechani s ms by simulation and visual comparison to the experimental data A fitting program (DynaFit) was used to fit the experimental time course of the reaction with a leasts quare s regre ss ion methodology ( Kuzmic 1996 ).

PAGE 38

I 23 Computer modeling of the reaction kinetics provided a way to validate, and predict several of the mechanistic features deterrnined in this project

PAGE 39

CHAPTER2 LI I'ERA TURE REVIEW DNA Replication in Escherichia coli A complex assembly of DNA replication proteins fo1ms at each replication fork for synthesis of a nascent E s c h e ri c hia c ol; chromosome At the leading edge of the replication fork is a topoisomerase an enzyme that relieves torsional stress created by duplex DNA unwinding Opening up the replication fork and priming the leading and lagging strand templates is a machine called the primosome The primosome is made up of two proteins DNA helicase which unwinds parental DNA and primase, which synthesizes RNA primers, the sites of initiation of replication of the parent templates Stabilizing the single strand template DNA that is exposed upon unwinding by the helicase is single-stranded DNA binding protein Associated with the primosome is a complex of enzymes that synthesize and proofread the nascent DNA This machine is DNA polymerase III holoenzyme a dimer of the synthesizing and proofreading units that coordinate simultaneous high fidelity elongation of the leading strand and Okazaki fragments on the lagging strand of nascent DNA The dimeric DNA polymerase ill is physicall y linked to the parental template leading and lagging strands by ring-shaped sliding clamp processivity proteins s uch that it cannot easily dissociate from the template A clamp loading machine located within dimerized DNA polymerase ill utilizes the energy of ATP binding and hydroly s is to load the circular sliding clamp on the leading strand template and on each Okazaki fragment of the lagging strand template DNA polymera s e I and DNA ligase follow at the tail end of the replication fork, where 2 4 I

PAGE 40

I 25 DNA polymerase I is poised to excise the RNA primers and replace them with DNA and DNA ligase seals the remaining nicks produced between the Okazaki fragments on lagging strand DNA As a whole, this assembly of replication machines has been termed the replisome. The mass of the replisome approaches I million Daltons, and together two replisomes move along the parental chromosome in opposite directions simultaneously replicating the nascent daughter chromosome from a single point of initiation and meeting at the I point of tertnination. This process of replicating of the entire chromosome lasts about 1 hour for a pair of replisomes ; however under some conditions dichotomous replication of the chromosome can occur reducing the time of chromosomal replication to nearly 20 minutes Up to six replisomes at six separate replication forks can be simultaneously synthesizing three daughter chromosomes from the single original parental chromosome during this process DNA Polymerase Ill Holoenzyme At the heart of the replisome is the DNA polymerase III holoenzyme (pol III holoenzyme ). DNA polymerase III was identified as the essential polymerase for E. coli chromosomal replication distinct from the two previously discovered activities of DNA polymerase I and II by analysis of mutants temperature sensitive for DNA synthesis and for cell viability (Gefter et al 1971 ; Hirota et al ., 1972) Pol Ill holoenzyme replicates DNA in a semidiscontinuous manner Pol III holoenzyme is capable of megabase processivity for continuous synthesis of the leading strand, and responsible for controlled 1 2 kilo base Okazaki fragment synthesis of the lagging strand Pol III holoenzyme performs these activities simultaneously at speeds approaching 1 kilobase per second and has an extraordinary fidelity of a single

PAGE 41

26 nucleotide misincorporation in 1 x 10 9 nucleotides polymerized (Kornberg and Baker, 1992 ). Through purification and biochemical characterization of DNA polymerase III and accompanying proteins found to be involved in synthesis elongation the individual subtinits of this machine began to be identified Pol ill holoenzyme consists of 10 distinct subunits : a, e 0 -r 'Y, o o' X, o/ and~ Five of these subunits are present in two copies bringing the total number of pol ill holoenzyme subunits to 20 The core of pol III holoenzyme (pol ill core) was later purified and resolved into individual subunits : a s 0 (McHenry and Crow 1979). The a sub11nit encoded by the dnaE gene (Mr = 140,000 Daltons) was found to be the subunit responsible for catalysis of 5' -3 DNA synthesis activity (Maki et al ., 1985; Spanos et al 1981). The E subt1nit encoded by the dnaQ gene (Mr = 25,000 Daltons) is the 3 -5 exonuclease or proofreading subunit ( Scheue1mann and Echols, 1984) The 0 subunit encoded by the holE gene (Studwell-Vaughan and O'Donnell, 1993) (Mr = 10,000 Daltons) is tightly bound to a and s in pol III core but a 0-specific function has not been identified. The a subunit was further characterized after overexpression and purification of the dnaE gene product (Maki et al ., 1985). Gap-filling replication assays and complementation assays were perfonned to show that the a sub11nit was responsible for the 5 -3 polymerase activity of pol ID holoenzyme and that elevated levels of a in vivo did not increase the amount of pol ill holoenzyme in the cell Within pol III core the a and s subunits are tightly bound and complement each other s function with the overall effect of increasing the fidelity of chromosomal replication. Using purified a and s subunits (Maki and Kornberg, 1987) showed that a

PAGE 42

I 27 complex formed of a-B bad increased 5 '-3 polymerase activity over a subunit alone and highly increased 3 -5 B-exonuclease activity The affmity for DNA of the a sub1Jnit in pol III core resulted in an increase in apparent affinity of e for the 3 '-hydroxyl ter1ninus thus stimulating the exonuclease activity In the cell this proofreading activity during synthesis was found to represent a 5-fold stimulation compared to exonuclease activity uncoupled from synthesis which suggested that the fidelity of DNA replication may be controlled by the relative abundance of the a and B subunits I Polymerase ill core is not processive in DNA replication whereas polymerase ill holoenzyme is highly processive Using a kinetic assay along with product size determination assays it was originally shown that pol III holoenzyme was processive over thousands of nucleotides, and pol III core had distributive activity, capable of synthesizing 10-30 nucleotide stretches only (Fay et al ., 1981) It was found that pol III holoenzyme could be isolated from cells in two distinct f orrns other than pol III core each form catalyzing synthesis of a characteristic length of product DNA (Fay et al ., 1982 ; LaDuca et al 1983) Polymerase Ill was the first of these smaller forms of pol III to be purified At the time pol III was purified and characterized the t subunit was discovered and predicted to dimerize the pol Ill core assemblies for1ning pol III (McHenry 1982) The processivity of pol Ill was increased 6-fold over pol ill core and pol III exhibited greater ability in synthesizing long single stranded templates coated with spermidine a polybasic amine that stabilizes the helical structure of DNA. The Polymerase III* form was also purified and contained all of the same subunits found in pol III holoenzyme except the subunit-dimer The activity of pol III* showed an increase in processivity of at least 20-fold over pol III core (LaDuca et

PAGE 43

28 al .; 1983) When pol III* was reconstituted with the P dimer, the characteristic processivity of the holoenzyme was restored, suggesting P as the major factor in I conferring proce s sivity on pol III holoenzyme I Pol in holoenzyme must place the p dimer on primers formed by primase in order to f ortn preinitiation complexes The formation of these preinitiation complexes requires ATP binding and hydrolysis by pol III holoenzyme, and is absolutely required for initiation of processive synthesis ( Burgers and Kornberg 1982) The P subunit binds directly to the a subunit within pol III holoenzyme (Stukenberg et al 1991), and pol III holoenzyme is dimerized by the 't subunit by direct interaction also with a (Studwell Vaughan and O'Donnell 1991) Through these interactions, pol III holoenzyme simultaneously and processively synthesizes DNA Therefore preinitiation complexes must be formed at least once on the leading strand for its continuous synthesis and mariy times on the lagging strand for Okazaki fragment synthesis Fragmented synthesis of the lagging strand by pol III holoenzyme is in conflict with the level of processivity gained through binding the P processivity sub11nit. The polymerase mu st detach from each completed Okazaki fragment and rapidly cycle to the next preinitiation complex on the lagging strand (O'Donnell 1987) During chromosomal replication, anywhere from 2 000 to 4 000 Okazaki fragments are synthesized at a rate of approximately 1 per second ( Kornberg and Baker 1992 ). Such polymerase cycling activity requires preci s e coordination of protein protein and protein DNA interactions at the replication fork The coordinated efforts of several subunits including processivity proteins within pol III holoenzyme are re s ponsible for this rapid and ordered activity and consequentially give the holoenzyme structural asymmetry I

PAGE 44

I I 29 while the DNA synthesis activity of the polymerase cores remain symmetric between the leading and lagging strands The -r Subunit is the Coordinator of Pol ID Holoenzyme Function and Processivity The dnaX gene codes for both the t and 'Y subunits of pol III holoenzyme. The fulllength dnaX gene product 1 is the t sul, unit (M r= 71,000 Daltons) The 'Y subunit (Mr = l 47 500 Daltons) is created by a 1 translational frameshift adjacent to a hairpin-loop structure in the mRNA that causes a stop codon (UGA) to appear approximately two' thirds the way through the dnaX gene ( Flowei: and McHenry, 1990 ; Tsuchihashi and Kornberg 1990) Therefore the 'Y polypeptide is identical in sequence with the frrst two thirds oft This translational frameshift occurs with about 50% yield oft and 'Y polypeptides Although they are extensively identical the larger t polypeptide has several important functions specific to its C-terminus, which greatly distinguishes it from 'Y (Gao and McHenry 2001) A dimer oft subunits ( t 2 ) acts as the '' glue '' of pol ill holoenzyme, holding it together and providing a structural scaffold for the asymmetric function ofholoenzyme on the leading and lagging strands A tight interaction between t 2 and the a subunit of core occurs through the C-terminus oft bridging two molecules of pol ID core (Kim et al ., 1996b ; Studwell-Vaughan and O'Donnell 1991) The C-ter1ninus oft also binds to the DnaB helicase physically linking the replication machine of pol ill holoenzyme with the primosome machine composed of DnaB and primase The physical and communications link between pol ill holoenzyme and the primosome enhan ces the DNA unwinding activity ofDnaB helicase and connects pol III holoenzyme to the RNA priming activity involved in Okazaki fragment length determination and polymerase cycling on the lagging strand ( Dallmann et al 2000 ; Kim et al 1996a ). A single DnaB

PAGE 45

30 helicase couples with both the leading and lagging strands, and through interaction with the 't-~ridged polymerase may help to keep both strands closely associated with the replication fork I The t subunit protects the p processivity protein from removal off of the leading strand indicating a direct role of 't in the high processivity of the leading strand (Kim et al. 1996c ). This activity is most likely communicated through the close proximity of 't-a, and P a binding sites on the C -terminus of a Within this proximity 't could potentially contact providing direct protection from removal or the 't-O. interaction may cause a rearrangement of the P-core complex preventing removal of p. On the lagging strand 't still protects the p processivity protein but mu.st be able to switch between protective and non-protective states to allow polymerase cycling Two distinct triggers for polymerase cycling on the lagging strand have been identified (Li and Marians 2000). The dynamic action of primase binding the replisome through DnaB helicase and synthesizing a primer is thought to trigger polymerase cycling and collision of the lagging strand polymerase with the 5 -end of the previously synthesized Okazaki fragment also is thought to trigger cycling With respect to the second case a processivity switch requiring the -r subunit wa s identified ( Leu et al ., 2003 ; Wu et al ., 1992) This '' -r processivity switch' is ''off' during processive s ynthesi s, conferring protection of th e P-core interaction with high processivity for completed synthesis of the Okazaki fragment The processivity switch is turned ' on '' only upon incorporation of the fina] dNTP of the Okazaki fragment when the 5 -end of the previously synthesized primer is reached The resulting nick in DNA activates the processivity switch, and through action s of the C-terminus of 't core i s released from P and allowed to cycle to the next preinitiation complex

PAGE 46

' I 31 Pol ill holoenzyme contains the processivity proteins necessary to f or1n preinitiation complexes at newly primed sites on template DNA The t subunit is also part of this complex of processivity proteins that coordinates the protein-protein and protein-DNA interactions required for preinitiation complex formation. Unlike the functions of the t C-terrninus t activity in preinitiation complex formation is located in the N-terminal region of the protein identical to they subunit A DnaX-complex forms within pol III holoenzyme which functions in loading onto primed template DNA. For I I preinitiation complex formatio~ this '' clamp loader '' is discussed in detail below The DnaX complex within holoenzyme contains two t subunits (i.e same two that dimerize core) in a complex with y and the o, o ', x,, & 'I' subunits (stoichiometry : t 2 yoo X'I') (Pritchard et al 2000). The DnaX complex binds and hydrolyzes ATP for1ning preinitiation complexes on leading and lagging strand primers in what may be an ordered activity In their report, (Glover and McHenry 2001) showed that for1nation of the lea ding strand preinitiation complex required ATP binding but not hydrolysis by DnaX complex and that ATP hydrolysis was required for fo11nation of the lagging strand preinitiation complex Further a non-hydrolyzable analogue of ATP (ATPyS) caused removal of the polymerase presumably bound to a lagging-strand template extending the knowledge of pol III holoenzyme as an intrinsically asymmetric dimer with distinguishable leading and lagging strand polymerases. Structure of the p Sliding Clamp Processivity Protein When the X-ray crystal structure of the~ processivity protein was solved it was immediately clear how this homodimer conferred processivity to polymerase ill

PAGE 47

32 holoenzyme (Kong et al. 1992). The~ sliding clamp is composed of two identical crescent-shaped monomers arranged in head-to-tail fashion (Figure 2-1 ) Figure 2-1. The crystal structure of p sliding clamp. Two views of the clamp composed of crescent-shaped protomers (red / yellow ) are shown. A) Front view of P showing continuous P-sheet structure surrounding an inner core of a-helices. Asterisks de11ote the dimer interfaces. Green lines drawn through the left protomer represent imaginary boundaries between structural subdomains. From the top of the left (red) protomer the subdomains are numbered counter clockwise : 1 (N-terminal ), 2 ( middle) and 3 C C-terminal). B) Side view of the p dimer showing the asymmetry of the faces. On the left face is the extended loop region to which y complex and pol ill a. subunit compete for binding. Images were composed using Deep View / Swiss-Pdb Viewer Ver. 3.7, http // www.expasy org/spdbv / with structural coordinates downloaded from the Protein Data Bank (Berman Westbrook et al 2000 ), http // www.rc s b.org/pbd/. (PDB code : 2POL ) The P clamp topologically links the polymerase to template DNA in a non-specific manner providing a tight interaction with DNA while allowing essentially unlimited two dimensional diffusion along the template. Several structural characteristics of p clamp confer its extraordinary ability to provide processivity to DNA polymerase E ach p

PAGE 48

I 33 monomer is formed from three structurally similar domains containing P sheets on the outside and a. helices on the inside As a homodimer the six domains form an inner pore lined with 12 a. helices that are surrounded by an essentially continuous P sheet structure The axis of the a. helices lining the inner pore are almost exactly perpendicular to the local direction of the phosphate backbone of DNA facilitating rapid movement along the duplex For example the a helices traverse the major and minor grooves so as not to fall into them The height of the p clamp is ~ 80 A The width of P clamp( ~ 34 A) would cover about one full turn of duplex B-for1n DNA or ~ 10 base pairs. It was shown biochemically that an 11-base pair primer was required by p to fo1m a productive preinitiation complex synthesized by pol III (Yao et al 2000 ) The diameter of the inner pore is ~ 35 A enough space to encircle either duplex B-fonn DNA ( ~ 20 A) or a RNA DNA hybrid duplex (i e. a RNA-primed DNA template)( ~ 21 A ). The extra space within the inner pore between the clamp and DNA, is expected to be filled by one or two layers of water molecules The two faces of the p clamp are asymmetric (figure 2-lB ). One face has extended loop C-terminal structures and the other face is essentially flat giving the clamp an overall shallow cone-shaped thickness The extended loops on the extended C-terminal face contain the binding sites for both the DnaX clamp loader and the a subunit of pol ill core The presence of overlapping binding sites for both the clamp loader and polymerase determine the proper orientation of the p clamp when loaded onto primed template DNA and result in competition for the binding sites DNA mediates this competition for overlapping binding sites In the absence of DNA p clamp binds the

PAGE 49

34 clamp loader but when is loaded onto DNA, the DNA causes the clamp loader to lose affinity for the clamp and polymerase binds (Naktinis et al 1996 ). Calculation of the electrostatic field generated by the clamp revealed that the protein has an overall negative electrostatic potential. However the surface lining the inner pore has a focused positive electrostatic potential, precisely where the negatively charged DNA molecule would be. The calculated electrostatic features suggest that the inner pore of the clamp has inherent affinity for DNA This electrostatic interaction is most likely lubricated by water molecules allowing a great degree of freedom for linear diffusion while still maintaining some affmity for DNA The dimer interfaces appear as a continuation of the sheet structure from the outer surface of each monomer across a molecular boundary and contribute at least four hydrogen bonds at each interface In addition two distinct sets of interactions between neighboring a -helical side chains stabilize the dimer interface A small hydrophobic core is fo1med in the center of the interface by the packing of Phe and Ile side chains with Ile and Leu side chains between monomers Six potential intermolecular ion pairs between Glu Arg and Lys side chains surround the small hydrophobic core The~ dimer consequentially is very stable with a monomer-dimer dissociation constant of < 60 pM and a half-life on closed-circular DNA of ~ 100 minutes (Yao et al 1996) Considering that the cellular concentration of~ is ~ 250-500 nM, no monomeric would be present in vivo (Kornberg and Baker 1992 ). The number of specific and potentially strong interactions at the dimer interfaces underscores the requirement for a clamp loading machine to load this clamp on DNA

PAGE 50

I 35 The DnaX Clamp Loading Machine Three major parts make up the pol III holoenzyme within the E. c oli replisome. The replicative polymerase III core the DnaX complex that contains the t subunit the cement of pol III holoenzyme and the sliding clamp proce s sivity protein. The DnaX complex has an additional function in polymerase processivity by loading the P clamp I onto primed-template DNA for preinitiation complex formation Originally identified as the y-o complex or y complex (Maki and Kornberg 1988 ; O'Donnell 1987 ) this DnaX complex was shown to load~ onto DNA in an ATP-dependent manner (Onrust et al ., 1991) The t and y subunits are expressed at roughly equal levels in cells due to a translational frameshift in the DnaX gene (Flower and McHenry 1990) Therefore it was expected that a mixed t / y containing DnaX complex would reside in the pol III holoenzyme In fact, several DnaX complexes expressed from an artificial operon were isolated from cells including two forms of mixed t / y complex and a y only complex ( Pritchard et al ., 2000 ). Not-only complex was isolated from cells in that study One of the two isolated forms of mixed t / y complex had the stoichiometry oft 2 1 1 of the DnaX gene products and this forrn was predicted to function within the pol ill holoenzyme based on the requirement of the t subunit dimer for holoenzyme function The separate y 3 only complex and / or the mixed t 1 / y 2 complex were predicted to function separately from holoenzyme for example in DNA repair activity with DNA polymerase II (Bonner et al ., 1992 ). Identification of all five of the subunits of y complex ( Maki and Kornberg 1988) and the genes encoding them (Dong et al ., 1993 ; Xiao et al ., 1993a) lead to in vitro reconstitution and use of this y-only complex as the model~ sliding clamp loader ( Onrust

PAGE 51

36 et al ., 1995) Recently, sequence and structural analysis have identified several subunits of y cqmplex as AAA + superfamily members and y complex as a AAA + molecular machine (Davey et al ., 2002) These studies have provided significant insight into the mechanism of opening the stable ring-shaped dimer structure of p clamp and loading it onto DNA at the speed required by polymerase III catalyzed Okazaki fragment synthesis The 'Y complex contains five sub11nits with the stoichiometry: y 3, 61, 6' 1, X,1, and '1'1 The 'X and 'fl subunits bind a 'Y subunit through 'fl ( Glover and McHenry, 2000), and greatly increase the affinity for the 66 subunits to the complex (Olson et al ., 1995) The 'Y subunit, alone in solution was found to exist in a monomer-tetramer equilibrium, however when fo1med in a complex with 66 became a trimer of y3 (Pritchard et al., 2000). The structural asymmetry derived by single copies of the 6 o', x,, and 'I' subunits of the DnaX clamp loader within holoenzyme imparts structural asymmetry in the holoenzyme and implicates their function with the lagging strand polymerase. The 6 and 6 subunits, products of the holA and holB genes respectively, were purified and characterized for their interactions and function in y complex (Dong et al ., 1993). These subunits b ave similar mass (o: Mr = 38 700 Daltons and 6' : M r= 36 900 Dal tons), and high affinity for each other and were usually co-purified as a consequence Theo subunit binds p through a ''P interaction element '' ( PIE ), while 6' binds tightly to they subunit. During the clamp loading cycle, an '' inactive '' fortn of y complex, the o subunit is thought to occlude o from binding panda conformational change upon binding of ATP toy complex causes exposure of the PIE of the 6 subunit (Naktinis et al 1995) Genetic knockouts of the ho/A or holB genes are not viable revealing that the function of o in clamp loading absolutely requires the o subunit ( Song et al ., 2001 ). Song Pham et I

PAGE 52

I 37 al (2001) also showed that o and o' are required not only for ATP-dependent preinitiation complex fo11nation, but are essential for DNA synthesis elongation as well They participate in the DnaX clamp loader complex function in coupling to DnaB helicase at the replication fork to pol III holoenzyme for leading and lagging strand I synthesis. The 6 subunit alone was found to have the ability to unload P from DNA without any requirement for ATP binding or hydrolysis (Leu et al., 2000) In this study, the approximate quantity of each subunit of y complex was dete11nined from whole cell lysates by protein absorbance measurements and western blot analyses Surprisingly they discovered a ~ 4-fold excess of the o sub11nit present separate from any within y complex. In Okazaki fragment synthesis about 2000 to 4000 clamps are used for complete replication of the lagging strand. However with only about 300 molecules of P present in the cell there is a need to recycle the clamps left behind on the DNA as the lagging strand polymerase cycles between Okazaki fragments To keep up with the pace of Okazaki fragment synthesis p must actively be unloaded at a rate of at least 0 01 s 1 It was found that the quantity of y complex within the cell was limited to ~ 140 molecules by the o subunit Since pol III holoenzyme and any free y complex are present in low quantities and perform several DNA metabolic functions such as replication and repair it was envisioned that the excess o subunit aided in removal of P clamps abandoned by pol ID at the end of each Okazaki fragment. In their investigation ( Leu Hingorani et al ., 2000) also showed that in vitro removal of p from circular DNA substrates by purified o happened at a rate of 0 011 s1 This rate was comparable to measured rates of P unloading by y complex ( 0 015 s 1 ) and pol III holoenzyme ( 0.007 s 1 ) in similar assays

PAGE 53

38 As 1 all of the 8 subunit is expected to be stoichiometrically associated with y complex, the 8 subunit would only regulate the activity of 8 in clamp loading leaving free-8 subunit to recycle leftover B clamps keeping a sufficient level of free s liding clamp available for rapid Okazaki fragment s ynthesis Although not nece s sary for in vitro clamp loading activity y complex contains the x, and 'V subunits The x, ( M r~ 16 600 Daltons ) and 'V (M r~ 15 000 Daltons ) subunits are the products of the ho!C and h o lD gene s, respectively and have important functions adapting the lagging strand polymerase for Okazaki fragment synthesis ( Onrust et al ., 1995 ; Xiao et al 1993a ). As stated above the 'V subunit binds toy bridging the interaction of the x subunit to the complex The X,-'V di peptide forms a rod like structure (Kelman et al ., 1998 ), however it is not known where on the r subunit x-'V binds in the clamp loader complex ( Jeruzalmi et al ., 2001a ). A sub complex of 'Y-x'V or x-'V alone had no independent function in promoting DNA s ynthesis activity by pol III (Xiao et al ., 1993b ). The x, subunit through the 'V structural bridge to the y subunit in the DnaX complex strengthen s DNA polymerase III holoenzyme interactions with the single-stranded DNA binding protein ( SSB ) -coated lagging s trand template facilitating preinitiation complex for1nation and processive synthe s i s elongation Isolated pol III core or pol III ', which are without the x-'V subunits are inhibited in s ynthe s izing D N A on SSB-coated template DNA ( Glover and McHenry 1998 ). Removal of x-'V from pol III holoenzyme results in reduced efficiency of clamp loading activity and an increa s e in salt sensitivity in the physiological range ( 40-150 mM) (Kelman et al ., 1998 ). Inve s tigation of a common point mutation in SSB ( SSB-113 ) revealed that the direct interaction with the x subunit is I

PAGE 54

I 39 located at the C-terminus of SSB and that this interaction is most likely hydrophobic in nature Through this direct x-SSB contact on template DNA the x subunit was thought to provide re s istance to elevated ionic strength for proper clamp loading activity as well as possibly keeping the lagging strand polymera s e localized to the SSB-coated template for greater efficiency in synth~sis elongation The x subunit has been found to be required in a primase to polymerase handoff for Okazaki fragment synthesis A '' three-point switch '' was proposed where the x subunit I I through direct interaction with SSB causes primase to dissociate from a newly fo1med primer allowing initiation complex formation and synthesi s of the Okazaki fragment to proceed (Yuzhakov et al ., 1999 ). Primase (DnaG) interacts distributively with DnaB helicase in fo11nation of the primosome for controlled RNA primer synthesis (Tougu and Marians 1996 ). After synthesizing a primer primase remains attached to the nascent primer and is thought to protect it from exogenous exonuclease activity Primase is also directly attached to SSB and therefore blocks initiation complex formation. Through the x-SSB interaction, but not a direct x-primase interaction, primase is displaced, perhaps through a conformational change in SSB The t-DnaB helicase interaction provides a contact point to hold pol ill holoenzyme to the replication fork while lagging strand polymera s e cycle s ( Kim et al ., 1996a ). Contact of pol ill holoenzyme to the newly primed template and bringing the x subunit through the DnaX clamp loader complex into the vicinity of the SSB-coated lagging strand to displace prima s e through SSB would allow preinitiation complex forrnation for polymerase cycling for rapid lagging strand Okazaki fragment synthesis

PAGE 55

40 The Dna X (tor y) subunits of the clamp loader bind ATP (Tsucbihashi and Komqerg, 1989) In the reconstituted 'Y complex clamp loader interaction with the oo I subunits causes a solution-monomer-tetramer equilibrium of y subunit to form a trimer giving the clamp loader a stoichiometry ofy 3 0 1, o 1 X 1, and '1'1 (Pritchard et al ., 2000) They subunits in isolation (i.e. in monomer-tetramer equilibrium) do not hydrolyze ATP at an appreciable rate with respect to activity needed for replication of the lagging strand However, when reconstituted into a complex with the o o ; x and 'I' subunits or just the o and o subunits the rate of ATP hydrolysis is much higher and further enhanced in the presence of~ to a rate that would be rapid enough for preinitiation complex f or1nation for Okazaki fragment synthesis (i.e. ~ 2 s1 ) (Onrust et al ., 1991). This means that the clamp loader can bind and hydrolyze a maximum of three molecules of ATP to power the clamp loading reaction Since the y / t subunits of the clamp loader are the only subunits which bind and hydrolyze ATP to power the clamp loading reaction they are known as the motor subunits of the clamp loader. The AAA+ Superfamily of Motor Proteins Through sequence analysis and ultimately structural analysis the 'Y o, and o' subunits of the clamp loader have been identified as members of the AAA + superfamily of ATPases (Davey et al ., 2002; Neuwald et al ., 1999) As the name of this superfamily implies : '' ATPases Associated with a variety of cellular Activities '', proteins of this class are involved in many cellular processes Whole genome analyses have indicated that the AAA + class is ancient and has undergone substantial functional divergence prior to the emergence of the major divisions of life thus this class is large and diverse (Neuwald et al ., 1999 ). Proteins of the class generally assist in protein transitions such as remodeling

PAGE 56

I 41 assembly and disassembly AAA + proteins assist not only in protein-protein transitions but also in protein-nucleic acid transitions AAA + proteins including GTPase proteins, which are mechanochemical proteins that transduce the energy of nucleotide hydrolysis into useful work are becoming recognized as a quickly growing group of ' energase '' enzymes (Puri ch, 2001 ) An energase can simply be defined as an enzyme that couples the change in energy of a covalent bonding state directly to changes in non-covalent substrateand product-like I states For AAA + machines such as they complex clamp loader the chemical energy of ATP hydrolysis activity is transduced into mechanical changes in the clamp loader structure. As a AAA+ machine y complex clamp loader could be described as a molecular matchmaker They complex modifies a protein (i e ,~ clamp) such that it becomes enabled for interaction with DNA in such a way that it would not normally interact The AAA + superfamily includes chaperone-like ATPases such as regulatory components of proteases transcriptional regulators vesicle synthesis and fusion proteins, and dynein motor proteins to name a selected few Aside from AAA + proteins of y complex, other important replication proteins are members of the AAA + class For example bacterial DnaA DnaC and RuvB proteins and the subunits of the eukaryotic origin recognition complex, Mcm27 helicase and replication factor-C subunits of the eukaryotic clamp loader (Erzberger et al ., 2002 ; Neuwald et al ., 1999) Increasing investigation of AAA + proteins reveal that adaptor proteins may modulate their functions As a consequence, a growing list of these structurally unrelated adaptor proteins are being identified for many of the known AAA + proteins (Dougan et

PAGE 57

42 al .~ 2002) Generally smaller than their AAA + partner AAA + adaptor proteins provide a simpl~ and effective way to modulate the function of the AAA + machine Adaptor proteins have been found to give the AAA + protein better control over substrate ' specificity allowing quick response to changing local conditions and redirection of AAA + activity The 'Y complex has two proteins x and \JI that are not related in sequence or structure to the other AAA + class subunits of the clamp loader Questions concerning the function of x and 'ti in the clamp loading reaction by 'Y complex are of major importance in this dissertation and it is attractive to propose that they are in fact adaptor proteins in complex with the AAA + clamp loading machine As described above the x subunit, through \JI, has important interactions with SSB at the replication fork for pre initiation complex formation. As will be discussed in the results of this dissertation, x and \JI are also important for activity beyond interaction with SSB at the replication fork, and are important for optimal activity of the clamp loading machine itself This dissertation explores the structural and conformational stability provided to 'Y complex by the x and \JI proteins and therefore may reveal a novel example of how adaptor proteins modulate the activity of their partner AAA + machines The X-ray crystal structures of a minimal y 3 66' clamp loader and a x-'t' complex have been solved but it still remains a mystery as to where the x-'tl dipeptide binds to the 'Y subunit of the clamp loader The limited examples of AAA + -adaptor protein complexes have shown that the adaptor proteins generally bind to the least conserved N-ter1ninal domain of their AAA + partner This fact may provide direction for further structural analysis of the complete 'Y complex including the x and \JI proteins I

PAGE 58

43 Figure 2-2 The crystal structure of a AAA + motor protein. The y 1 243 crystal structure (Podobnik Weitze et al 2003 ) is shown here as a representative AAA + motor protein. This structure was truncated at the C-te1minus and only the AAA + domains I (red) and II (yellow ) are shown A molecule of ATPyS bound to the NTP-binding site is shown in space-filling representation The conserved structural and functional features of AAA + motors are indicated : Sensor-2 (blue ), P-loop (green ), and Sensor-I ( pink ). Also marked are the conserved Arg ( y-215 ) of sensor-2 and Tor ( y-155 ) and (SRC ) '' Arginine (y 169 ) -finger '' of sensor I A C ys-coordinated Zn ++ atom is also present in Domain I Images were composed using Deep View / Swiss-Pdb Viewer Ver 3 7 http // www expasy org/spdbv / with structural coordinates downloaded from the Protein Data Bank (Berman Westbrook et al 2000) http // www rcsb or g/ pbd /. (PDB code : lNJF ) AAA + proteins have several common structural features that define their function ( Figure 2 -2 ). All AAA + proteins have an approximately 220 amino acid conserved structural ATPase core although seque11ce is not always conserved ( Jenizalmi et al. 2001b ; Neuwald et al 1999 ). The AAA + conserved structure has two domains ( I and

PAGE 59

' ' 44 II), and a third oligomerization domain (ill) is common in multimeric complexes of AAA + protein subunits AAA + proteins generally contain the phosphate-loop (P-loop ) type NTP-binding site having the Walker-A and Walker-B motifs that are common in ATPase and GTPase proteins The Walker-A motif (GVxxxGKT) is involved in the phosphate binding of ATP ( or GTP) and the Walker-B motif ( DExx ) is involved in metal ( Mg J binding and catalysis ( Walker et al 1982) The P-loop a p-fold provides a platform upon which other AAA + structural motifs I are mounted Above the P-loop in domain-II is the sensor-2 motif containing a highly conserved Arg residue ( Arg-215 in the r subunit ). This Arg residue is predicted to interact with the~and r-phosphate of bound ATP ( Podobnik et al. 2003 ). Domain II containing the sensor-2 motif is typically a hinge-like domain around which movements in domains-I and III are made In domain-I flanking the P-loop from the underside is the sensor-I motif The sensor-1 motif contains the highly conserved Ser-Arg-Cys ( SRC ) sequence that is predicted to interact with they-phosphate of the bound ATP through hydrogen bonding The SRC sequence includes an '' arginine-finger ''. This arginine-fmger is thought to stimulate catalysis of ATP hydrolysi s by analogy to the similar conserved switch II region of GTPase activating protein s ( Ahmadian et al ., 1997) In an oligomeric AAA + complex such a s y complex, the SRC-sensor-1 motif of one AAA + s ubunit interact s with the nucleotide-bindin g site of a neighboring AAA + subunit and is implicated in interfacial s timulation of ATP hydrolysis ( Jenizalmi et al ., 2 001a ; Podobnik et al ., 2003 ). The X-ray crystal structure of they complex AAA + clamp-loading machine is described below It has five AAA + proteins that make up three part s of the clamp loading machine :

PAGE 60

45 three y-ATPase-motor subunits the 6 subunit harboring the interaction element (~IE) and the 6 '' stator '', upon which movement of the other subunits is supported I X-ray Crystal Structure of the Clamp Loading Machine The '.X-ray crystal structure of a reco nstituted minimal clamp loader complex ( y 3 66 ') was solved at a resolution of 3 0 A using multiwavelength anomalous diffraction methodology (Jeruzalmi et al ., 2001a) The s ubunit s crystallized as a heteropentameric circle having the stoichiometry (6 1-'Y 3 -01) ( Figure 2-3 ). While the amino acid composition of the 6 and o subunit s was complete all three 'Y subunits were truncated by 57 amino acids from protease-labile C-terminii. All subunits showed a three domain 'C '' shape previously observed in the structure of the o' subunit alone ( Guenther et al ., 1997) giving the complex an overall form analogous to five fingers extending down from the palm of a hand Both of the AAA + domains I and II were observed for all five subunits This was a surprising observation for the 6 subunit, as it shared only 7-8 % sequence identity with other AAA + proteins including those within the clamp loader All of the ATPase motor 'Y subunits crystallized in nucleotide free form, and the 6 and 6 subunits were already known not to bind ATP The X-ray cry s tal structure of a truncated for1n of the 'Y s ubunit ( y 1 243 ) including the AAA + domains I and II only was later solved in the presence of nucleotide (Podobnik et al., 2003 ) and will be used for the discussion of the nucleotide binding site s and consequences of nucleotide binding the y motor subunit The C-terminal domain III of all five subunits of the clamp loader forms a tight circular collar at the '' top '' of the complex (Figure 2-3B ). The subunits are arranged where the 6 and 6 subunits surround the three 'Y subunits I

PAGE 61

Figure 2-3 The crystal structure of the r 3 00 clamp loader The clamp loader forms a circular heteropentameric structure, each subunit having three domains (I II and ill ). A) ' Front '' -view: The point of view is directly into the '' open '' interface between the o' (white ) and o (blue) subunits The three y subunits (different shades of red ) are present behind o and o Structural domains are indicated : Domain-ill (the C-ter1ninal '' collar '' ) Domain-II ( '' hinge '' ) and Domain-I (N-te11ninal ' functional ') B) The clamp loadeJi has been tilted forward 90 for a slab-view of the 'top '' showing the tight pentameric '' collar '' of the terminal domain-III only Subunit nomenclature is indicated C) The clamp loader has been tilted back 90 from the view in ( A ), and domains-II and ill have oeen dimmed for the 'bottom '' view The te1minal domain-I of each subtinit is colored for emphasis on their asymmetrical disposition The NTP binding sites of the y subunits are occupied with sulfate molecules from crystallization ( space-filling representation) and the Zn atoms (yellow ) bound too ', 'Y 1 'Y2 and 'Y 3 sub11nits are shown The ~-interaction element (~IE) and the conserved hydrophobic-wedge residues of the PIE (o-Leu-73 and-Phe74) are shown (green ). Images were composed using Deep View / Swiss-Pdb Viewer Ver 3 7 http // www expasy org/spdbv /, with structural coordinates downloaded from the Protein Data Bank (Berman Westbrook et al 2000 ), http // www rcsb org/pbd/ (PDB code : IJR3)

PAGE 62

47 Hydrophobic interactions within a helical packing arrangement strengthen subunit interactions in this oligomerization domain Only the C-terminal domain of the o subunit appeared 'loose '' within this oligomerization domain and was proposed to have consequences for movement of the o sub11nit within the complex The N-terrninal domains of the five subunits containing the AAA + structural domains-I and II are highly asymmetric with respect to each other extending down from the C-te1 mioal collar (Figure2-3C) Each N-te11ninal domain is increasingly splayed out

PAGE 63

48 with respect to the circular C-tertninal collar forming an '' open '' a-shape Splaying-out of N-ter1ninal domains in this s tructure re s ulted in con s iderable exposure of the 6 subunit ' and thu s the PIE Ba s ed on biochemical s tudie s, a nucleotide-free structure would be ' predicted to be more '' closed ' where the 6 subunit-~ IE would be obstructed by the 6' subunit The authors s peculate that the observed s tructure of the clamp loader may be an artifact of the crystal packing forces but overall would reflect that of an '' open '' or activated complex The nucleotide binding sites of the 'Y subunits are buried within the subunit interfaces as is common with other oligomeric AAA + ATPa s es The AAA + domains are arranged such that domains-I and II of one subunit cradle domain-I of the neighboring subunit providing a mechanism for confo11r1ational communication based on nucleotide binding status Three interfacial nucleotide binding sites are between the 6 -r1 r 1 -'Y 2, and r 2 -r 3 subunits The N-terminal AAA + domains of the 'Y subunits were highly asymmetric in the complex, resulting in asymmetry in these interfacial nucleotide binding sites. The 6 -y 1 interface and y 2 ..ry 3 interface were open in this nucleotide-free structure while the r1y 2 interface had the most extensi v e contact and the ref ore appeared inaccessible to nucleotide Structure of the Nucleotide Binding Site and the Proposed Conformational Change of the 'Y Subunit The X-ra y cry s tal s tructure of C -tetminal truncated y 1 243 wa s solved in the presence of A TPyS to a resolution of 2. 2 A u s ing multi wa v elength anomalous dispersion methodology ( Podobnik et al ., 2003 ) The resulting structure had four 'Y subunits arranged in the asymmetric cry s tal unit One of these 'Y subunit s had complete electron density corresponding to A TPyS in the nucleotide binding s ite and another was missing I

PAGE 64

49 electron density corresponding to the y-phosphate, and therefore was taken as the structure of ADP-bound y Two other y subunits in the asymmetric 11nit had no nucleotide bound Slow hydrolysis of ATPyS through the crystallization procedure was thought to result in producing the ADP-bound y This structure allowed identification of several as yet unknown features of nucleotide binding and the conf 01 tnational changes induced due to nucleotide binding The results have important implications for the mechanism of communication between the AAA + domains within the clamp loader and therefore the overall clamp loading mechanism The structure showed that the nucleotide was bound in the expected position in the cleft between domains-I and II formed by the Walker-AP-loop (see figure 2-2) The adenine ring was located in a non-polar environment and f 01111ed hydrogen bonds from the N-6 position with the main chain carbonyl groups ofVal-19 and Val-49 This feature provides a mechanism for distinguishing ATP from GTP by the 'Y subunit Both the 2 and 3 -hydroxyl oxygens of the ribose moiety were hydrogen-bonded to the carbonyl oxygen of Ala-7 of the N-terminal helix extending from domain I Three residues of the conserved Walker-AP-loop motif coordinate the~and yphosphates of the nucleotide The Lys-51 residue fo1ms a salt-bridge and Thr-52 and Ser-53 form hydrogen bonds to the phosphate groups from the sensorI loop Comparison of the nucleotide-bound y 1 24 3 structure with the nucleotide-empty y 3 oo structure reveals that the 'Y subunit domain-II a.-helix containing the sensor-2 motif blocked the region corresponding to the position of the ribose ring This observation was extended to all of the empty nucleotide-binding sites in the y 3 oo structure Thus it was predicted that nucleotide binding to a given 'Y subunit alters the position of domain-I with

PAGE 65

50 respect to domain-III by confortnational change The reorientation of domain-I would open p the nucleotide binding site from a collapsed state, and clearly affect the positions I of the sensor-I and sensor-2 motifs within the complex Also rotation of the sensor-2 region containing the conserved Arg-215 residue occurs upon nucleotide binding and provides space for the phosphate groups of the nucleotide The sensor-I motif does not move with respect to the P-loop, and therefore follows the general movement with the tenninal domain-I upon nucleotide binding Conformational changes induced by nucleotide binding to the P-loop region are directly linked to movement of the catalytic '' arginine-finger '' of sensor-I thereby coupling (i e communicating) changes in nucleotide binding status to the neighboring subunits of the complex In the y 3 oo structure the sensorI motif containing the arginine-finger was either too far away from the neighboring nucleotide binding site that it is thought to stimulate in the case of the 6 -y 1 interface or in steric-clash with the neighboring nucleotide binding site as in the case of the Y 1 -Y 2 interface Therefore, conformational changes in individual subunits must be made to correctly position the sensorI arginine-fmger with the neighboring nucleotide for catalysis These confo1mational changes that occur upon ATP binding not only bring the subunits into proper orientation for ATP hydrolysis, but more importantly cause exposure of the o subunit from occlusion by the o stator so that it can bind~ clamp '' Crude' modeling of the clamp bound to the clamp loader structure was perfo1med using information from the 6-~ structural interaction (described in detail below ). This '' simplistic' docking of dimeric with the clamp loader revealed that the orientation of the 6-~ interaction would cause the clamp to make contact with all subunits

PAGE 66

51 in the clamp loader (Jeruzalmi et al., 2001a) One of the key points addressed in this dissertation is the question of how affects the clamp loader in the clamp loading mechanism The multipoint contact suggested by the modeling of~ bound to the clamp loader provides a structural basis for several conclusions concerning individual subunit confo1mational changes an(.i how may ''trap'' an ATP-bound '' activated'' clamp loader. This contact may potentially explain how the clamp loader stabilizes the open ring while a single dimer interface remains open (see below) until a primer template junction is located. The exact nature of how the clamp loader binds to DNA is not yet fully understood A cysteine-coordinated zinc module is present in the o and all three y subunits of the complex (figure 2-3) and may provide a interaction zone for duplex DNA, however, the function of the zinc modules in the clamp loader complex has not been investigated Calculation of the surface electrostatic potential of the clamp loader structure invites some interesting speculation on the nature of where DNA binds and stimulates the activity of the clamp loader Two extensive regions of positive electrostatic potential were observed on the structure. Both were seen on the N-ter1ninal asymmetric ''bottom'' side of the clamp loader that is predicted to interact with the 13 clamp One of these regions was on the o subunit, adjacent to the PIE presumably where DNA would thread through the P clamp when bound too The other region was observed as a partially continuous belt that tracks along the outer surface of the N-terminal domains of the y subunits Each of these regions of positive electrostatic potential predict that when DNA is encircled within the P clamp under the clamp loader single-stranded DNA could be positioned to interact with the outer regions of the ATP binding domains causing

PAGE 67

52 additional r,earrangement of sensor-2 and sensor I motifs stimulating hydrolysis of ATP The r~quirements for formation of the DNA binding site ofy complex are further explored in the results of this dissertation The x-ray crystal structure of the o subunit bound to a P monomer was also solved, and provides a possible mechanism of clamp opening by the 8 subunit as well as how the p clamp may interact with y complex through binding the o subunit-J3IE X-Ray Crystal Structure of the 6 Subunit Bound to a p Monomer and the Mechanism for Opening the p Sliding Clamp The X-ray crystal structures of two complexes between the o subunit and a mutant monomeric fo1rr1 of P (P 1 ) were solved by multi wavelength anomalous diffraction (Jeruzalmi et al 2001b) Attempts to crystallize the 6 subunit with wild-type P 2 were unsuccessful and therefore a mutant p that remained monomeric due to mutations within the dimer interface was used ( Stewart et al 2001) Theo : P 1 structures either contained full-length o sub11nit composed of all three domains observed in the r complex structure including the AAA + regio~ or truncated 8 subunit (amino acids 1 140) 8 1 1 4 0 containing the N-te1minal domain I only Both complexes of 6 : P 1 and o 1 1 40 : p 1 crystallized in 1 : 1 complexes at resolutions of 2 9 A and 2.5 A respectively (Figure 2-4) Examination of both structures revealed a mechanism by which P clamp is opened upon the 6 subunit inducing or trapping a confor1national change in p such that a stable dimeric interface cannot be f 01med Some possibilities for ring opening were that o forcibly breaks the dimer interface, but due to the fact that ATP is not required by 6 to open p this was not very probable Another possibility was that the p clamp could oscillate between conformations and the o subunit could have high affinity for a confortnation inconsistent with ring closure I

PAGE 68

53 Figure 2-4 The crystal structure of the o-P 1 complex The full structure of the~ subunit ( blue ) is shown bound to ~ 1 monomer Domains (I IT and III ) of o are indicated The subdomains of ~ 1 are colored : N-te1111ina.1 subdomain-1 ( white ), middle subdomain-2 (pink), and C-terminal subdomain-3 (red ). The conserved hydrophobic-wedge ~IE (green ) Leu73 and Phe74 of o subunit are marked The a-helix of the p subunit involved in the '' spring-loaded '' confo1mational change is colored yellow Images were composed using DeepView / Swiss-Pdb Viewer Ver 3 7 http // www expasy org / spdbv / with structural coordinates downloaded from the Protein Data Bank {Bet man, Westbrook et al ), http // www rcsb org / pbd/ (PDB code : IJQJ) This also was not a very probable mechanism due simply to the high stability of the dimer alone on circular DNA A third possibility was that the o subunit could induce and stabilize a conformation in p that is inconsistent with ring closure The results from analysis of these structures are consistent with the third possibility and it was proposed that o subunit binding induces a conformational change in the p clamp that results in

PAGE 69

' I 54 straightening of a crescents haped monomer such that a single dimer interface is opened to allow DNA passage into the clamp. The ~IE is on the N-terminal domain of the o subunit and consi s tent with earlier biochemical studies makes contact with the C-te1minal-extended-loop face of the~ subunit (Naktini s et al ., 1995 ). Only one o subunit bound ~ 1 in the full length o: ~ 1 structure and it was observed that domains II and III of the o subunit impeded binding of a second molecule of o to p 1 In the 0 1 14 0 : Pi structure showed that the ~IE forms a ' hydrophobic wedge that binds to a hydrophobic pocket on the ~ 1 s ubunit between subdomains 2 and 3 ( of the p subunit ) (Figure 2-4 ) Subdomains 2 and 3 are the middle and C-tenninal structural domains of the p protomer (Kong et al ., 1992 ). The Met-71, Leu-73 and Phe-74 residues fotm the hydrophobic wedge on the o subunit T he hydrophobic pocket on p, into which this wedge is inserted, is fotmed by Leu-177, Pro242 Val-247 Val-361 and Met-362 of p The ~IE hydrophobic wedge of the o subunit is highly conserved among all bacterial o subunit sequences examined Likewise the residue s fotming the hydrophobic pocket on p are also highly conserved across evolution, and therefore found on sliding clamps of bacteriophage eukaryotes and archaea ( see below ). Conservation of this interaction between the clamp loader and sliding clamp suggests a common mechani s m for clamp opening in all known organisms. These structures have revealed that interaction of o with requires conf 011national chan g es in~ that weaken the dimer interface and importantly o doe s not interact directly with the dimer interface The structure of the P dimer interface consists of a hydrophobic core surrounded by ionic interaction s ( Kong et al ., 199 2). An a-helix participating in these interactions at the dimer interface is distorted in the wild-type dimeric p clamp such

PAGE 70

55 that additional hydrophobic residues contained on this distorted helix do not extend into the int~rface The hydrophobic pocket binding site where the a-subunit-PIE binds is near j a 5 -re sidue loop adjacent to the distorted a-helix in p. The binding of o is thought to ' impose strain on the distorted helix that causes its relaxation into a more ideal a-helical confo11nation This results in extension of the hydrophobic residues from the previously distorted portion of the a-helix directly into the dimer interface Removal of dimer interface stability was proposed to trigger release of spring-like tension in the crescent shaped p monomer such that it becomes straightened. Thus the clamp is not simply an inert ring but contains structura l inf or1nation for its own opening The crescent shape of the p subunit to which 6 bound was calculated to straighten by 12 at the unopened interface and by 5 at the opened interface by comparison with the structure of dimeric p Comparison of the o subunit in the 6 : P1 structure with the structure of the o subunit from the y 3 66 structure revealed that the N -terminal domain of 6 is in a considerably different position when bound top An a helix (a4) containing the PIE, in domain-I I translates by ~ 5 5 A and rotates ~ 45 with respect to the rest of domain-I and the P IE or hydrophobic wedge is extended further away from the a4-helix for contact with p The '' reverse'' conforrr1ational change in 6, from the P-bound fortn to the P:free fortn of results in exposure of specific hydrophobic residues that are important for interaction with the 6 stator of the clamp loader Although the two structures were solved with mutants of p that do not for1n dimers the above conclusions regarding release of spring-like tension in opening an interface were consistent with molecular dynamics simulations performed on the dimeric p clamp In these simulations a P protomer was simply released from dimeric constraints I

PAGE 71

56 dete1mined from the original ~ 2 clamp structure. The protomer followed a trajectory in the simulation that resulted in straightening of its crescent-shape such that it superimposed well with the structure detertnined from the 6-bound monomer DNA structural requirements for p clamp loading by y complex The clamp loading mechanism and preinitiation complex formation for processive synthesis by pol III holoenzyme has been studied on several different DNA substrates including synthetic poly-dT-primed poly-dA-templates as well as circular phage genomes in different replicative fortns ( j e singleor multi-pnmed nicked-duplex etc .). It was shown that short synthetic primed-template DNA (pt DNA) substrates ( i e an 80-105 nucleotide template with a 30 nucleotide primer) whose sequence was based on the Ml 3 phage where sufficient for proficient elongation by pol III core in the presence of~ clamp and reconstituted r complex, and the ref ore for the study of the clamp loading mechanism (Bloom et al ., 1996) Using these short synthetic pt DNA substrates in experiments along side circular phage genome substrates containing supercoiled or relaxed-structures the DNA structural requirements for~ clamp loading where investigated (Ason et al ., 2000 ; Ason et al ., 2003 ; Yao et al ., 2000 ) For preinitiation complex fo11nation at the replication fork it was known that RNA primers were required The exact structural nature of these primers were unknown except that a 3 -hydroxyl group was annealed at the single-stranded DNA (ss DNA ) I double-stranded RNNDNA ( ds RNA/DNA ) junction where the preinitiation complex was f or1ned In experiments with supercoiled DNA and closed relaxed circular DNA substrates it was shown that reconstituted r complex did not require a 3 -end to load ~ as it was able to load the clamp onto supercoiled DNA but not closed relaxed

PAGE 72

57 circular DNA (Yao et al., 2000) It was hypothesized that supercoiled DNA may produce unwound regions due to superhelical tension and at these ds RNA/DNA / ss DNA I junctions 'Y complex could load p fo11ning the preinitiation complex Using circular single-stranded Ml3 phage DNA primed with synthetic DNA primers differing in specific structural features such as length and unannealed 15nucleotide '' flaps , at 5 or 3 -ends, or both 5 and 3 ends it was shown that 'Y complex can load p onto a wide range of these substrates ( Yao et al 2000) The minimal primer length requirement was found to be 10 base pair s, consistent with the size of the inner pore width of p, and with the known fact that in vivo primers fo1med by primase are ~ 10-12 nucleotides in length (Kitani et al ., 1985 ~ Kong et al., 1992). Yao, Leu et al. also showed that 'Y complex could load p onto primers with 5 and/or 3 15-nucleotide-long unannealed flaps supporting their conclusion that y complex only requires a ds DNA / ss DNA junction to load the clamp By placing proteinor DNA secondary-structural blocks on or within the primer in replication assays the polarity and primer spatial requirement for clamp loading were determined Using physical (i e protein) blocks tightly bound at the annealed primer 5' or 3 '-ends it was shown that y complex exhibits polarity in loading the clamp specifically at the 3 '-end of the primer This result was expected due to the fact that polymerase synthesis elongation extends from the primer 3 -end in the 5' to 3' direction Placement of DNA secondary structural elements at different distances upstream from the 3 -end of the primer, allowed detern1ination of the spatial requirement of the primer for P-bound y complex and P-bound pol III core It was shown that with y complex 14-16 base pairs of primer were needed to load the clamp and 20-22 base pairs were needed for t

PAGE 73

I 58 to bind pol III core This result indicated that 'Y complex interacts directly with 4 to 6 base pairs of the primer and that the clamp is pushed back an additional ~ 6 base pairs when pol III core binds~ at the ds DNA / ss DNA junction ( Yao et al ., 2000) Utilizing fluorescence-based steady-state and pre-steady-state kinetic anisotropy assays with a X-Rhodamille-labeled short synthetic pt DNA in solution, a DNA-triggered switch in y complex was discovered (Ason et al ., 2000) This DNA-triggered switch caused a change in affinity of 'Y complex for DNA, and was found to be pt DNAand I ATP hydrolysis-dependent Initially in stead:Y-state assays it was observed that y complex had higher affmity for ss DNA than pt DNA Even more surprising, was that the 5 -single-stranded template overhang of the pt DNA was the same sequence and length as the ss DNA substrate examined in these experiments However pre-steady state real-time assays revealed DNA binding kinetics suggesting that y complex bound transiently with high affinity to the pt DNA substrate Use of non-hydrolyzable ATPyS in both steady-state and pre-steady-state assays with pt DNA substrates removed this dynamic switch in y complex affinity for DNA resulting in a high affinity state only. It was therefore proposed that ATP hydrolysis by y complex was required for cycling between high and low affinity states and that the low affinity state may have been an ADP-bound y complex With ss DNA substrates y complex with~ maintained high affmity for the DNA hydrolyzed ATP but did not load~ (Bloom et al 1996 ~ Turner et al ., 1999 ). The dynamic interaction with pt DNA and the DNA triggered switch in y complex accomplishes two major goals in the clamp loading reaction The primer-template ds DNA I s s DNA junction provides the proper site for clamp loading and preinitiation

PAGE 74

59 complex foIIDation, and modulates the DNA binding affmity of the clamp loader, so as not to allow y complex competition with pol III core bound to at previously formed initiation complexes The nature of the low affmity state of y complex remains a mystery ~d this dissertation addresses several possibilities [and questions] including the dynamics of conformational changes defming different states of y complex with respect to the clamp loading reaction A recent investigation addressed the possibility that the pt DNA-triggered modulation of y complex provides a dynamic mechanism for recognition of appropriate sites for clamp specific for proficient DNA synthesis This was accomplished by investigation of the DNA structural features required to trigger y complex into the low affinity state and release~ (Ason et al., 2003) In fluorescence-based steady-state and pre-steady-state anisotropy assays in solution, y complex binding and clamp loading were studied with elongation-proficient DNA substrates (i.e synthetic template DNA primed on its 3 -end or center ) or elongation-deficient DNA substrates (i e synthetic template I DNA primed such that a blunt duplex was formed at the 5 -end of the template, or unprimed-ss DNA ) In steady-state titrations of r complex to the elongation-proficient or deficient DNA substrates results similar to the previous investigation defining the DNA triggered switch were observed The y complex showed high affinity for elongation deficient DNA substrates but did not appear to bind well to the elongation-proficient DNA substrates Pre-steady-state assays revealed biphasic '' up-down '-kinetics (i.e ., a rapid rise to a defined peak followed by a decay phase into steady-state DNA binding activity ), representing transient high affinity of y complex for the elongation-proficient DNA substrates as well as characteristic changes in anisotropy for the clamp loading I

PAGE 75

I 60 reaction when~ was present For elongation-deficient DNA substrates, pre-steady-state binding kinetics were monophasic suggesting a simple high affinity bimolecular equilibrium whether~ was present or not Additionally a correlated DNA-binding and ATP hydrolysis assay with y complex and elongation-proficient pt DNA clearly showed that a binary complex consisting of y complex with pt DNA transiently formed just prior to DNA-triggered ATP hydrolysis and release of y complex without any further binding Overall the results show that y complex uses a dynamic mechanism driven by ATP I binding and hydrolysis for targeting~ clamp only to DNA that can serve as a template for synthesis elongation by pol Ill core and prevent further interaction with polymerase bound ~ Mechanism of the p Clamp Loading Reaction Cycle by 'Y Complex Formation of a processive pol III holoenzyme requires the formation of preinitiation complexes on the leading and lagging strands at the replication fork. The ring shaped sliding clamp must be opened and loaded onto the circular E. c oli chromosome in order to fo1m a topological link between pol III and DNA The clamp loading machine perfo11ns this task through dynamic protein-protein and protein-DNA interactions Some 26 years ago Sue Wickner presented a model for the mechanism of DNA elongation catalyzed by DNA polymerase III that still generally holds true today ( Wickner 1976 ). Wickner s model described a mechanism where primed-template DNA was activated by the ATP-dependent transfer of '' DNA elongation factor I '' (the sliding clamp) by '' DNA elongation factor III '' (the clamp loader ), to which DNA polymerase III then bound in an ATP-independent manner followed by DNA elongation Combined biochemical analysis and structural details have in recent times given us a detailed yet

PAGE 76

61 still incomplete view of the clamp loading mechanism required for processive synthesis by pol III holoenzyme The 'Y complex ( 1 3 8 8' X,,'V) or a minimum complex of five subunits ('Y 3, 8 8') is the energase c omplex that transduces the energy from ATP binding and hydrolysis into mechanical work to load~ onto DNA (Figure 2-5) Within this machine, the three 'Y subt1nits serve as the motor subunits, o has the ~interaction element (~IE ) and o is the '' stator '' upon which movement of the other subunits is thought to be supported In general terr11s ATP binding and hydrolysis by the 'Y sub11nits promote confoimational changes that modulate the dynamic protein-protein and protein-DNA interactions involved in clamp loading To work at the speed and efficiency required at the replication fork within the cell there must be precise communication between all subunits of the clamp loading machine to power and regulate its activity Initially ATP binding to the 'Y subunits, but not hydrolysis, promotes changes in the clamp loader that modulate the binding affmity for the clamp and DNA, and therefore powers most of the steps in the clamp loading reaction (Bertram et al., 1998 ; Hingoraoi and O'Donnell, 1998 ). Asymmetry of the interfacial nucleotide binding sites in the clamp loader structure provided a model for activation upon ATP binding for clamp loading Theo -"{1 and -y 2 -"{ 3 interfacial nucleotide binding sites were open in the structure ad could provide space for the first one or two molecules of ATP to bind. These conformational changes could also result in the opening of the y 1 -"{ 2 ATP binding site, and further '' splaying-out '' of the 'Y subunits from the '' backbone '' of the o' stator, and ultimately exposure of the o subunit ~IE (Figure 2-5B) This model would bring the I

PAGE 77

62 maximum number of ATP molecules needed for exposure of the o sub11nit ~IE to three in '' open '' y complex. 1-complex D. + A. I I primed-template DNA B. E. ATP Pi + ATP C. ~-clamp + .__p Pol III core F. ATP Figure 2-5 A schematic cartoon of the basic steps in the clamp loading reaction and initiation complex formation The cartoon of the clamp loader in '' closed '' and '' open '' states is based on the y 3 oo crystal structure and they complex subunits are colored as in figure 2-3 A) ATP binding to they subunits B ) ATP-dependent conformational changes occur within the clamp loader exposing the o subunit ~IE (green ). C) clamp binding to the clamp loader through the ~IE induces a conformational change in ~ opening the clamp at a single interface. D) The ~-bound clamp loader complex binds pt DNA and positions the clamp at the ds DNA / ss DNA junction at the 3 -OH of the primer E) pt DNA triggers ATP hydrolysis, the release of inorganic phosphate ( Pi ), and dissociation of ADP-bound clamp loader from loaded P clamp F) Polymerase III core ( a B and 9 subunits) can then bind and commence processive elongation of the template Originally it was shown that ATP binding caused a conformational change in y complex that resulted in characteristic proteolytic cleavage of the exposed o subunit and that this change did not require p (Hingorani and O'Donnell 1998 ; Naktinis et al ., 1995) The

PAGE 78

' ' 63 interaction of the exposed 6 subunit ~IE with~ then induce s ot stabilizes confor1national changes in that result in ring opening at a single dimer interface (Figure 2-5C ) The kinetics of ATP binding and the dynamics of the conformational changes within the clamp loader are a major component of this dissertation and will be discussed further in chapters 4 and 5 ATP binding toy complex is also required for interaction with DNA in the clamp loading reaction ( figure 2-5D ) ( Bloom et al 1996 ; Stukenberg and O Donnell 1995 ) I Studies described in thi s dis s ertation show that none of the individual subunits of the clamp loader have significant DNA binding affinity and it is hypothesized here and that the interaction of all clamp loader subunits form the DNA binding site With a clamp bound and opened, the clamp loader then binds DNA at a ds DNA / ss DNA junction specifically on elongation proficient primed-template DNA ( Ason et al ., 2003 ; Yao et al 2000 ) The DNA binding kinetics and positioning of~ at the 3 -hydroxyl of the primer happen in less than I 00 ms with an approximate bimolecular binding constant on the order of2 0 4 0 x 10 8 M 1 s 1 just prior to the hydrolysis of one molecule of ATP for each y sub11nit in the clamp loader ( Ason et al. 2000 ) Primed-template DNA triggers ATP hydrolysis and dissociation of the clamp loader ( Figure 2-5 E) Earlier investigations of ATP hydrolysi s by the clamp loader showed that mutations of the conserved lysine in the y s ubunit Walker-A motif of domain-I to alanin e or arginine ( K51A or K51R ), resulted in complete abrogation of ATP hydrolysis activity by the clamp loader ( Xiao et al 1995 ) Our laboratory has also shown that these mutations of the con s erved lysine in y complex abolished both J3 and DNA binding in s olution ( unpublished ), therefore it is likely that the lysine-mutants

PAGE 79

64 cannot bind ATP Use of non-hydrolysable ATPyS allowed 'Y complex to bind P and DNA , consistent with the notion that nucleotide binding powers most of the steps in the I clamp loading reaction In these reactions, the comple x of P-bound 'Y complex remained in a dynaniic steady-state interaction with DNA for periods of time extending to 90 minutes (Bertram et al ., 1998 ; Bloom et al ., 1996 ). These investigations showed that although ATP binding can bring ~-bound 'Y complex to DNA ; ATP hydrolysis was I absolutely nece s sary for release of 'Y complex and completion of the clamp loading reaction The pre-steady state ATP hydrolysis a s sa y s s howed that the ATPs were hydrolyzed at a minimal rate of 20-34 s 1 (Bertram et al ., 2000 ). Closure of the~ clamp on DNA was found not to require any further energy ( Turner et al ., 1999), therefore ATP hydrolysis by the clamp loader is not needed to close onto DNA The 'Y complex crystal structure predicted a mechanism wherein ATP h y drolysis drives final conformational changes in which p is pushed off the 6 subunit ~IE as 6 returns to its occluded interaction with the rigid 6 subunit This is consistent with an earlier investigation that showed ATP I hydrolysis was required for release of 'Y complex from on DNA leaving the clamp behind (Bloom et al. 1996 ). Dissociation of 'Y complex from the loaded clamp is essential and would provide space for the interaction of~ with polymerase ill which is known to bind the same surface of~ that 'Y comple x had occupied (Figure 2-SF) Pre steadys tate analysis of DNA binding activity and ATP hydrolysis have shown that a s ingle 'turnover '' of the clamp loading reaction takes approximately 300 ms before entering a steady-state that continues at a rate of ~ 2-2.5 s 1 ( Ason et al ., 2000 ). Ultimatel y ATP hydrolysis would then allow the clamp loading machine to reset itself for continued clamp loading most likely through nucleotide exchange and

PAGE 80

65 conformational changes The y complex released from the clamp loaded on DNA is presumably in some inactive '' closed '' state that would have to release ADP and become able to bind ATP again. ADP release could cause conformational changes that may transiently bring y complex through some state in which there is no nucleotide bound ATP binding would then cause the conf 011national changes that reactivate y complex for another clamp loading reaction In previous pre-steady-state ATP hydrolysis assays there was a '' pause '' in hydrolysis activity observed during the last 200-250 ms of the first I turnover of ATP during transition into the steady-state ( Ason et al ., 2000) Given the way these assays were initiated (i e ., with preincubation of y complex with ATP and~ before mixing with pt DNA), during this transition, the rate-limiting step of the reaction cycle was believed to occur The exact nature of the rate-limiting step of the clamp loading reaction is not yet known Possibilities include ADP-release ATP binding, confo11national dynamics within y complex, and clamp binding The steady-state and pre-steady-state kinetics of DNA binding and ATP hydrolysis by the clamp loader are further studied in this dissertation and reveal some novel kinetic features that predict that confo1mational changes within y complex are most likely the rate-limiting step in the reaction and regulate the clamp loading mechanism Mutations of the p Clamp, and 'Y Complex o' and 'Y Subunits: Effects on the Clamp Loading Mechanism Mutations in the clamp as well as in subunits of y complex have been studied to provide greater detail for the interactions modulating this clamp loading mechanism The steady-state and pre-steady-state kinetics of DNA binding and ATP hydrolysis activities were investigated using a~ clamp with mutations at two positions within the dimer interface The leucine to alanine (L273A L108A) mutations were thought to

PAGE 81

66 weaken the dimer interfaces, although this mutant was still able to f or1n dimers in solution (Bertram et al. 1998) The~ interface mutants were found to bind 'Y complex I and DNA similar to wild-type ~, but they remained bound to DNA in a ternary complex. The DNA binding assay results appeared similar to assays perfonned in the presence of non-hydrolyzable A TPyS where the ~-bound clamp loader appeared '' stuck '' in a dynamic steady-state interaction with DNA, without loading~ In correlated pre-steady-state DNA binding and ATPase assays the interface mutants bound to DNA and hydrolyzed ATP with nearly identical kinetics as wild-type-~ However the clamp loader steady state A TPase activity with the mutants was significantly decreased, coincident with extremely slow dissociation of y complex from mutant~ on DNA (Ason et al., 2000) The results confirmed the idea that the clamp loader must cycle off of~ that is loaded onto DNA and undergo a rate-limiting step that is separated from the DNA-bound state in order to continue loading clamps as previously suggested by Bloom et al. 1996 The results of these investigations also showed that although~ does not require any energy from ATP hydrolysis to close on DNA ATP hydrolysis is tightly coupled to release of the clamp on DNA It seemed interesting that mutations within the dimer interface so greatly affected the steady-state DNA binding and ATP hydrolysis activities of the clamp loader Now with better knowledge of the interaction between the clamp loader-6 subunit and clamp it is more clear how interface mutations would cause these effects in the clamp loader Perhaps the confo1mational change of~ cannot be properly promoted by the 6 subunit-~IE interaction disallowing loading onto DNA This possibility could result in a decrease in the interaction of y complex with DNA, and reduced DNA stimulated ATP hydrolysis activity I

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I I 67 To gain further insight into the AAA + machine activity of 'Y complex, mutations in the sensor-I ( SRC) motif were made and steady-state ATP binding hydrolysis, and clamp loading activities were examined (Johnson and O'Donnell 2003 ). In one of these mutations arginine-158 of the o stator subunit (o -R158A ) SRC motif was changed to alanine. This mutation removed the '' arginine-fmger '' from the o -y1 nucleotide binding interface The results showed that the reconstituted o -R158A-y complex still bound approximately three molecules of ATP, but had reduced ATP hydrolysis activity and I I diminished ability to load in DNA synthesis assays. These results implied that the ' arginine-finger '' contributed from the o subunit to the o -'Y 1 nucleotide binding site was in fact catalytic in nature and therefore this '' stator '' subunit was coupled with the motor function of the clamp loader, as well as performing as the motor support subunit Recent results from our laboratory have shown that this reconstituted o -RI 58A-y complex mutant has little ability to bind~ as well as significantly reduced DNA binding and clamp loading abilities ( Snyder A K ., personal communication ). This is consistent with the reduced DNA synthesis activity observed in the original study T he '' arginine-finger '' was removed from they subunits to create a second clamp loader mutant (Johnson and O'Donnell 2003 ). The y-R169A mutation removed the '' arginine-finger '' from the y 1 -y 2 and y 2 -y 3 nucleotide binding interfaces thus onl y affected two nucleotide binding sites in the clamp loader This reconstituted 'Y 3 -Rl69A complex s till maintained the ability to bind three molecules of ATP and ~ but had no ATP hydrolysis activity clamp loading activity and was unable to stimulate DNA synthesis Our laboratory ha s since shown that this y 3 -R169A-complex can bind pat a level near that of wild-type y complex in an ATP-dependent manner in solution, but

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68 showed no DNA binding activity in steady-state and pre-steady-state solution-based assay~ ( Snyder A K ., personal communication). Johnson and O'Donnell discussed the 8 and y-subunit mutants in terms of an ordered ATP binding and hydrolysis mechanism ' where ATP molecules are hydrolyzed in the reverse order than they were bound requiring proper positioning of the ' arginine-fingers '' for catalysis Our laboratory's results indicate that there is more than just a disruption in catalysis of ATP hydrolysis in these mutants Taken together all of the mutation analysis results exemplify the need for precise subunit conformational communication within the clamp loading machine for binding and also for formation of the putative DNA binding surface on the clamp loader. The Clamp Loading Machine Within Polymerase ill Holoenzyme Most of the biochemical analyses and all of the structural analyses of the clamp loader and clamp loading mechanism have been perfortned with reconstituted y complex How do these results apply to the clamp loader within pol III holoenzyme? The clamp loader within holoenzyme contains both the t and y subunits encoded by the dnaX gene In the t subunit the region of the DnaX protein structure extending from the C-terminus of the y subunit region into the C-ter1ninal domain oft contains many pro line residues and is thought to be unstructured and therefore a highly flexible region (O'Donnell et al 2001) This flexible region divides the DnaX (t ) protein into they-AAA + motor region, and the t-C-terminal replisome interaction region Within holoenzyme, there are at least two -r subunits and a single y subunit (Figure 2-6 ). The structural arrangement of the DnaX subunits of the clamp loader within holoenzyme is likely to be [81 t 2 ,Y 1 ,8 1, X,1,\jf 1 ]or [8 1, Y 1, t 2, 8 1 ,X.1 \jf 1 ] The X and \jf subunits only bind to they subunit through 'V and the 8 subunit also has been shown to bind only to they subunit with high affinity ( Glover and McHenry 2000 ). I

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Fork direction Pol III core Leading strand 69 t Lagging strand Previous~thesized OkaviJci ent Figure 2-6 Architecture of the polymerase ill holoenzyme at the replication fork organized by the DnaX clamp loading machine The leadingand lagging strands are unwound by hexameric DnaB helicase (green), shown bound to the SSB-coated (yellow) lagging strand. The DnaX clamp loader containin g two t subunits (violet) (0'1,'Y1, -r 2, 0 1 x,1 \j/1) is connected within the holoenzyme through the flexible linker regions of the -r subunit C-tenninal domains that also dimerize pol III core (a e and 9), and further cement the holoenzyme through contact with DnaB The clamp loader x, and v subunits are not shown for clarity Two P rings (red) clamp the pol ill cores to the templates and a third is shown bound to the clamp loader In this schematic primase would bind and synthesize a new RNA-primer on the lagging strand Then it is predicted that the clamp loader would ''swing'' over to the nascent primer displace primase through the x, subunit, and load a fresh clamp f or111ing a preinitiation complex When the lagging-strand polymerase reaches the 5 end of the previously synthesized Okazaki fragment it would then release p and cycle to the newly for1ned preinitiation complex and commence elongation of DNA The two -r subunits cement the array of proteins at the replication fork by dimerizing the core polymerases in the holoenzyme binding to the replicative DnaB helicase, and protecting the subunit from removal when it is attached to the polymerase a subunit

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70 Through the flexible linker region, these functions of the t subunits are connected to the y-AAA + motor region of the clamp loader In this way it is envisioned that the clamp loader associated with the holoenzyme in vi1 1 0 has a swinging range of motion on the flexible tether loading at least one clamp on the leading strand and providing continuous clamp loading activity on ~e lagging strand for Okazaki fragment synthesis (O'Donnell et al., 2001 ). Duplex DNA is anti-parallel and therefore the core polymerases of the holoenzyme I must work in some asymmetric fashion to simultaneously complete replication of the chromosome (Figure 2-6) The single 'Y subunit of the DnaX clamp loader and consequential asymmetric distribution of the o o x.,and \j/ subunits gives the holoenzyme structural asymmetry. The 'Y subunit is the only clamp loader subunit to which 'V and therefore x, adaptor proteins bind This provides the holoenzyme clamp loader both structural and functional asymmetry through t-DnaB helicase contact, and the x, SSB interactions necessary for lagging strand synthesis Recently it was proposed that the asymmetry in the clamp loader provides asymmetry to the core polymerases through nucleotide binding to the clamp loader (Glover and McHenry, 2001) Use of non-hydrolyzable ATPyS was sufficient to allow the DnaX complex to load a clamp for only one polymerase pre s umably the leading strand polymerase ATP hydrolysis was absolutely required for clamp loading on the '' lagging strand '' polymerase in their assays and subsequent addition of ATPyS was able to disassociate this '' lagging strand '' polymerase Whether the nucleotide binding status of the clamp loader or simply the structural asymmetry of the clamp loader provide distinct leading and lagging strand functions to the core polymerases will require further

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71 investigation Overall the structurally and functionally asymmetric holoenzyme has the means to stay continuously linked to the leading strand while perfo11ning discontinuous synthesis on the lagging strand at the replication fork by the distinct structural and t functional characteristics of the DnaX clamp loader Polymerase processivity is required for DNA replication in all free living cells, and nature has provided a consistent mechanism to provide the means for polymerase processivity though conserved evolution of these replication proteins across all branches of life Clamps and Clamp Loaders of Bacteriophage, Eukaryotic, and Archaeal Organisms The combined biochemical and structural investigations of the E. coli clamp and clamp loader have yielded many great details into the mechanism of processivity clamp assembly on DNA These intimate details have allowed complementary studies of clamps and clamp loaders of other organisms to thrive due to the evolutionary conservation of these processivity proteins across all branches of life and suggest a common mechanism of clamp loading in all life fo11ns It is well known that sliding clamps contact polymerases with DNA for processive synthesis of complete chromosomes or replicons in a fundamentally similar way (Ellison and Stillman 2001; Hingorani and O'Donnell 2000) Now structural evidence is revealing the extraordinary functional similarities in clamps and clamp loading machines of viral replicases prokaryotes eukaryotes and archaea for processive DNA elongation (O'Donnell et al 2001) All organisms through these evolutionary branches of ljfe appear to utilize AAA + proteins as the energase of their clamp loading machines (Table 2-1 ) Of those organisms studied each clamp loader appears to or is predicted to utilize a AAA + clamp loading machine like r complex that contains several A TPase motor subunits a clamp interacting subunit, and a supporting stator subunit (Davey et al ., 2002 ). This

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I 72 conservation of sequence and, to a greater extent, structural similarities of these AAA + proteins illustrate the evol u tionary importance of these clamp loading machines Table 2-1. Clamps and clamp loaders through evolution Evolutionary Processivity AAA + Function a Branch Proteins p r otein Prdkaryotic clamp Bacteriophage (viral) Eukaryotic Archaeal y I "' o' 0 X 'V gp44 gp62 PCNA RFC-2 d RFC-3 RFC-4 RFC-5 RFC-1 PCNA RFC-S RFC-L + + + + +(?) + + + + + + + -motor stator -clampinteracting subunitc AAA+ adaptor -AAA+ adaptor clamp -motor ( stator?) -clamp interacting subunitc clamp motor -motor motor -stator -clamp interacting subunitc -clamp motor ( stator?) motor I clamp interacting subunit a by analogy to the y complex clamp loader b gp gene product Some Characterized Organisms Escherichia colz.e Bacillus subtilus BC1cillus staerothermophilus Aquif ex aeolicusf Thermus thermophilusf T4 phage T7 phage RB69 phage Homo sapiens Saccharomyces cerev is ia e Schizosaccharomyces pombe Cae norhabditis e legans Drosophila melanogaster Arabidopsis thaliana Oryza sativa (rice) Pyrococcus furiosus Archaeoglobus fulgidus Sulfolobus solfataricus Methanobacterium thermoautotrophicum Lili c has conserved hydrophobic wedge residues of clamp interaction element d nomenclature used is for yeast replication factor C (RFC) e clamp loader structure known 'The11nophile bacteria

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73 Bacteriophage T4 Clamp and Clamp loader When infecting bacteria the bacteriophage has the necessary components to fo1m I its own replication machinery The replisome of T4 bacteriophage contains gene product (gp) gp43 polymerase gp41 / gp59 helicase and its accessory factor respectively gp61 primase and gp3 2 single-stranded DNA binding protein T4 also provides the processivity clamp gp45 and clamp loader complex gp44 / 62 to complete the replisome ( Salinas and Benkovic 2 000 ) The gp45 clamp interacts with gp43 polymerase through a C-te1 rninal region of the polymera s e This interaction between the clamp and polymerase is highly conserved across evolution (Ellison and Stillman 200 I ) and the structure of a T4 clamp bound to polymerase on DNA has been modeled based on the nearly identical RB69 bacteriophage sliding clamp bound to polymerase (Alley et al ., 2001 ; Shamoo and Steitz, 1999) To date these are the only structural views of a polymerase bound to its clamp on DNA The gp45 processivity protein is a ring-shaped clamp formed of six similar structural domains like clamp but unlike dimeric ~ is a trimeric clamp Although there is less than 10 % sequence identity between gp45 and~ ( and PCNA see below), the clamp structure is amazingly similar ( Jeruzalmi et al 2002) The crystal structure of gp45 shows that its propertie s are similar to clamp and PCNA ( Moarefi et al. 2000) The gp45 clamp has an overall negative charged ~ -s heet outer surface surrounding an helical inner pore of positive electrostatic potential The dimensions of the gp45 clamp show an inner pore diameter of ~ 35 A, and a width of ~ 25 A however the clamp has an overall triangular topology compared to the nearly circular structures clamp and PCNA Consistent with data that s how gp45 clamp is the least stable of the known sliding clamps on DNA (Y ao et al ., 1996 ), the gp45 clrunp i s predicted to be slightly open in solution

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I I 74 through a pucker which gives it a '' lock-washer '' -type structure ( Alley et al., 1999). It is still possible that the clamp loader must open the clamp further for the loading reaction (Alley et al ., 2000 ). The T 4 clamp loader is a pentameric complex of the gp44 and gp62 subunits with a subunit stiochiometry of four gp44 subunit s to one gp62 subunit (Jarvis et al ., 1989 ). The T4 gp44 / 62 complex binds and loads gp45 onto DNA in an ATP-dependent manner and requires hydrolysis of bound ATP In earlier work, it was shown that four molecules of I I ATP were hydrolyzed by gp44 / 62 during forrnation of the T4 holoenzyme and that some step following ATP hydrolysis was rate limiting in the reaction ( Sexton et al ., 1998 ). A highly detailed investigation using fluorescent resonance energy transfer techniques with fluorescent-labeled gp45 clamp combined the pre-steady-state kinetic analysis of clamp opening and closing by the gp44 / 62 clamp loader with analysis of binding and hydrolysi s of ATP ( Alley et al ., 2000 ). This investigation showed that hydrolysis of ATP wa s required to open the gp45 clamp and then additional hydrolysis was required to load the clamp on DNA A more recent study on the ATP hydrolysis activity of gp44 / 62 in clamp loading conflicted with previous work and showed that ATP binding alone is sufficient for gp44 / 62 to bind gp45 and at least one ATP is required to complete the loading reaction ( Pietroni et al ., 2001 ). This study also revealed that ATP hydrolysis was not required to open the gp45 clamp and that the rate-limiting step in the loading reaction wa s either ADP release from gp44 / 6 2, or a confor1national change before clamp loading This investigation is more consistent with the detailed study of the 'Y complex in clamp loading

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75 The gp44 subunits of the complex have homology with AAA + proteins and contain the Wfllker-A P-loop and sensor-I SRC motifs needed for ATP binding and hydrolysis I ( Davey et al ., 2002 ; Neuwald et al ., 1999) By analogy toy complex the gp44 subunits are expected to be the motor subunits of the T4 clamp loader that undergo ATPdependent conformational changes that drive gp45 binding and interaction with DNA in the loading reaction The gp62 subunit shares no sequence homology to other AAA+ subunits and does not bind or hydrolyze ATP Until its structure is known it can only be predicted that gp62 is a AAA + homologue and the subunit that interacts with the gp45 clamp during the clamp loading reaction (i e like the o subunit of y complex) There is no support-like stator subunit in the gp44 / 62 complex as all of the gp44 '' motor '' subunits are identical However, even in y complex the o stator subunit has been shown to be directly involved in the catalytic motor function of the clamp loader through precise positioning of its SRC motif by conformational changes (Johnson and O'Donnell 2003) One of the gp44 subunits of the T4 clamp loader may work in a similar manner and it I may tum out that this putative distinct gp44 subunit may also be incapable of ATP hydrolysis like o' in y complex These predictions await further biochemical and structural analyses of the T4 clamp loader Eukaryotic PCNA Clamp and Replication Factor-C Clamp Loader The essential replication machinery of eukaryotic organisms was originally identified by investigations using the SV 40 DNA virus as a model system SV 40 uses host cell machinery in replication of its DNA and requires only its own '' T-antigen'' for replication initiation and DNA helicase activity (Waga and Stillman 1994 ) Processivity proteins proliferating cell nuclear antigen (PCNA) and replication factor-C (RFC) were I

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76 identified as the sliding clamp and clamp loader, respectively for the replicative polymerases o I E (Waga and Stillman, 1998) Like the 'Y complex clamp loader RFC has DNA-dependent ATP hydrolysis activity that is further stimulated by the presence of the PCNA clamp (Lee et al ., 1991) RFC is also directly involved in a primase to polymerase switch similar to 'Y compl~x and influences the length of primer synthesis by polymerase a primase ( Mossi et al ., 2000 ; Tsurimoto and Stillman, 1991) The PCNA sliding clamp structure was solved from budding yeast S. ce revisia e I (Krishna et al 1994) Once again, even though sequence homology between the bacteriophage T4 clamp prokaryotic p clamp and PCNA was low, the structure of PCNA is nearly identical to the other clamps (Moarefi et al 2000) Unlike the p clamp but like T4 gp45 clamp, PCNA found to be trimeric All three clamps have six structurally similar subdomains, dimensions (i.e. the PCNA inner pore is -35 A) and similar electrostatic characteristics on their structurally conserved outer P-sheets and inner a-helices The overall topology of the PCNA trimer is more hexameric or circular than gp45 remains closed in solution, and is more stable on DNA than gp45 but not as stable as dimeric P clamp (Yao et al ., 1996). RFC has been extensively studied from yeast and human origin and has been identified as a heteropentameric complex consisting of one large subunit and four small subunits of different mass The subunit nomenclature is : RFC(yeast/human) : 1 / p140 2 / p37 3 / p36 4 / p40 and 5 / p38. For simplicity the yeast RFC nomenclature will be used here. Deletion analysis of RFC subunits showed that the C-terminus of each subunit was required for forrnation of the pentameric complex, and that this complex must fo11n to allow DNA stimulated ATP hydrolysis activity (Uhlmann et al ., 1997) It was also

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77 shown that none of the individual subunits alone had DNA binding activity (Uhlmann et al ., 1996) Using surface plasmon resonance ( SPR ) and filter magnetic-bead binding I I assays it was shown that ATP mediated all interactions of RFC with PCNA and DNA ( Gomes ruid Burgers 200 I ) This study also showed that ATP binding but not hydrolysis was required for the interaction with DNA and that use of non-hydrolyzable ATPyS resulted in a '' stuck' complex of PCNA-bound RFC on DNA These results are consistent with complementary investigations performed with y complex The DNA structural requirements for PCNA loading by RFC were also tested and showed that RFC could load PCNA on ds DNA I ss DNA junctions not requiring an annealed 3 -OH similar to the DNA structural requirement for y complex catalyzed clamp loading activity Recent studies with yeast RFC (Schmidt et al 200 la ), as well as previous investigations with human RFC (Cai et al 1998) identified the subunits involved in ATP hydrolysis activity and those essential for DNA recognition by mutational analysis of the I conserved Walker-A lysine residue in each subunit The results of these mutational analyses revealed that RFC subunits 2 3, and 4 were required for ATP binding and hydrolysis in efficient PCNA loading Mutations in RFC-1 the large subunit had no effect on PCNA binding and loading abilities RFC-5 has modified Walker-A and B sequences suggesting that it does not bind or hydrolyze ATP similar to the 6' subunit of y complex (Cai et al ., 1998 ; Schmidt et al. 2001a ). Consequentially these Walker-A mutations ofRFC-5 did not effect PCNA binding and loading activities As in the T 4 clamp loading reaction cycle the use of ATP by RFC in PCNA loading is also not fully understood. This may be due to limitations of the SPR and filter I

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I I I 78 binding analyses that were being perfonned along side steady~state ATP hydrolysis analysis of these reactions Despite these limitations a pathway of multiple stepwise ATP binding events is thought to be required for RFC loading PCNA onto DNA, and that this proposed reaction resembles the well studied E. coli clamp loading reaction pathway ( Gomes et al ., 2001 ) In t];le yeast PCNA loading reaction, 2 ATP molecules initially bind RFC, PCNA then binds RFC, and RFC gains affinity for a third ATP Conforrnational changes within RFC are most likely taking place as each ATP binds an I I RFC subunit Finally RFC-PCNA binds pt DNA, and apparently one more molecule of ATP binds RFC perhaps to the RFC-1 subunit Upon interaction with pt DNA PCNA is released, coincident with hydrolysis of up to all four bound ATP molecules It remains unknown how many ATP molecules are used for this reaction The four small subunits of RFC (2 -5 ) form a functional core and are used in other cellular processes The large RFC-1 subunit swaps with another protein, specific for functions such as replication tertnination (Kouprina et al 1994 ), and cell cycle checkpoint control (Green et al. 2000) Differing the nature of ATP binding affmities and putative confo11national changes in the RFC-2-5 core may provide differential reaction mechanisms for these diverse functions. Perhaps, the first two ATP molecules bind and cause conforrnationa] changes that allow a presentation of general binding site for PCNA and DNA only when RFC-1 is present, or form and expose different binding sites when another protein takes the place of RFC-1 Then other A TPs could bind and produce confo1rnational changes specific for each function Another possibility could be that when a specific protein such as PCNA binds, it alters the affinity for ATP at additional sites in RFC specific for in this case clamp loading

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79 All five RFC subunits are AAA + homologues and are related in sequence to the y and o subunits of y complex (Neuwald et al ., 1999 ; ODonnell et al ., 1993) The heteropentameric stiochiometry of the RFC s ubunits predicts that this eukaryotic clamp loader wi11 have structural similarity to r complex The RFC-1 subunit is the least conserved of the RFC subt1n.its and its specificity in clamp load.ing with the RFC-2-5 core and d.ispensability from the RFC-2-5 core in functions other than clamp loading suggest that it may be the PCNA clamp interacting subunit of the RFC complex by analogy to the o subunit ofy complex ( see Table 2-1 ). Natural modifications of the Walker A and B nucleotide-binding domain of the RFC 5 subunit suggest that this subunit may not bind or hydrolyze ATP but it still has a sensor-I SRC motif that could interact with the other RFC subunits ( Cullmann et al 1995 ). Like the o subunit of y complex RFC-5 is predicted to function as the stator in this eukaryotic clamp loading machine (Davey et al ., 2002) The remaining RFC 2 3 and 4 subunits, when mutated in the conserved Walker-A sequence for ATP binding and hydrolysis show the most severe in vitro defects and in vivo phenotypes (Schmidt et al ., 2001a; Schmidt et al 2001b) Therefore RFC-2, 3 and 4 were proposed to be the motor subunits of this machine In fact as heterotrimeric complexes both yeast and human RFC-2-3-4 complexes had DNA-dependent ATPase activity (Ellison and Stillman, 1998 ). This functional analogy of the different RFC AAA + subunits to they complex subunits for prediction of RFC clamp loader structure and mechanism of clamp loading awaits solution of an atomic structure of the RFC clamp loader Although, as described below the atomic structure of part of the archaeal RFC clamp loader which has

PAGE 95

I I 80 significant homology to eukaryotic RFC has been solved and closely resembles the structure of y complex motor subunits Human RF C alone and in a complex with PCNA has been viewed by transmission electron microscopy ( T E M ) and atomic force microscopy ( AFM) ( Shiomi et al ., 2000 ) The TEM results showed ~hat RFC forms a '' closed-U-forrn '' s tructure in the absence of ATP When viewed in the presence of ATP the R F C molecules appeared as a more ' openC -form '' structure demon s trating that ATP caused RFC to open Partial I I proteolysis results with ATP non-h y drolyzable ATPyS or ADP confi-rtned that there was induction of a distinct s tructural change in RFC only in the presence of ATP Additional TEM imagining also showed that ATPyS or ADP had virtually no effect on the structure of RFC ( i e RFC remained in '' U-fo1m '' similar to that without nucleotide) Although the images were low resolution and did not allow specific distinction of RFC or PCNA molecules in the imaged RFC-PCNA complexes the author s concluded that the '' open ' ATP-bound RFC '' C-form '' was most likely bound to PCNA in the TEM images. Archaeal PCNA Clamp and Replication Factor-C Clamp Loader Biochemical analyses and recent structural analysis of the clamps and clamp loaders of several archaeal organisms are providing important information that is helping bridge the gap between the prokaryotic structural and functional clamp loading characteristics and eukaryotic structural and functional clamp loading characteristics Archaeal PCNA clamps and RFC clamp loaders share ~ 30-40 % sequence homology to their eukaryotic counterparts (Seybert et al ., 200 2). Archaeal organisms have a PCNA clamp and a RF C clamp loader complex ( Table 2-1 ). Archaeal RFC and PCNA have been biochemically characterized for S ulfol o bu s s olfatari c us ( De Felice et al ., 1999 ; Pisani et al 2000 ), Me than o ba c t e rium th e rm oa ut o tr o phi c um AfI ( Kelman and

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81 Hurwitz, 2000), Archaeoglobus fulgidus (Seybert et al ., 2002) and Pyro cocc us furiosus (Oyama et al ., 2001) The RFC clamp loading machine from each of these archaeons is composed of two subunits RFC-L ( large ), and RFC-S (small), and each has AAA + homology (Davey et al ., 2002) Biochemical analyses of RFC and PCNA of these organisms have identified several general characteristics and these characteristics are consistent with the clamp loading mechanisms of both eukaryotic and prokaryotic organisms Each archaeal RFC must fotm a complex to acquire DNA-dependent ATP hydrolysis activity. This ATPase activity is associated with the RFC-S subunits and has shown preference for ds DNA I ss DNA primer-template junctions (Kelman and Hurwitz, 2000; Pisani et al ., 2000). For the Archaeoglobus fulgidus ajRFC clamp loader it was further shown that the ATP hydrolysis activity was enhanced 2 to 3 fold by ajPCNA, and that ATP binding not hydrolysis was required to stimulate the interaction of ajPCNA with DNA (Seybert et al ., 2002). Each archaeal RFC clamp loader was also shown to support processive DNA I synthesis with its respective PCNA and pol B-family archaeal DNA polymerase The major difference in the characterization of these clamp loaders was their subunit stiochiometry All have been shown to form pentameric complexes similar to the eukaryotic and prokaryotic clamp loaders except the Methanobacterium thermoautotrophi cu m MI RFC which has been shown to fotm a hexameric complex of two RFC-L subunits with 4 RFC-S subunits The general pentameric subunit stoichiometry resemble s the T 4 bacteriophage clamp loader and the eukaryotic RFC, having four RFC-S subunits with 1 RFC-L subunit

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82 I About the same time the y complex clamp loader crystal structure was solved, the crystal structure of a RFC-S subunit complex of Pyro cocc usfuriosus was solved at 2 8 A resolution (Oyama et al 2001 ) The RFC-S subunit formed a '' dimer-of-trimers '', not necessarily the functional assembly of the clamp loader E ach RFC-S subunit showed significant AAA + structural homology Superimposition of a RFC-S subunit with the y complex o subunit structure had a root-mean-square displacement of only 3 A for the two conserved AAA + domain s ( I and II) and showed marked overall similarity The I RFC-S subunits had the Walker-AP-loop and Walker-B moti f s flanked by sensor-2 and sensor-1 motifs for1r1ing the AAA + -structurally conserved nucleotide binding site and also had the SRC '' arginine-fmger '' motif Each RFC-S subunit had an a-helical terminal Domain-III corresponding to the circular '' collar '' oligomerization domain of the y complex clamp loader. The subunit interfaces within each trimer of RFC-S subunits was arranged such that each nucleotide binding site was in contact with the neighboring sub\1nit and the interfaces differed between each subunit suggesting asymmetry among the subunits in the crystal a feature distinguishing they complex structural-functional characterization ( Jen1zalmi et al ., 2001a ) The RFC-S subunits are AAA + proteins and based on the extraordinary structural similarity with they AAA + subunits of the prokaryotic clamp loader suggest that the mechanism of ATP-dependent confo1mational changes that drive they complex is most likely the same mechanism driving the archaeal clamp loader The RFC-S s ubunit has the highest sequence similarity to the eukaryotic RFC-3 AAA + subunit (Cann and Ishino 1999 ), and therefore two predictions can be made First the RFC-S subunit of archaeal organisms i s the '' motor '' subunit of the clamp loader and second that the mechanism of

PAGE 98

83 the eukaryotic clamp loader may be closer to the mechanism of the prokaryotic clamp loade~ than originally expected To add to this the RFC-L subunit generally lacks an SRC motif and shows highest sequence similarity to the eukaryotic RFC-1 subunit (Cann and Ishino ~ 1999) Thus it has been proposed that like the eukaryotic RFC-1 and prokaryotic o subunit RFC-L is the clamp interacting subunit of the archaeal clamp loading machine (Davey et al 2002) The functional conservation of the sliding-clamps and clamp loaders in all branches of life is readily understandable considering the importance of their functions in DNA replication and therefore in cell division for propagation of any given organism. These are ancient proteins that have not changed considerably for billions of years The use of the E. coli model system for studying the mechanisms of clamp loading for processive replication has thus given us the fundamental basis for study of the clamp loading mechanisms in all for111s of life In this dissertation project the conformational dynamics of the E. c oli clamp loader were under investigation, and it is hoped that the results can be applied for understanding of other AAA + based clamp loaders and provide additional functional insights for other AAA + motors

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CHAPTER3 MATERIALS AND METHODS NOTE : The anisotropy binding assays and MDCC-PBP ATPase assays were I perfor1ned with both 'Y complex and the minimal complex clamp loader usually in 'back' to-back '' ( i e hours apart ) assays. Therefore the term '' clamp loader '' will be used throughout this chapter to describe both except where different concentrations of each were used or where only the activity of 'Y complex was studied Proteins, Reagents, and Oligonucleotide Substrates DNA Polymerase ID Proteins DNA polymerase III proteins were a generous gift from Mike O Donnell s laboratory at The Rockefeller University New York NY Proteins were stored in 20 mM Tris-HCl pH7 5 2 mM DTT 0 .' 5 mM EDTA, and 10% glycerol Assay buffer for all experiments contained 20 rnM Tris-HCl pH 7 5 50 mM NaCl 40 / mL Bovine serum albumin (lnvitrogen Corp ., Carlsbad CA) 5 mM DTT and 8 rnM MgCl 2 (Table 3-1 ). Concentrations of 'Y complex were determined from absorbances at 280 nM in 6 M guanidine hydrochloride and the calculated extinction coefficient This concentration was verified by amino acid analysis following acid hydrolysis of the protein (perfo11r1ed by the Protein Chemistry Core Facility, Biotechnology Program University of Florida ) A sample for amino acid analysis was prepared by dialyzing 'Y complex against 20 mM sodium phosphate buffer pH 7 5 at 4 C to remove glycerol and other buffer components that interfere with the analysis. The concentration of this dialyzed protein was also 84

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85 determined by three other methods for comparison ; 1) its absorbance at 280 nM in 6 M guanidine hydrochloride pH 6 5 2) its concentration in a Bradford assay using prepared ' reagents from BioRad and a BSA standard and 3) its concentration in a Bradford assay I using an igG standard The amino acid analysis and absorbance measured under denaturing conditions yielded concentrations of 1 6 Mand 1 7 M, respectively, within experimental error. Both Bradford-type assays overestimated the concentration of protein by a factor of 1 2 using the BSA standard and a factor of 3 0 using the IgG standard The concentration of~ was deterrnined from its absorbance at 280 nm and the extinction coefficient for native protein of (17 900 M 1 cm 1 ) (Johanson et al 1986) Reagents ATP was from Sigma-Aldrich Co (St Louis, MO) and 5 -0-(3-thiotriphosphate) (ATPyS) was from Roche Molecular Biochemicals Bacterial purine nucleoside phosphorylase (PNPase) and 7-methylguanosine (MEG) were from Sigma-Aldrich and stored as solutions at 80 C Table 3-1 Assay and protein buffers Standard assay buffer 20 mM Tris-HCl pH 7 5 50 mMNaCl 40 g/mL BSA 5mMDTT 8mMMgCl 2 Oligonucleotide Substrates Protein storage 20 mM Tris-HCl pH 7 5 0 5 mMEDTA 2mMDTT 10 / o glycerol Protein dilution 20 mM Tris-HCl pH 7 5 40 g/mLBSA 5mMDTT 0 5mMEDTA 10 % glycerol Synthetic oligonucleotides were made on an ABI 392 DNA/RNA synthesizer using standard P-cyanoethylphosphoramidite chemistry and reagents from Glen Research Corp (Sterling VA) Denaturing polyacrylamide gel electrophoresis was used to purify new DNA. The 30-mer primer has the sequence : 5'-GAG CGT CAA AAT GTA GGT ATT I

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I 86 TCC ATG AGC GTT TTT CCT GTT GCA ATG GC-3 and the 105-mer template has the sequence: 5 GAG CGT C AA AAT GTA GGT ATT TCC ATG AGC GTT TTT CCT GTT GCA ATG TCG GGC GGT AAT ATT GTT CTG GAT ATT ACC AGC AAG GCC GAT AGT TTG AGT TCT TCT-3 '. A 5 -C2-dt amino linker (Glen Research Corp ., Sterling VA ) wa s ipcorporateq onto the 105-mer during synthesis. Following synthesis and purification, the 5 -amino group was covalently labeled by addition rhodamine isothiocyanate (RhX) ( Molecular Probes Inc ., E ugene OR) as described I (Bloom et al ., 1996 ). Recently, it was demons,:trated that the position of the RhX fluorescent probe on the 105-mer template had no effect on anisotropy measurements in pre-steady-state experiments ( Ason et al ., 2003). For the MDCC-PBP A TPase assay a 105-mer of the same sequence was synthesized, but was not functionalized with the 5 C2-dt amino linker as it did not require fluorescent labeling The 30-mer primer was annealed to the X-rhodamine-labeled 105-mer (or unlabeled 105-mer) by incubating 1 2 mol equivalent of the 30-mer with 1 mol equivalent 105-mer in a water bath at 80 C then allowed to slowly cool to room temperature Excess 30-mer was not further purified from the p i t DNA since it has been previously shown that it had no effect on clamp loading reactions in anisotropy assays (Ason et al ., 2000). Purification of Escherichia coli Phosphate Binding Protein E col i phosphate binding protein (PBP ) was purified and labeled with N-[2-(lmaleimidyl ) ethyl]-7( diethylamino ) coumarin-3-carboxamide ( MDCC ) (Molecular Probes Inc ., E ugene OR) using the method described by (Brune et al ., 1998) without the use of the '' enhanced P i -mop ''. Using this procedure approximately 90 mg of the mutant Al97C phosphate binding protein was recovered from a 2-L culture LB medium ( 10 mL ) containing 12 5 mg / L tetracycline was inoculated with E.coli strain ANCC75

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87 harboring the plasmid pSN 5182 / 7 with the mutated phoS gene ( a gift from Martin Webb) This culture was diluted to 100 mL in the same medium after 6 hat 37 C This culture was then grown for 16 hat 37 C and used for inoculation (i.e. 10 mL added) of ' four 500-mL cultures containing high-phosphate TG plus medium with supplements ( Table 3-2) containing 12 5 mg/L tetracycline (Brune et al ., 1994) Table 3-2 TG-plus media contents Buffer 120 mM Tris HCl pH 7 2 4 g/L glucose 0 640 mM KH 2 P04 pH 7 5 a 0 064 mM KH 2 P0 4 pH 7 5 b a for high-potassium phosphate b for low-potassium phosphate Essential amino acids 20 mg/mL L-arginine-HCl 10 mg/mL L-leucine 8 mg/mL L-histidine-HCl 4 mg/mL L-methionine 4 mg/rnL L-tryptophan 4 mg/mL L-adenosine 2 mg/mL L-thiamine Salts 120mMNaCl 30mMKCl 4 5 mMNa2S04 0 3 mMMgS04 0 3 mMCaCl2 1 5 M FeS04 For optimum growth of cells it is advantageous to use 2.8-L Fembach culture flasks for the 500 mL cultures with vigorous shaking throughout to obtain maximum aeration of the cultures The cultures were incubated at 3 7 C for ~ 6 h at which point the optical density at 600 run was measured The optical density at 600 nm should approach 2.0 for optimum growth in these cultures Cells were then pelleted by centrifugation at 1 670 x g (JLA-10 500 rotor ) at room temperature in autoclaved centrifuge bottles for 30 min Cells were resuspended in 500 mL TG plus containing the same supplements except low phosphate was used (Table 3-2) It was important to wash the pellets in the low phosphate medium to remove any carryover of the high phosphate from the original TG plus medium Cells were grown for another 16 hat 37 C and then harvested by centrifugation at 3 000 x g (JLA-10.500 rotor) for 25 min at room temperature

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88 An osmotic shock procedure was used to release PBP from the cells periplasm (Willsky and Malamy, 1976) Supernatant was discarded and pellets were resuspended in 650 mL total volume containing IO mM Tris-HCl pH 7 6 and 30 mM NaCl at room temperature This was pooled into two 500-mL centrifuge bottles and spun at 3 000 x g I (JLA-10 500 rotor) for 25 ~in at room temperature. The supernatant was discarded and the cells were resuspended in 350 mL of the same buffer The cells were centrifuged as above, the supernatant removed and the pellet weighed Typically 6 IO g of cells were I I obtained from the 2-L culture. Cells were then resuspended in 100 mL 33 mM Tris-HCl pH 7 6 at 25 Cina centrifuge bottle containing a magnetic stir bar While stirring rapidly on a magnetic stir plate, 100 mL of 40% sucrose, 0 .1 mM EDTA, and 33 rnM Tris-HCl pH 7.6 was rapidly added at 25 C After the mixture stirred for 10 min at 25 C cells were spun at 18 600 x g ( JLA-10 500 rotor) for 20 min at 4 C After discarding the supernatant the cells outer membranes were burst by osmotic shock by rapid resuspension in 200 mL ice cold 0 5 mM MgCl 2. After rapid stirring for 15 min on an ice bath the cells were spun at 18 600 x g (JLA-10 500 rotor) for 10 min at 4 C. The supernatant ( 'shockate '' ) was then dialyzed against the column equilibration buffer : 10 mM Tris-HCl pH 7 6 and determined by absorbance at 280 nm to be ~ 200 mg unpure periplasmic protein The Osmotic shock procedure was repeated once again on the pelleted cells by another resuspension in ice cold 0 5 mM MgC1 2 Generally this second osmotic shock produced an additional significant quantity of PBP from the cells for purification ( ~ 70 mg). PBP wa s purified from the shockate by FPLC anion exchange purification on a 75mL High-Trap Q-Sepharose column (Amersham Biosciences Corp Piscataway NJ)

PAGE 104

89 equilibrated with 10 mM Tris-HCl pH 7 6 and 1 mM MgCl 2. 'the column was loaded with 200 mg protein, and washed with 5-column volumes of equilibration buffer A 1, L linear gradient of Oto 200 mM NaCl was initiated at a flow-rate of 8 mL / min The protein eluted in a single broad peak at ~ 30 mM NaCl The peak fractions were pooled and concentrated using centricon-centriplus YM-10 centrifuge tubes (10 mL each) (Millipore Corp ., Bedford, MA) Protein concentration was determined by absorbance readings at 280 nm using an extinction coefficient for PBP (E o I % at 280 nm, 60 880 M 1 cm 1 ). The identity of purified PBP was confir1ned using denaturing polyacrylamide gel electrophoresis analysis with molecular weight standards and previously purified PBP as markers Purified A197C PBP (69 mg) was stored at 80 C Labeling of Phosphate Binding Protein with MDCC The purified Al97C PBP protein was labeled in a solution (4 mL total volume) of 20 mM Tris-HCl pH 8 1 containing ~ 100 M PBP 200 MMDCC in dimethylformamide 168 M MEG 0 2 U / mL PNPase and 5 M MnCl2 The labeling I reaction was incubated for 30 min at room temperature while gently turning on a rotisserie with protection from light After the labeling reaction PBP was concentrated in a Centricon-Centriplus YM-10 centrifuge tube (Millipore Corp ., Bedford, MA) to a volume of approximately 1 mL Free MDCC and other low molecular weight impurities were removed a with P6 gel desalting column ( 39-mL ) (Bio-Rad Laboratories Inc ., Hercules CA ). Labeled-protein separated from free MDCC and eluted in the void volume of the column MDCC-PBP was pooled and dialyzed against equilibration buffer ( 10 mM Tris-HCl pH 8 0) overnight MDCC-PBP ( typically 11-12 mg in ~ 9-13 mL ) was then loaded onto a 5 X 5 mL High-Trap Q-Sepharose column ( Amersham I

PAGE 105

90 Biosciences Corp ., Piscataway NJ ) equilibrated with 10 mM Tris-HCl pH 8 0 After washing with 5-column volumes of equilibration buffer a 200-mL linear gradient from 0 to 50 mM NaCl in equilibration buffer was initiated This purification typically gave ~ 70 % recovery of > 80 % -labeled / responsive MDCC-PBP Pooled fractions were concentrated in an Amicoij stirred-cell pressure concentrator ( Millipore Corp ., Bedford MA) using a Biomax( PB) polyethersulfone membrane (NMWL 5000 Dalton s) (Millipore ) at ~ 44 PSI 4 C C oncentrated MDCC-PBP was then dialyzed overnight I against Mono-Q column equilibration buffer ( lOmM Tri s -HCl pH 8.0 100 M KH 2 P0 4). In general ~ 9 mg MDCC-PBP was loaded onto the equilibrated 8-mL Mono-Q column (Amersham Biosciences Corp ., Piscataway NJ ). The column was then washed with 5column volumes of equilibration buffer, and an 80-mL linear gradient from O to 30 mM NaCl was initiated Two peaks were eluted ; the earliest peak fractions contained P i responsive protein, and the latter non-responsive protein Both peaks were pooled separately and concentrated using an Amicon stirred-cell pressure concentrator (Millipore Corp ., Bedford, MA ) using a Biomax tm ( PB) polyethersulfone membrane (NMWL 5000 Daltons) (Millipore ) at ~ 44 PSI 4 C The concentrated P i -responsive protein was dialyzed overnight against 1 L 10 mM Tris-HCl pH 8 0 ( changing the dilution buffer two times ), and then s tored in 80 C There is some remaining P i contamination in the purified MDCC-PBP but this was removed with the 'P i -mop '' before each experiment ( see below ). Characterization of MDCC-Labeled Phosphate Binding Protein 1vIDC C -PBP was analyzed by three methods 1 ) UVMS absorbance for concentration calculation and determination of the efficiency of labeling 2 ) a

PAGE 106

91 responsivity test to dete1n1ine the extent of molar response to P i, and 3) active site titration with P i standards I Removal of Free-Inorganic Phosphate (Pi) Contamination with the "Pi-mop" The '' Pi-mop '' utilizes purine nucleoside phosphorylase (PNPase) to catalyze the p;hosphorolysis of 7-methylguanosine ( MEG ) for removal of P i from buffers, quartz cuvettes and the stopped-flow instruments Phosphorolysis of 7-methylguanosine is essentiall y irreversible toward formation of ribose-1-phosphate product (Nixon et al ., 1998 ). To minimize free-P i contamination disposable plasticware was used, and rinsed only in deionized water to clean. At room temperature this '' P i -mop '' removes the free-P i contamination to levels below the threshold of sensitivity of MDCC-PBP ( < 0.1 M) (Brune et al ., 1994 ). Generally quartz cuvettes and the stopped-flow apparatus were preincubated in buffer containing the '' P i -mop '': PNPase ( typically 0 1-0 2 U / mL) and MEG ( typically ~ 160 Concentration and Efficiency of Labeling of MDCC-PBP The concentration ofMDCC-PBP wa s determined by its absorbance at 280 nm after correcting for the absorbance of MDCC at 280 nm ( 0 164X of the MDCC absorbance peak at 430 nm was subtracted from the absorbance at 280 run) using an absorbance scan from 250 to 460 run Us ing the molar extinction coefficient for MDCC at 430 nm ( 46 800 M 1 cm1 ) ( Brune et al ., 1994) the concentration of MDCC was determined. Using the value of:MD C C concentration, the percent labeled-PBP ( i e efficiency of labeling ) was detemrined ( typically 86-90 /o). This percent-labeled qliautity wa s used as the concentration ofMDCC-PBP all :rvIDCC-PBP ATPase assays since it is only this labeled-PBP that undergoe s an increase in fluorescence upon binding Pi I

PAGE 107

I I 92 Therefore, the only fluorescence response observed in the MDCC-PBP ATPase assays always corresponds to the fraction (percent) labeled-PBP It is noteworthy that the dissociation constant for the labeled-PBP is an order of magnitude lower (i e has greater affinity for Pi) than for any unlabeled-PBP which may have come through the purification (Brune et al ., 1998) Characterization of the fluorescence-molar response of MDCC-PBP to Pi A ' P i -responsivity test '' was performed by scanning the fluorescence emission of lvIDCC-PBP in standard assay buffer (Table ~ -1) using the QuantaMaster QM-1 fluorimeter described below with excitation and emission monochromator band passes of 2 nm Approximately 4 M MDCC-PBP (either Pi -responsive or non-responsive) in assay buffer containing MEG was initially scanned, then ''Pi-mop' PNPase was added to begin the removal of P i contamination. Emission scans were performed at 5 min intervals until the peak fluorescence intensity reached a minimum value (typically took 15-20 min) and was completely red-shifted from ~ 464 nm to --4 72 run The minimum peak intensity at 472 nm was recorded as the intensity value for Pi-free MDCC-PBP To this was added a solution containing potassium phosphate (KH 2 P0 4) giving a final concentration of 200 M in order to observe the saturated intensity of completely Pi bound MDCC-PBP The fold-difference between the P i -free and P i -bound intensities is noted as the Pi-response and was generally 7to 10-fold for Pi -responsive MDCC-PBP and 1to 2-fold for non-responsive MDCC-PBP The observed intensities of the P i -free and P i -bound peaks for non-responsive 1v1DCC-PBP were characteristically at least an order of magnitude lower than the observed intensities for responsive MDCC-PBP

PAGE 108

93 1.2e+6 1.De+6 I 8.0e+S 3 C G) (,) 6.0e+5 I 0 4.0e+S ::, u.. 2.0e+5 0.0 ' I I l I I I I I 0 5 10 1 & 20 25 30 35 40 45 50 [P l ] M Figure 3-1 P i titration analysis ofMDCC-PBP 5 MlvIDCC-PBP was subjected to a range of potassium phosphate (P i ) from O to 50 Min Pi-mopped assay buffer The average fluorescence emission intensity values at 464 nm from 30 s measurements are plotted as a function of concentration of potassium phosphate standards Linear regressions for the rising and saturated data points intersect at a point reflecting the apparent concentration of the fraction oflabeled-MDCC-PBP ( ~ 4 5 M) ( vertical line ) Active Site Titration of MDCC-PBP MDCC-PBP was titrated with P i of known concentrations Potassium phosphate was used as the s ource of P i in the titration The experimental setup of this analysis was designed to mimic the steady-state MDCC-PBP A TPase assay described below. Fl uorescence intensities at 464 nm were recorded for solutions containing 5 M MDCC PBP subjected to a range of potassium phosphate from O to 50 M. The potassium phosphate ( P i) standards were added to separate assays in random order to prevent any systematic error in the analysis A 76-L sample containing 5 M MDCC-PBP in assay buffer ( Table 3-1 ) was placed in a quartz microcuvette ( internal dimensions 3 X 3 mm ), (Hellma Cells Inc

PAGE 109

94 Plainview, NY) Fluorescence emission intensity of Pi-free :t\IDCC-PBP was recorded for 30~s Then a 4-L solution containing varied concentrations of Pi was mixed into the I cuvette and another 30 s measurement of P i -bound emission intensity was recorded The ' averaged values of the 30-s measurements of P i -free and P i -bound fluorescence intensities were used to analyze the nature of P i binding to :MDCC-PBP Figure 3-1 shows a typical P i titration This characterization has great significance for the quantitation of P i in the :MDCC PBP A TPase assays. The rising and saturated data points of this titration were separately subject to linear regression and the point of their intersection relates to the concentration of MDCC-PBP in the experiment since the fluorescenc.e of MDCC-PBP in response to Pi saturates at stiochiometric quantities of P i (Brune et al ., 1994) Control titrations containing an equimolar amount of unlabeled-PBP showed no change in the fluorescence response (not shown ). Together the active site-P i titration shows that only the labeled fraction of PBP ( ~ 4 5 ) actually binds Pi Thus it is proper to use the concentration I of the fraction of MDCC-labeled PBP in the calculation of molar P i in the MDCC-PBP A TPase assays Fluorescence Anisotropy Binding Assays Calculation of Anisotropy In each anisotropy binding assay vertically and horizontally polarized emission intensities were measured and corre s ponding background intensities were measured for solutions containing assay buffer only The background intensities were subtracted from experimental intensities for background correction To correct for any polarization bias a '' G-factor' was detennined by measurin g vertical and horizontal emission intensities I

PAGE 110

I 95 when the fluore s cent-labeled s pecies was excited with horizontally polarized light (Lakowicz 1999 ) Anisotropy values were calculated using background corrected intensities and the equation r = ( I vv gl vh) / ( I vv + 2gl vh), where r i s anisotropy and I vv & I v h are the background corrected intensities of vertically and horizontally polarized emission respectively measured when samples are excited with vertically polarized light and g is the G-factor The anisotropy values determined in titration as s ays were generally converted to the fraction-bound of fluorescent-labeled sub s trate and fit to the quadratic i equation for estimation of the binding constant (Ki ) : Fraction bound X b = [AB] [AB] = + (A] + (B] ~~ + [A] + [B)) 2 4 [A][B] ( r Ii \+ Ii brun d &eel free 2[B] Where [A] and [B] are the concentrations of the clamp loader and fluorescent-labeled DNA or 13 respectively and r oo un d and r rree are the characteristic steady-state anisotrop y values for boundand free-labeled DNA or 13 respectively Steady--state Measurement of Clamp Loader RbX-DNA Binding Kinetics Steady-state fluorescence-polarized emission intensity measurements for clamp loading assays were recorded using a Quantamaster QM-1 fluorimeter ( Photon Technology International Inc Lawrenceville NJ) equipped with a 75-W xenon arc lamp an excitation monochromator dual emission monochromators photon-counting detectors with Hamamatsu R928 PMTs (Hamamatsu Photonic System s Bridgewater NJ ), and Gian-Thompson polarizers Samples were excited with vertically polarized light at 580 nm ( 5 nm band pass ) and both vertically and horizontally polarized emi s sion at 610 run ( 5 nm band pass ) were simultaneously mea s ured in a ' T-fo11nat ''. Reaction final volumes

PAGE 111

96 wete 80 Lin a quartz microcuvette (internal dimensions 3 X 3 mm, (Hellma Cells Inc., Plainview NY) Determination of clamp loader RhX-labe l ed DNA binding constants ' In fluorescence-based titration assays for the calculation ofRhX-pt DNA an.isotropy, the Quantamaster fluorimeter setup described above was used A solution of RhX-labeled DNA, either single-stranded (ss DNA) or primer-template (pt pNA) substrates, at a concentration of 50 nM in assay buffer (Table 3-1) was placed in a cuvette Initially, background polarized emission intensities were recorded with the free RhX-DNA substrate. When p was included in titration assays it was added to the RhX DNA solution after background fluorescence measurements The final concentration of P was 500 nM in these assays Next the RhX-DNA was titrated by addition of a constant volume (8 L) of a solution containing clamp loader, at differing concentrations, in assay buffer ATP was present either originally in the assay buffer or added later giving a final concentration of 0 3 rnM or 0 5 mM The amount of ATP was high enough such that its concentration was essentially constant over the short time-course of the titration experiment Typically 30 s measurements of polarized emission intensities were averaged for each step in the titration assays ; including background measurements, after addition of p ( when present) and after addition of each solution of clamp loader for calculation of anisotropy values In some assays the steady-state polarized emission intensities were observed over longer time-courses and anisotropy was calculated per each second of the experiment Steady-state anisotropy values are a function of both the lifetime and rotational correlation time of a fluorophore (Lakowicz, 1999 ; Otto et al ., 1994) It was previously determined that the fluorescence lifetime of X rhodamineI

PAGE 112

I I 97 I labeled DNA did not change when clamp loader and clamp were present in assays with RhX-DNA demonstrating that the measured changes in steady-state anisotropy were the result of change s in rotational dynamics of the fluorescent probe on the DNA substrate (Bloom et al ., 1996 ). Determination of the effect of ADP on clamp loader RhX-labeled DNA binding The steady-state fluorescence emission intensities of RhX-DNA ( either ss DNA or pt DNA ) in assay buffer were measured for calculation of anisotropy as described above I Initially background measurements were recorded for a solution containing either ss or pt RhX-DNA in the presence of differing concentrations of ADP ADP was varied over a range of 0 2 mM to 0 8 mM in this solution. When present J3 clamp (final concentration 500 nM) was then added This was followed by addition of a solution containing clamp loader (final concentration 500 nM) ATP was finally added to the cuvette giving a final I concentration of 0 3 mM At all steps, reactions were monitored for 30 sand resulting polarized emission intensities were averaged for the calculation of anisotropy Determination of clamp loader J3pyrene clamp binding constants Fluorescence-based titration experiments were perf onned using the Quantamaster fluorimeter in a similar manner to those above for RhX-DNA except that the excitation light was at 345 nm and the polarized emission inten s ities were measured at 375 nm (5 nm band pass ). Initially background intensity measurements were recorded for a solution containing pyrene-labeled J3 clamp ( J3 pyre n e) in assay buffer ( final concentration 50 nM) Then, a constant volume (8 ) of a solution containing clamp loader was added to the cuvette Concentrations of the clamp loader were varied in separate assays After addition of the clamp loader solution, ATP-independent J3 pyrene _clamp loader

PAGE 113

98 binding activity was measured Finall y, ATP ( final concentration, 0 3 mM) was added to the re~ction to induce ATP-dependent binding of (3p yrene and clamp loader. At each step I in the assay a 60-s measurement of polarized emission intensities was recorded, and the average intensity values for these 60-s measurements was u s ed in the calculation of anisotropy Determination of the apparent binding constant of ATP to the clamp l~ader In fluore s cence-based titration assays with (3 pyrene, similar to those described above, the apparent binding constant of ATP to the clamp loader was estimated Initially background intensity measurement s were recorded for a solution containing pyrene labeled f3 clamp ( f3 pyrene) (final concentration 65 nM ) in assay buffer To this was added a solution of clamp loader ( final concentration 400 nM ) in assay buffer To induce binding of f3 pyrene and clamp loader a constant volume ( 4 ) of a solution containing ATP was added to the reaction T he concentration of ATP was varied in separate assays At each step in the assay a 60-s measurement of polarized emission intensities was recorded, and the average intensity values for the s e 60s measurements was used in the calculation of anisotropy The concentrations of ATP stocks were determined from the average of at least three absorbance measurement s at 259 nm using the molar extinction coe f ficient at 259 nm ( 15 400 M 1 cm 1 ). Determination of the effect of ADP on clamp loader (3pyrene binding A constant volume ( 8 ) of a solution containing clamp loader was added to a cuvette containing a solution of f3p yrene in assa y buffer following initial background intensity measurement s Concentrations of clamp loader were varied while each separate a s say contained 55 nM (3 pyrene in assay buffer Next a solution of ATP ( final

PAGE 114

99 concentration, 0 3 mM) was added to induce clamp loader interaction with '3 p)'Iene_ Finally, a solution containing an excess amount of ADP (fmal concentration, 0 8 mM) in a small volume ( 2 L) was added to the cuvette At each step in the assay, a 60-s measurement of polarized emission intensities was recorded and the average intensity values for these 60-s measurements was used in the calculation of anisotropy . Pre-Steady-State Measurement of Clamp Loader RhX-DNA Binding Kinetics A Biologic SFM-4 stopped-flow (Molecular Kinetics Pullman WA) equipped with I I four independently driven reagent syringes and a 31-L cuvette (model# FC-15) with a 1 5 mm path length was used to record polarized emission intensities in real-time Dichroic sheet polarizers (380 770 run model # 27346) (The11noOriel Corp ., Stratford, CT) were mounted directly onto the cuvette A QuantaMaster QM-1 fluorimeter, described above was used as an excitation source by focusing the output fr~m the excitation monochromator (580 nm 1.0 1 5 nm band pass) onto a fused silica fiber optic consisting of a bundle with dimensions of 0 25 X 2 56 mm containing nine 250-M fibers (Fiberguide Industries, Stirling, NJ) The excitation fiber optic was mounted against the stopped-flow cuvette ''sandwiching'' the dichroic sheet polarizer Vertically and horizontally polarized emission were collected simultaneously in a '' I-format '' through UVNIS liquid light guides with a 5-mrn core diameter (ThermoOriel Corp ., Stratford, CT) mounted directly against the stopped-flow cuvette '' sandwiching'' the dichroic sheet polarizers Fluorescence emission was detected with a photon-counting detection system consisting of two channels each with a filter holder containing a 610 nm cut-on ftlter (CVI Laser Corp., Albuquerque NM), mounted onto an ambient photomultiplier housing (Products for Research Inc ., Danvers MA) with a R4457P PMT

PAGE 115

100 (Hamamatsu Corp ., Bridgewater NJ ) Signal s from both PMT s were detected via a SR45~ preamplifier a SR400 gated-photon counter ( Stanford Research Sunnyvale CA ) I and MCS-II multichannel scalar cards ( Oxford Research Oakridge TN) The external I synch from the SFM-4 s topped-flow unit triggered data acquisition by the MCS-11 cards All assays were performed at 20 C F or e ach a s say run vertically and horizontally polarized emission intensitie s were recorded at 1-ms intervals and 18 20 stopped-flow runs were s ignal averaged. The cuvette was flushed out prior to each run with assay buffer ( Table 3 -1 ). Following each assay the s teadys tate anisotropy ofRhX-DNA free in assay buffer ( r free) was measured using the s etup described above for steady-state anisotropy calculation Since the excitation polarizer is '' sandwiched '' between the cuvette and fiber optic in the Biologic SFM-4 stopped-flow, it is not easily rotated for horizontal excitation to deter111ine the G-factor polarization bias of the system However since there are no emission monochromators in this system there is only modest polarization bias The G-factor was solved for with the steady-state anisotropy value of free RhX-DNA left over from the assays using the equation : G = [(I vv)/ (I vh) ] / [(1 r &ee ) l ( 2r rree + 1 ) ] where r rree is the steady-state anisotropy of free RhX-DNA, and I vv & l vh are the background corrected intensities of vertically and horizontally polarized emission, respectively measured when s amples are excited with vertically polarized light Pre-steady-state kinetics of clamp loading initiated at different reaction steps Three different mixin g s chemes for pre-steady-state anisotropy binding assays were achieved by changing the reagents in the stopped-flow drive syringes All reactions were initiated by mixing 80 Leach from two s yringes at a 10-mL / s flow rate The stopped flow reaction dead time under the s e conditions was 3 7 + / 0 7 ms determined using the

PAGE 116

101 method of (Peterman 1979) See below for description of the dead-time detennination For scheme-I mixing one syringe contained 500 nM clamp loader, 0 5 mM ATP and 1 2 M J3 in assay buffer The second syringe was loaded with 100 nM RbX-pt DNA and 0 5 mM ATP in assay buffer For scheme-2 mixing one syringe was loaded with 500 I nM clamp loader and 0 5 mM ATP in assay buffer. The second syringe was loaded with 100 nM pt DNA 1 2 2 4 or 4.8 M J3 and 0 5 mM ATP in assay buffer For scheme-3 mixing, one syringe contained 500 nM clamp loader only The second syringe was l loaded with 100 nM pt DNA 1 2 or 4 8 M J3 and 0 2 0 5 or 1 0 mM ATP in assay buffer In control reactions without enzyfile one syringe was loaded with assay buff er only, and the second syringe was loaded with 100 nM pt DNA 1 2 M J3, and 0 5 mM ATP in assay buffer Pre-steady-state kinetics of clamp loading on different RhX-DNA substrates The activity of y complex with RhX-pt DNA versus RhX-ss DNA was compared by analysis of pre-steady-state kinetics using anisotropy binding assays For these experiments schemeI mixing was utilized One syringe was loaded with 480 nM y complex 0 5 mM ATP and 1 M J3 ( when present) in assay buffer The second syringe was loaded 100 nM RhX-pt DNA or RhX-ss DNA and 0 5 mM ATP in assay buffer Reactions were initiated as above Fluorescence-based MDCC-PBP ATP Hydrolysis (ATPase) Assay Steady-State Kinetics of ATP hydrolysis The steady-state change in fluorescence of MDCC-PBP was measured using a Quantamaster QM-1 fluorimeter (Photon Technology International Inc ., Lawrenceville

PAGE 117

102 NJ ) '. Samples were excited at a wavelength of 425 nm ( 2 nm band pass) and emission was seJected at 464 nm ( 2 nm band pass ). A 76-L sample of clamp loader clamp ( when present) pt DNA, and MDCCI PBP in standard assay buffer (Table 3-1 ) was placed in a quartz microcuvette (internal dimensions 3 X 3 mm ( Hellma Cells Inc Plainview NY) A 10-minute timebased scan was initiated and approximately 14-s later a 4-L solution of ATP was add~d to initiate the reaction The P i -mop ( described above ) wa s used to remove P i contamination separately from the assay buffer ( 0 06 U / mL PNPase 160 M :MEG ) and the solution containing MDCC-PBP clamp loader pt DNA and p ( when present) ( 0 1 U / mL PNPase, 160 M MEG ). Both mopped solutions were diluted 4-fold when added to the cuvette before starting the reaction This dilution effectively reduced the PNPase concentration such that it had only a minimal effect on the steady-state measurement of P i -binding MDCC-PBP in these assays Initial velocities for reactions containing 41 nM y complex 50 nM pt DNA, 50 nM p ( when present ), and 3 4 MMDCC-labeled phosphate binding protein in assay buff er were measured for reactions containing a range in molarity of the substrate ATP ( 0 6 1 1 2 6 7 10 20 40 and 77 ). For analysis of the minimal complex reaction s contained 100 nM y 88 ', 100 nM pt DNA 312 5 nM p In all experiments the emi s sion intensity mea s ured prior to the addition of ATP was recorded as the intensity of P i -free MDCC-PBP. Following e ach reaction 1 L of potassium pho s phate ( 16 .2 M) was added to give a final s aturating ex c e ss concentration of P i (200 The saturated emis s ion intensity was recorded a s the inten s ity of completely P i bound :MDCC-PBP I

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' 103 The concentrations of ATP stocks were determined from the average of at leas t 3 absorbance measurements at 259 nm using the molar extinction coefficient at 259 nm ( 15 The mole fraction ofMDCC-PBP bound to Pi Xt,(t) was calculated by subtracting the intensity of free MDCC-PBP (Ir) from observed intensities at time t loh s (t) and dividing this difference by the difference of fully saturated Pi -bound MDCC-PBP (lb) and free MDCC-PBP Xb(t) = I:lo bs( t)Ir] / [l b Ir]. Reactions were perfo11ned 3 times and the initial velocities were calculated as the linear slope of P i release (M) from clamp loader ATPase activity (:S 10% product). Initial velocities were plotted versus ATP concentration then analyzed using the Michaelis-Menten equation to determine steady state ATP hydrolysis parameters, where [S] is the concentration of ATP Vmax[S] vt + [S] Michaelis-Menten equation Effect of (3 concentration on the steady-state ATP hydrolysis activity of the clamp loader The steady-state MDCC-PBP ATPase assay was perfo11ned as described above with different concentrations of (3. For analysis of y complex a range of 50, 100, 200 and 740 nM '3 was used For analysis of y 3 88' minimal complex, a range of 200 ,3 10 600, 1500 nM (3 was used The concentrations were 41 nM y complex 50 nM p / t DNA, and 3.4 M :tvIDCC-labeled phosphate binding protein in assay buffer or for the minimal complex, reaction s contained 100 nM y 3 88 ', 100 nM pt DNA, and 3 4 M MDCC-PBP.

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104 Steady-state A TPyS-chase assay Steady-state ATP hydrolysis activity of the clamp loader was chased by addition of I non-hydrolyzable ATPyS after initiation of the MDCC-PBP ATPase assay The ATPyS' chase assay was performed by initiating a timebased fluorescence emission measurement of a solution containing 50 nM clamp loader 150 nM p ( when present), 150 nM pt DNA, and 3 4 M MDCC-PBP in assay buffer that was P i -mopped as described above for the steady-state MDCC-PBP ATPase assay After 14 s 4-L of a solution containing ATP was added to this solution giving a final concentration of 77 M ATP A constant volume of ATPyS ( 4 L) was then added after ~ 10-s into the reaction time course The A TPyS concentration was varied to produce differing final concentrations over the range of : 20, 40 80 160 and 800 Min separate chase reactions Pre-Steady-State Kinetics of ATP Hydrolysis Measurement of the change in fluorescence of MDCC-PBP in real-time was carried out on a SX 18MV Stopped-Flow Reaction Analyzer (Applied Photophysics Ltd., UK) Excitation light from a 150-W xenon arc source was at 425 nm through a monochromator with a 0 5 mm band pass Fluorescence emission intensity was recorded through a 455 nm cut-on filter (CVI Laser Corp ., Albuquerque NM) by an analogue PMT-detection device Data points were collected every 1 ms for the first 2 s of the reaction time course then every 5 ms for the remaining 10 s using a split-timebase Al] experiments were performed at 20 C The stopped-flow apparatus and all reagents were P1-mopped (0.14 U / mL PNPase, 168 M MEG) for ~ 45 min prior to each experiment Generally a sequential-mix '' thr ee sy ring e'' experiment was performed where 80 L each of the contents of two syringes were mixed and preincubated for 1 s The I

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' 105 preincubated solution ( 80 ) wa s then rapidly mixed with 80 L of the contents of a third syringe This action both initiated the reaction and triggered the detection system The stopped-flow reaction dead time under these conditions was 1 47 ms determined using the method of (Peterman 1979 ) as described below E ach assay typically had three I steps 1 ) An initial measurement of the fluore s cence intensity of P i -free MDCC-PBP in the presence of pt DNA, clamp loader and P (when present ), and no ATP 2) One or more experimental ' shots '' in the pr~sence of ATP 3 ) A final shot under conditions of saturating excess P i for measurement of the fluorescence intensity of completely P i -bound MDCC-PBP Standard pre-steady-state MDCC-PBP A TPase assay In a s tandard sequential-mix ' three-syringe '' assay one s yringe was loaded with 1 08 M clamp loader and 4 Mp (when present) in assay buffer ( Table 3-1 ). A second syringe was loaded with 400 M ATP in assay buffer For initial mea s urement of the fluorescence intensity of Pi-free MDCC-PBP as described in step one above this second syringe initially contained assay buffer onl y then was replaced with the solution containing ATP for the experimental shots The content s of these two syringes were mixed and preincubated for 1 s as above then mixed with the contents of a third syringe loaded with 5 4 M MDCC-PBP and 2 M pt DNA in assay buffer Final reaction concentrations were 270 nM clamp loader 1 MP ( when present ), 1 M pt DNA 2 7 M MDCC-PBP, and 100 M ATP For the fmal saturated emis s ion inten s ity measurement of completely P i -bound MDCC-PBP, the syringe containing ATP was removed and 14 L potassium phosphate ( 16 2 mM) was added to the syringe m i xed

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106 with the remaining ATP solution, and then flushed into the stopped-flow A fmal experiplental shot added ~ 200 M excess Pi to the reaction mixture Pre-steady-state MDCC-PBP ATPyS-chase assay ' ''Back-to-back '' pre-steady-state :MDCC-PBP ATPase assays were perfo1med in the presence and absence of J3 where ATP hydrolysis activity was chased by mixing with ATPyS at initiation of the reaction For these sequential-mix '' three-syringe.,, ATPyS chase assays, one syringe was loaded with 1 08 M clamp loader, and 4 M J3 (when present) in assay buffer After the initial measurement of the fluorescence intensity of Pi free MDCC-PBP as described in step one above, the second syringe, originally containing assay buffer only, was exchanged with a syringe loaded with 400 M ATP in assay buffer The contents of these two syringes were mixed and preincubated for I s, then mixed with the contents of a third syringe loaded with 2 mM A TPyS, 5 4 M MDCC-PBP and 2 M pt DNA in assay buffer Final reaction concentrations were 270 nM clamp loader, 1 M J3 (when present) I M pt DNA, 2.7 MMDCC-PBP 100 M ATP and I mM ATPyS (a 10-fold excess over ATP) For the final saturated emission intensity measurement of completely P i -bound MDCC-PBP, the syringe containing ATP was removed, and 14 L potassium phosphate (16 2 mM) was added to the syringe, mixed with the remaining ATP solution, and then flushed into the stopped-flow A fmal experimental shot added ~ 200 M excess P i to the reaction mixture Measurement of the effect of y complex concentration in the pre-steady-state MDCC-PBP ATPase assay The above standard pre-steady-state sequential-mix ''three-syringe' assay was performed with a range of concentrations of y complex in the presence of (3 In three I

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i I 107 separate assays one syringe was loaded with 0 4 1 08 or 1 8 My complex and 6 4 M J3 in assay buffer After the initial measurement of the fluorescence intensity of Pi-free MDCC PBP as described in step one above the second syringe originally containing assay buffer only was exchanged with a syringe loaded with 1 6 mM ATP in assay I buffer The contents of these two syringes were mixed and preincubated for 1 s, then I mixed with the contents of a third syringe loaded with 5 4 M MDCC-PBP and 3 2 M I pt DNA in assay buffer. Final reaction concetrations were 100 270 or 450 nM y complex 1 6 M (3 1 6 M pt DNA, 2.7 M MDCC-PBP and 400 M ATP For the final saturated emission intensity measurement of completely Pi-bound MDCC-PBP the syringe containing ATP was removed and 14 L potassium phosphate (16 2 mM) was added to the syringe, mixed with the remaining ATP solution, and then flushed into the stopped-flow A final experimental shot added ~ 200 M excess Pi to the reaction mixture. P r e-steady-state MDCC-PBP ATPase assay with varying preincubation periods A standard pre-steady state sequential-mix ''three-syringe ' assay was performed for y complex in the absence of J3 where the preincubation period after mixing the contents of the first two syringes was varied One syringe was loaded with 1 08 My complex in assay buffer. After the initial measurement of the fluorescence intensity of Pi-free MDCC-PBP as described above the second syringe, originally containing assay buffer only was exchanged with a syringe loaded with 1 6 mM ATP in assay buffer In separate experimental shots the contents of these two syringes were mixed and preincubated for 15, 50 100,250,500 or 1000 ms then mixed with the contents of a third syringe loaded with 5 4 M MDCC-PBP and 2 M pt DNA in assay buffer. Final

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108 reaction concentrations were 270 nM y complex 1 M pt DNA 2 7 M MDCC-PBP and 400 M ATP For the final saturated emi s sion intensity measurement of completely t P i -bound MDCC-PBP, the syringe containing ATP was removed and 14 L potassi11m phosphate (16 2 m.M ) was added to the syringe mixed with the remaining ATP solution and then flushed into the stopped-flow A final experimental s hot added ~ 200 M exces s P i to the reaction mixture Pre-steady-state MDCC-PBP ATPase assay with no preincobatioo period This assay is a single-mix experiment utilizing only two syringes of the stopped flow apparatus By using this simpl e '' tw o sy rin ge'' experimental setup there was essentially no preincubation period of clamp loader with ATP prior to reacting with pt DNA in the presence ofMDCC-PBP The stopped-flow reaction dead time under these conditions was 1 6 ms dete1mined using the method of (Peterman 1979) described below One syringe was loaded with 540 nM y complex in assay buffer and the other syringe was loaded with 5 4 M MDCC-PBP 2 M pt DN~ and 800 M ATP in assay buffer The contents of the two syringes were rapidly mixed to initiate the reaction and trigger the detection system. Final reaction concentrations were 270 nM y complex, 1 M pt DNA 2 7 M MDCC-PBP and 400 M ATP For the final saturated emission intensity measurement of completely P i -bound MDCC-PBP the syringe containing ATP was removed and 14 L potassium phosphate (16.2 m.M) was added to the syringe mixed with the remaining ATP solution and then flushed into the stopped-flow A final experimental shot added ~ 200 M exces s P i to the reaction mixture t

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I 109 Determination of the ATP concentration dependence on y complex ATP hydrolysis activity The single-mix '' two-syringe' MDCC-PBP ATPase assay with no preincubation period was also used to explore the concentration dependence of ATP on the kinetics of ATP hydrolysis by y complex In separate assays one syringe was loaded with 540 nM y complex in assay buffer and the other syringe was loaded with 5 4 M MDCC-PBP 2 M pt DNA, and 200 500 or 1 mM ATP in assay buffer The contents of the two syringes were rapidly mixed to initiate the reaction Final reaction concentrations were 270 nM y complex 1 M pt DNA 2.7 M MDCC-PBP and 20 80 100,200,250,400 or 500 M ATP For the final saturated emission intensity measurement of completely Pi-bound MDCC-PBP the syringe containing ATP was removed and 14 L potassium phosphate (16.2 mM) was added to the syringe, mixed with the remaining ATP solution and then flushed into the stopped-flow. A final experimental shot added ~ 200 M excess P i to the reaction mixture Computer Modeling of ATP Hydrolysis Kinetic Data Kinetics data in Figure 4-9 were fit to the model to shown in Figure 4-1 OA using the program DynaFit (Kuzmic 1996). The DNA binding rate has been measured pre viously (Ason et al ., 2000 ; Ason et al ., 2003 ). The forward rate constant for DNA binding was set to a constant value of 23 5 M 1 s 1 determined by fitting the previously published data to our model This va lue is consistent with the 300 400 1 s 1 rates that were reported based on simulation of these data The concentration of MDCC PBP P i as a function of time was calculated by including experimentally dete1mined rate constants for MDCC-PBP binding Pi (Brune et al ., 1994) as constants The total

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110 concentration of 'Y complex was treated as an adjustable parameter to account for the differ~nce between total and active protein concentrations as well as the experimental I accuracy of concentration and volume measurements The fit yielded a conce ntration of I 0 25 M (93o/o active ) 'Y complex for assays containing an experimentally determined of 0 27 M 'Y complex Some steps were modeled using a single forward rate constant because these data do not provide sufficient inf or1nation to calculate a unique pair of forward and reverse rate constants. This does not mean to imply that these steps are irreversible Data in Figure 4-lOC were fit to a version of the model in Figure 4-lOA that included an additional initial step converting an enzyme species PITT to the species ETTI For this fit all of the rate constants shown in Figure 4-lOA were held constant; the rate of conversion of PITT to ETTT kpe and the total concentration of 'Y complex were the only parameters fit This fit yielded a concentration of 0 26 M 'Y complex For simulation of the different species of y complex in a 1-s equilibration (Figure 4-10B) using the model in Figure 4-lOA, the program KinTekSim was used (Barshop et al ., 1983 ; Dang and Frieden 1997 ) Appendix A contains all DynaFit program scripts, and analyses indices used in the computer modeling, as well as the KinTekSim simulations Correlated Pre-Steady-State MDCC-PBP ATPase Assays and Fluorescence Anisotropy Binding Assays The standard sequential-mix '' three-syringe '' assay described above for the SX 18MV apparatus was used for pre-steady-state MDCC-PBP ATPase assays as well as pre-steady-state fluorescence anisotropy measurements under nearly identical experimental conditions The measurement of anisotropy on the SX 18MV stopped-flow apparatus required the use of the FP-1 attachment that housed optics for the polarization

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I 111 of the excitation light at 580 nm and measurement of polarized emission intensities from RhX-pt DNA Also fitted on the apparatus were 610 nm cut-on filters (CVI Laser Corp. Albuquerque NM) specific for the X-rhodamine fluorescent probe Other than these instrumental modifications required for performing anisotropy measurements the standard '' three-syringe '' reaction was unchanged between the MDCC-PBP ATPase assay and anisotropy binding assay For each assay one syringe was loaded with 900 nM clamp loader and 2 M (3 l (when present ) in assa y buffer A second syringe contained 800 M ATP in assay buffer The contents of these two syringes were mixed and pre incubated for a period of 1 s then mixed with the contents of a third syringe loaded with either 10 M MDCC-PBP, and 900 nM pt DNA in assay buffer for the tvIDCC-PBP ATPase assays or 900 nM RhX-pt DNA in assay buffer for anisotropy binding assays Final reaction concentrations were 225 nM y complex, 500 nM p ( when present ), 450 nM pt DNA, 200 M ATP The MDCC-PBP ATPase assays contained a final concentration of 5 M MDCC PBP In the MDCC-PBP A TPase assays the second syringe initially contained assa y buffer only for the initial measurement of the fluorescence intensity of P i -free tvIDCC PBP This second s yringe was then exchanged with a syringe loaded with 400 M ATP in assay buffer For the fmal saturated emission intensity measurement of completely P i bound MDCC-PBP the syringe containing ATP was removed and 14 L potassium phosphate ( 16.2 mM) was added to the syringe mixed with the remaining ATP solution and then flushed into the stopped-flow. A final experimental shot added ~ 200 M excess P i to the reaction mixture

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112 For the anisotropy binding assays RhX-pt DNA with assay buffer only was initialJy placed into the cuvette for setup of the PMTs and software calculation of the G, factor The stopped-flow software ultimately calculates anisotropy in the real-time output I over the time courses of each experimental reaction shot Up to 14 separate experimental rcordings of calculated anisotropy were signal averaged to reduce the signal-to-noise ratio Stopped-Flow Dead Time Determinations Determination of the Dead Time for the Biologic SFM-4 Stopped-Flow The dead time for the Biologic SFM-4 stopped-flow described above was determined using the method of ( Peter1nan, 1979 ). The quenching of N-acetyl tryptophanamide (N-AcTrpNH 2 ) fluorescence by N-Bromosuccinamide (NBS) in sodium phosphate buffer ( pH 7 0) was used as a reaction for dead time determination Rapid mixing of differing concentrations of NBS quencher results in single-exponential decays in N-AcTrpNH 2 fluorescence at different amplitudes and rates N-AcTrpNH 2 fluorescence was excited at 280 run, and emission intensity was measured through 320 nm cut-on filters (CVI Laser Corp ., Albuquerque, NM) Thin-film polarizers were removed from the stopped-flow apparatus in this experimental setup In each reaction one syringe contained 20 M N-AcTrpNH 2 in sodium phosphate buffer ( monobasic) ( pH 7 0 ). The second syringe was loaded with varying concentrations of NBS in sodium phosphate buffer ( roonobasic) ( pH 7 0 ). The contents of each syringe were rapidly mixed in 80-L shots at a 10-mL / s flow-rate Generally 13 reaction shots were signal averaged for each concentration of NBS In between reactions differing in concentration of NBS quencher the stopped-flow syringe containing NBS was

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113 thoroughly rinsed with deionized water The final reactant concentrations were 10 M N-AcTrpNH 2 50 mM sodium phosphate ( monobasic) pH 7 0 and a range of NBS : 312 5 500, 625, 750 I 000, 1250 1500 and 2000 Each fluorescence decay curve was fitted as a single exponential decay in the program SigmaPlot (SPSS Inc ., UK) for determination of the experimental decay amplitudes and rates. Figure 3-2 shows a fit defining the expe rimentally determined rate constants plotted as a function of NBS concentration. This graph shows that the increasing rate of the quenching reaction is linear as a function of NBS concentrations used in this analysis .. ti) .., C !! tn C 0 (,,) ti> l! >ftS j ,, 700 600 600 400 300 200 100 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 [NBS] M Figure 3-2. A plot of observed reaction decay rate s as a function of NBS concentration. The experimentally determined fluorescence decay amplitudes as a function of observed decay rates were fitted to the equation : A = Ao *exp ( -l...--t. ) for the calculation of dead time in Figure 3-3 where tis the dead time A represents the amplitude of the reaction, Ao is the calculated initial amplitude and k is the experimentally observed rate constant

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114 2500 --------------, 2000 .g 1500 ::, a. f; 1000 500 0 4------.---.--...-----.-----.----,----,--------I 0 100 200 300 400 500 600 700 800 decay rate constants (s 1 ) Figure 3-3 Fluorescence decay amplitudes plotted as a function of experimentally ob s erved decay rate constants to detecmine dead time of the SFM-4 stopped flow using a IO mL / s flow rate The red curve is the fitted data to the equation : A = A 0 *e x p < -Jct )_ The calculated dead time ( t ) is 3 3 ms in this example Determination of the Applied Photo physics SX.18MV Stopped-Flow Reaction Analyzer Sequentialand Single-mix Dead Times The dead time for the Applied Photophysics SX l 8MV s topped-flow described above was determined using the method of ( Peterman 1979 ). T he quenching ofN acetyl-tryptophanamide (N-AcTrpNH 2 ) fluorescence by N-Bromosuccinamide (NBS ) in s odium pho s phate buffer ( monobasic ) ( pH 7 0 ) was used as a reaction for dead time determination N-AcTrpNH 2 fluorescence wa s excited at 280 nm and emission intensity wa s measured through a 3 20 run cut-on filter T h e se qu e nt i al m ix t hr e e sy rin ge'' se t u p dead ti,n e wa s determined in an experiment wh e re one s yring e contained a solution of 40 M N-AcTrpNH 2 in sodium phosphate buffer (pH 7 0 ) A second syrin g e wa s loaded with a solution of s odium phosphate buffer only ( pH 7 0 ). The content s of the s e two s yringe s were mi x ed and

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115 preincubated for 1 s, then mixed with the contents of a third syringe loaded with varying concentrations of NBS quencher in sodium phosphate buffer (pH7.0). The third syringe and stopped-flow apparatus were rinsed thoroughly with deionized water in between reactions differing in NBS concentration The fmal reactant concentrations were 10 M N-AcTrpNH 2 50 mM sodium phosphate pH 7 0 and a range of NBS : 312.5 500 625 750 1000 1250 1500 and 2000 Using the data analysis function of the SX 18MV software, the observed ' fluorescence decay reactions were fit to a single exponential equation for deter111ination of the experimental amplitudes and decay rates The '' X-datum '' initial data point was set to 2 4 ms in this analysis as suggested by the manufacturer Changing this initial data point to 2.2 or 2 3 ms had no significant effect on the dead time calculation Figure 3-4 shows the experimentally determined fluorescence decay amplitudes as a function of observed decay rates were fitted to the equation : A = A o *exp < kt), for the calculation of dead time (t). As expected, analysis of the observed rate of the quenching reaction was linear as a function of NBS concentrations used (not shown) The dead time calculated using this method for the sequential-mix setup was 1 47 ms. Analysis of the reaction data recorded by the stopped-flow software for all MDCC-PBP A TPase experiments performed over the course of two years revealed that the average software-calculated dead time was 1 48 (+/0 16 ms) for 241 '' shots' This is in excellent agreement with the reaction dead time experimentally calculated by the Peterman method

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116 2.0 1.6 G) 't, ::, ... 1.2 C. i 0.8 0.4 0 200 400 600 800 1000 1200 1400 1600 1800 2000 decay rate constant (s 1 ) F igure 3-4 Sequential mix reaction fluorescence decay amplitudes plotted as a function of experimentally observed decay rate constants to dete1 rnine dead time of the SX 18MV stopped-flow The red curve is the fitted data to the equation : A = A 0 *exp < -kt )_ The calculated dead time ( t ) is 1 47 ms T h e s in g l e -m ix twosy rin ge' se tup d ea d tim e was detennined in a s et of experiment s where one syringe contained a solution of 20 M N-AcTrpNH 2 in s odium phosphate buffer (pH 7 0) The second s yringe was loaded with varying concentrations of NBS quencher in sodiu1n phosphate buffer ( pH7 0 ) The s econd syringe and stopped flow apparatus were rinsed thoroughly with deionized water in between reactions with differing NBS concentrations The final reactant concentrations were 10 AcTrpNH 2, 50 mM sodium phosphate pH 7 0 and a range ofNBS : 31 2 5 500 625 750 1000 1 2 50 1500 and 2000 M. The e x perimental fluorescence quenching decay amplitudes and rates were deter111ined a s described above using the SX 18MV s topped-flow software Figure 3 -5 shows the experimentally determined fluorescence decay amplitudes as a function of observed decay rates fitted to the equation : A = A 0 *exp < -kt ) for the calculation of dead

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117 time ( t ). The dead time calculated using this method for the single-mix setup was 1 6 ms Analysis of the reaction data recorded by the stopped -flow software for all single-mix MDCC-PBP A TPase experiments perf orrned over the course of two years revealed that the average software -calculated dead time was 1 30 ( +/ 0 13 ms) for 23 '' shots ,'. This is consistent with the reaction dead time experimentally calculated by the Petennan method CD ,, ::, Q. E ftS 2.8 2.4 2.0 1.6 1.2 0.8 0.4 0 200 400 600 800 1000 1200 1400 decay rate constant (s 1 ) Figure 3 -5 Single-mix reaction fluorescence decay amplitudes plotted as a function of experimentally observed decay rate constants to determine dead time of the SX l 8MV stopped-flow The red curve is the fitted data to the equation : A = Ao *exp ( -kt )_ The calculated dead time ( t) is 1 6 ms

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CHAPTER4 ATP-DEPENDENT CONFORMATIONAL CHANGE IN THE CLAMP LOADER Introduction The E sch e ri c hia coli DNA polymerase ill y complex clamp loader assembles the ring-shaped p sliding-clamp onto DNA Once bound to DNA, the clamp topologically links the core polymerase to the template that it is copying to increase the processivity of DNA synthesis ATP binding and hydrolysis promote conforrnational changes within the y complex that modulate its affinity for the clamp and DNA allow it to accomplish the task of assembling clamps on DNA The mechanical task of assembling clamps on DNA requires that proteinprotein and proteinDNA interactions change during a single cycle of a clamp loading reaction The clamp loader must initially have a high affinity for clamps and DNA to bring the clamp to DNA, but then must have a decreased affinity to release the clamp onto DNA and avoid competing with the polymerase for loaded clamps (Jeruzalmi et al., 200 la ; Nakti.nis et al., 1995) ATP binding to they subt1nits induces conformational changes (Podobnik et al. 2003) that expose this region of o allowing the clamp loader to bind with high affrnity and open the clamp ATP binding also increases the affinity of the clamp loader for DNA (Ason et al ., 2000; Bloom et al ., 1996) although a discrete DNA binding site has not yet been identified Subsequent binding to a primed DNA template triggers ATP hydrolysis reducing the affinity of the clamp loader for p and DNA (Ason et al. 2000 ; Ason et al ., 2003) This reduced affinity is likely to be produced by conf ortnational changes that mask and DNA binding 118 I

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I I 119 domains Dissociation of the ADP-bound clamp loader from and DNA allows the clamp to close and gives the polymerase access to the newly loaded clamp. The precise nature of the ATP-induced conformational changes within the clamp loader is not yet known. Successive binding of ATP to each of the three-'Y subunits could potentially promote a series of confotmational changes that ultimately produces an activated complex Structural data for a minimal E c oli clamp-loading complex (y 3 00 ') suggest that conformational changes that occur in each y s ubunit are translated to the o I subunit pulling it away from 6 and exposing the~ binding domain (Jeruzalmi et al ., 2001a ). The conformational flexibility that must be inherent in this complex to produce these structural changes and the presence of three distinct ATP binding sites suggest that several conformational states of the complex could exist in solution In this chapter ATP hydrolysis and clamp loading reactions were measured under steady-state conditions to study the affect has on the clamp loader affinity for DNA and ATP and also to analyze the clamp loader '' Micbaelis-Menten '' kinetics in the presence or absence of~ ATP hydrolysis and clamp loading reactions were also measured under pre-steady-state conditions to identify kinetic phases associated with conformational transitions in they complex ( y 3 o6'm ) on binding ATP A novel ATP hydrolysis assay was used to measure the kinetics of ATP-dependent conformational changes where y complex was incubated with ATP for a defined period of time 0 to 1000 ms to differentially populate confortnational states that are formed on binding ATP DNA was then added to trigger ATP hydrolysis and probe the relative populations of species present Computerized simulation and fitting of the experimental kinetic data were

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120 applied for estimation of the rates of confonnational changes between three species of 'Y comp~ex presented in a novel model Steady-State Characterization of 1 Complex ATP Hydrolysis, DNA Binding and Clamp Loading Activities Enhancement of Steady-State ATP Hydrolysis Kine tics of 1 Complex by P clamp The DNA-dependent A TPase activity of 'Y complex is stimulated by the ~-sliding clamp ( Onrust et al ., 1991 ; Stukenberg et al ., 1991 ) The steady-state kinetics of ATP hydrolysis triggered by primed-template (pt) DNA were re-examined here with a newly designed methodology (i e for study of y complex ) to compare the hydrolysis activity by y complex with pt DNA alone or in the presence of~The fluorescence-based f\.IDCC PBP A TPase assay was utilized for measurement of the initial velocities of ATP hydrolysis activity In MDCC-PBP ATPase assays the kinetics of ATP hydrolysis were measured as a function of inorganic phosphate release from the clamp loader and therefore establish a minimum rate for ATP hydrolysis ( Bertram et al 4 2000; Brune et al. 1998 ). Separate steady-state assays were initiated by addition of a range of molarity of the substrate ATP (from 0 6 to 77 M) to a solution in a cuvette containing 'Y complex pt DNA and MDCC-PBP in the presence or absence of~ The initial reaction velocities under these conditions were measured as a function of an increase in fluorescence of MDCC-PBP upon binding inorganic phosphate Three separate reactions were perfo1med for each concentration of ATP and resulting averaged initial velocities were plotted against ATP concentration and analyzed by fitting to the Michaelis-Menten equation giving the steady-state parameters shown in Table 4-1

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I 121 Table 4-1. Steady state ATP h y drolysi s kinetic s of y complex in the absence and resence of a V max s 0 07 ( 0 02 ) 0 02 ( 0 003 ) kca 1con 1 p ex Sc 2 0 ( 0 4 ) 0 6 ( 0 07 ) K m ( 11 ( 6 7 ) 6 0 ( 3 0 ) kcat comp ex/ Km S 0 2 0 05 0.1 0 04 a S eady-state MDCC-PBP ATPase assays were performed by addition of a range in molarity of ATP from 0 6 to 77 M to a solution of 41 nM y complex with 50 nM ptDNA and 50 nM ( when present ). All as s ays contained 2 4 M tvIDCC-PBP and P i -mopped assay buffer containing 20 mM Tris-HCl 50 mM Na C l 5 mM DTT 40 g/mL BSA and 8 mMMgCl 2. b Standard deviation s were calculate d from three separate steady-state MDCC-PBP ATPase assays c Since the clamE: loader has three subunits capable of hydrolyzing ATP the turnover number ( kca tcomp ex) reflects the activity of the combined activity of the complex These data are consistent with previous analyse s u s ing autoradiography 32 P-based methods for measuring the DNA-dependent ATP hydrolysis activity of y complex in the presence and absence of~ (Bertram et al ., 2000 ; Hingorani et al ., 1999 ). The kcat complex was increased approximately 3 -fold and the Km value roughly doubled in the presence of j3 The kca tcom p lex / K m '' specificity constant' value from this analysis can be understood as an apparent second~order binding constant for ATP Interestingly this value is not what would be expected for diffusion-controlled binding of a small nucleotide substrate such as ATP Therefore this result sugge s ts that A T P binding was followed by a s low s tep in the reaction before hydrolysi s activity The ATP specificity constant kcatcomplex / K m value was increased in the presence of~ clamp about 1 8-f old This measure of enhancement of ATP binding specificity in the presence of~ demonstrates that partitions a fraction of y complex toward s a more active clamp loading form po ss ibly increasing or bypassing the rate of the s low step preceding ATP hydrolysis The results of steady-state ATP hydrolysis activity are outlined and discussed in additional detail in the followin g chapter

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122 where 'Y complex activity is compared to the activity of the minimal clamp loader comp1ex (Table 5-1 ). Steady-State DNA Binding and Clamp Loading Activities of y Complex Examination of the steady-state DNA binding and clamp loading kinetics of y c,omplex was accomplished using the fluorescence-based anisotropy DNA binding assay The y complex was studied in the presence and absence of ~ to observe the DNA binding activity in solution based assays In original studies measurement of DNA replication activity was used as a gauge of clamp loading activity by y complex (Maki and Kornberg 1988 ; Stukenberg et al ., 1991 ). The solution-based anisotropy DNA binding assay offers study of 'Y complex activity under equilibrium conditions and was originally used for investigation of y complex by (Bloom et al ., 1996), and later extended for clamp loading assays with mutant~ clamps (Bertram et al ., 1998) In some labs gel filtration assays under non-equilibn11m conditions are still used to assay the clamp loading activity of y complex ( Johnson and O'Donnell 2003 ). Here the anisotropy DNA binding assay was used to monitor the reaction of y complex with X-Rhodamine (RhX ) -labeled pt DNA in the absence or presence of~ over extended periods of time In these assays the observed steady-state anisotropy of the RhX-probe on DNA is a population weighted average of the combined anisotropies for free DNA and protein-bound DNA Therefore the observed anisotropy increases as the population of protein-bound DNA increases (Bloom et al ., 1996) Polarized emission intensities of the RhX-probe covalently attached to pt DNA were measured and used in calculation of a nisotropy over time The s teady-state measurement of DNA binding activity or clamp loading activity was observed in assays where ATP was added to a solution ofRhX-ptDNA, clamp ( when present) and y complex to initiate the reaction.

PAGE 138

123 0.32 ~-----------, 0.30 Ycomplex + 0.28 e 0.26 0 U> C 0.24 Ycomplex tV 0.22 0.20 0 200 400 600 800 1000 time (s) Figure 4-1 Steady-state DNA binding and clamp loading activities of y complex The change in steady-state anisotropy of a 30-nucleotide-primed 105-nucleotide template DNA labeled with RhX at the 5 end is plotted against time for y complex ( blue ), and for the clamp loading reaction ; y complex in the presence of P ( black) Reactions contained 50 nM RhX-ptDNA, 500 nM y complex 500 nM P ( when present ), and 0 3 mM ATP in a solution of assay buffer : 8 mM MgCl 2, 20 mM Tris-HCI pH 7 5 50 mM NaCl 5 mM DTT 40 g/mL BSA Steady-state polarized intensities from the RhX probe were recorded once per second for 30 minutes. The plot shows a slightly expanded time s cale since both reactions no longer showed activity for the longer time period A 30-second plot of anisotropy ofRhX-pt DNA free in solution i s shown ( gray ). Figure 4-1 shows the resulting time courses for steady-state anisotropy of DNA binding b y y complex in the presence or absence of p. The steady-state anisotropy ofRhX-pt DNA was approximately 0 21 and was increased slightly (~ 0 23) in the DNA binding reaction with y complex in the absence of p T hi s low activity s lowly decayed back to the level of free RhX-pt DNA over the course of the assay In the presence of p y complex catalyzed the clamp loading reaction

PAGE 139

' I 124 on this RhX-pt DNA substrate (Ason et al ., 2000) A large increase in anisotropy was initially seen ( ~ 0 29) consistent with a dynamic interaction of 'Y complex and P with the RhX-pt DNA The loaded p clamps easily slide off of this short DNA substrate keeping their concentration essentially in constant equilibrium. This high anisotropy value was sustained for more than 200 s before decaying to the level of free RhX-pt DNA over and additional ~ 450 s indicating that the clamp loading reaction continued at a constant rate until all of the ATP was used up by 'Y complex It was also possible that a large amount I of the ADP product of hydrolysis built up and began to inhibit the reaction. The difference observed in these assays between 'Y complex DNA binding and clamp loading activities in the presence or absence of p, indicates that p enhanced that activity of the clamp loader consistent with the steady-state hydrolysis assays described above The sustained clamp loading reaction suggests that p clamp increased the affinity for ATP binding 'Y complex, or possibly increased the substrate (ATP) specificity of the clamp loader The steady-state anisotropy binding (DNA binding/clamp loading) assay was used for further analysis of the clamp loading reaction Assays were performed to identify the concentration of ATP at which the clamp loading reaction was no longer sustained Figure 4-2 shows the results of ten separate clamp loading reactions where the final concentrations of ATP were decreased from 450 to 25 The steady-state clamp loading reactions were again recorded over an extended time course after addition of varied concentrations of ATP to a solution ofRhX-pt DNA, 'Y complex and p. Polarized emission of the RhX-pt DNA was measured for calculation of anisotropy, and the assays

PAGE 140

125 were allowed to run until the reaction anisotropy reached a level equivalent to free RhX pt DNA indicating that all of the ATP was used up 0.32 0.45 r .. _, rtne y= r .. + :IE 0 40 1 x-x 1 l+exp b o E o 35 0.30 0.30 D. 0.25 <( 0 20 0.28 .., 0 15 0.10 0. 0.05 e 0 ... 0.26 0 0 100 200 300 400 500 600 700 800 900 "' inflection pt. (s) C C'G 0.24 0.22 0.20 .....__ ________ ...------.......---------.--J 0 200 400 600 800 1000 1200 1400 1600 1800 time (s) Figure 4-2 Steady-state kinetic s of clamp loading a s a function of ATP concentration Varied concentrations of ATP were added to a solution ofRhX-ptDNA y complex and ~ to initiate clamp loading reactions ATP concentrations were ( left-to-right in the plot ) ( : 25 (red ), 38 ( green ), 50 (blue ), 75 (pink ), 100 ( yellow ), 150 ( cyan ), 175 ( dark green ), 200 ( dark red ), 300 ( black ), and 450 ( magenta ) Final reactant concentrations were, 50 nM RhX-ptDNA 500 nM y complex and 500 nM in solutions of assay buffer : 8 mM MgCl 2, 20 mM Tris-HCl pH 7 5 50 mM NaCl 5 mM DT T, 40g/mL BSA Steady-state polarized intensities from the RhX probe were recorded once per second for 10 min or 30 min, depending on the ATP concentration. A 30 second plot of anisotropy of RhX-pt DNA free in solution ( r rree) is shown ( gray ). The curves were fitted to a 4-parameter sigmoid logistic equation in SigmaPlot 8 0 software shown in the inset The inset shows the calculated points of inflection of each curve ( i e where tangents of the curves meet at the center of the sigmoidal decay) plotted against the concentration of ATP added to each assay The red line is a linear fit to the data with they-intercept equal to 4 88 MATP

PAGE 141

126 It was observed that an ATP concentration between 25 and 38 M (Figure 4-2, red and green left-most curves) was unable to sustain the clamp loading reaction and the anisotropy quickly decayed to the level of unbound RhX-pt DNA In contrast, with increasing concentration of ATP the steady-state clamp loading activity was sustained for increasing amounts of time This indicated that the point at which the anisotropy transitions into a decay phase there was no longer enough ATP available for 'Y complex to continue loading ~ At the highest concentration of ATP ( 450 the clamp loading reaction was maintained up to roughly 12 min ( ~ 750 s). Each of the clamp loading reaction curves was fitted to a four-parameter sigmoid logistic function (see equation Figure 4-2 inset) Fitting the curves to this equation revealed that the slope of the decay increased with decreasing concentrations of ATP (b value) and that the point of inflection during the decay phase increased in time (Xo value) with increasing ATP concentrations The inflection point represents the midpoint anisotropy value of the decay phase for each reaction, presumably where there is no longer enough ATP available for clamp loading activity. By plotting the calculated inflection points as a function of starting ATP concentration (Figure 4-2 inset) and fitting these data to a linear regression, the concentration of ATP present at zero time intercept) was estimated to be 4.88 Since ATP binding is required for 'Y complex to bind with high affinity this value indicates the concentration of ATP bound to y complex at initiation of the clamp loading reaction and represents an apparent dissociation constant for ATP binding This apparent~ is consistent with previously the previously measured value ( ~ 2 M) (Hingorani and O Donnell 1998), and also with the apparent measured in this dissertation project with anisotropy binding assays using I

PAGE 142

I 127 fluorescent-labeled '3 clamp ( ~ 2 M) (see Figure 5-4). This apparent Kd for ATP was used in this research project for dete11nination of a appropriate molar-range of ATP in the design of the steady-state 1v1DCC-PBP ATPase assay presented above and again in the following chapter These assays were performed early in this dissertation project and the I resulting DNA binding and clamp loading kinetics were later addressed in additional detail by pre-steady-state analysis Pre-Steady-State Kinetics of DNA-Dependent ATP Hydrolysis by r Complex I I Pre-Steady-State MDCC-PBP ATPase Assays for r Complex in the Absence and Presence of p Clamp Pre-steady-state kinetics of ATP hydrolysis by 'Y complex were measured in assays with and without the clamp to dete11nine how alters the kinetics of ATP hydrolysis A real time fluorescence-based assay containing E. c oli phosphate binding protein (PBP) covalently labeled with MDCC was used to measure the amount of inorganic phosphate produced on hydrolysis of ATP (Bertram et al ., 2000; Brune et al ., 1994) When MDCC-PBP binds inorganic phosphate in a 1 : 1 stoichiometry an increase in the fluorescence of MDCC occurs. Sequential mixing assays were perfo1med by incubating y complex (0.27 ) with ATP (100 M) in the presence or absence of~ (1 M) for 1 s prior to adding a solution of DNA (1 M) to trigger hydrolysis and MDCC PBP (2 7 M) to give the final concentrations indicated in parentheses. The DNA substrate used in all experiments consisted of a 30-nt primer annealed to a 105-nt template and supports loading of the~ clamp and processive synthesis by pol III core (Ason et al ., 2000 ; Bloom et al ., 1996 ; Hingorani et al ., 1999) In assays with y complex alone (Figure 4-3, black trace) the first turnover of ATP is biphasic and is followed by a slower steady-state phase beginning in about 700 ms

PAGE 143

n ,,... .._ :i ::1. 0. I Q. al 0.. I 0 0 C :i 1 4 1 2 1.0 0 8 0.6 0 4 0 2 0 0 0 0 0 2 0 4 0 6 128 + ~ 0.8 time (s) no J3 1 0 1 2 1 4 1 6 F igure 4-3 Kinetics of ATP hydrolysis by y complex in the presence and absence of p The ATP hydroly s is kinetic s for 'Y complex in the ab s ence ( black) and pre s ence of~ ( red ) were mea s ured in real time Using a sequential-mix ~hree s yringe '' stopped-flow assay one syringe loaded with a solution containing 'Y comp l ex and p ( when present ) was mixed with the content s of a second s yringe loaded with ATP T he re s ulting mixture was preincubated for a period of 1 s and then mixed with the contents of a third syringe loaded with a solution-containing pt DNA and MD C C PBP All solutions were prepared in P i -mopped assay buffer : 20 mM Tri s -HCI 50 mM NaCl 5 mM DT T, 40 g/ mL BSA an d 8 rnM MgC1 2 Final reactant concentrations were 270 nM 'Y complex 1 0 M ( when present ) 100 M ATP 1 0 M pt DNA and 2 7 M MD C C-PBP The flat trace ( gray ) at the bottom of the figure showing no change was a negative control a s say performed in the absence of ATP Raw fluorescence data were transforined into the concentration of P i bound MDCC-PBP a s a function of time u s ing the equation X b( t ) = [I obs ( t If] / [I b I r ] to s olve for the fraction of P i -bound MD C C-PBP X b ( t ) then multiplying thi s value by the concentration ofMDCC PBP present in the as s a y to get the va l ue MDC C -PBP P i ( M) plotted I r was the fluorescence intensity of P i free MDCC-PBP in as s ay buffer and l b was the saturated fluorescence intensity of completel y P i -bound MDC C PBP in the presence of 200 M pota s sium phosphate ( P i source )

PAGE 144

129 The rapid pre-steady-state phase of ATP hydrolysis is complete within about 100 ms where~ slower phase lasts about 600 ms The amplitude of the rapid phase represents I about 60 o/ o of the total burst amplitude In contrast to assays with y complex alone, a ' single pre-steady-state phase of ATP hydrolysis is present in reactions with and the second slower phase is not Addition of~ (Figure 4-3B red trace) increases both the overall rate of the first turnover of ATP and the steady-state rate of ATP hydrolysis The burst of ATP hydrolysis in reactions with~ occurs at the same rate as the rapid phase in assays without p and its amplitude is equal to the sum of the amplitudes of the rapid and slow phases in assays without p ( Figure 4-3 black trace) Burst amplitudes indicate that 2 7 molecules of ATP are hydrolyzed per molecule ofy complex in the presence and absence of~ The presence of p increases the overall pre-steady-state rate of ATP hydrolysis by increasing the relative proportion of the rapid reaction Pre-Steady-State MDCC-PBP A TPase Assays at Different Concentrations of 'Y Complex in the Presence of P The pre-steady-state MDCC-PBP ATPase assay is essentially an active-site titration of the clamp loader enzyme, allowing quantification of the number of ATP molecules hydrolyzed during the first turnover of the clamp loading reaction During development of the assay it was questionable if the real time fluorescence response by MDCC-PBP in this assay was reliably measuring the molar release of inorganic phosphate product from hydrolysis of ATP by y complex At the time only one other group was using this pre-steady-state assay to quantify the number of molecules of ATP hydrolyzed by an enzyme (PcrA helicase) during its reaction (Dillingham et al 2000 ) I

PAGE 145

2.8 2.6 2.4 2.2 2.0 :i ::1. .. 1.8 0. 1.6 I 0. 1.4 m 0. 1.2 I (.) 1.0 (.) C 0.8 :e 0.6 0.4 0.2 0.0 130 Q) a. 3 E 0 CJ 2 >fl) 1 0.. 0 0 0 0.1 0 2 0,3 time (s) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 time (s) Figure 4-4 Quantification of the number of ATP molecules hydrolyzed by y complex in the frrst turnover of~ clamp loading The pre-steady-state ATP hydrolysis activity during clamp loading by y complex was measured at concentrations of 87 nM (black trace) 270 nM (blue trace middle) and 450 nM (red trace) using the sequential-mix 'three-syringe' stopped-flow MDCC-PBP A TPase assay One syringe loaded with a solution containing varying concentrations of y complex with was mixed with the contents of a second syringe loaded with ATP The resulting mixture was preincubated for a period of 1 s and then mixed with the contents of a third syringe loaded with a solution containing pt DNA and MDCC-PBP All solutions were prepared in P i mopped assay buffer : 20 mM Tris-HCI, 50 mM NaCl 5 mM DTT 40 g/mL BSA and 8 mM MgCl 2. Final reactant concentrations were 87 270 or 450 nM y complex 1 6 M ~, 400 M ATP 1 6 M pt DNA, and 2.6 MMDCC PBP The flat trace (gray) at the bottom of the figure showing no change was a negative control assay performed in the absence of ATP The inset shows the moles of ATP hydrolyzed per mole of y complex during the '' burst' phase for all three clamp loading reactions was the same (trace colors as indicated above ). Raw fluorescence data were transformed into the concentration of P i bound MDCC-PBP using the equation described in figure 4-3. To determine if our MDCC-PBP A TPase assay was responding specifically to the nwnber of ATP molecules hydrolyzed by y complex during the clamp loading reaction

PAGE 146

131 three separate assays were performed at different concentrations of y complex while the MDCC-PBP concentration was held constant Sequential mixing assays were performed where varying concentrations of y complex in the presence of~ were mixed with ATP then preincubated for 1 s before mixing with pt DNA and MDCC-PBP Figure 4-4 shows that the pre-steady-state '' burst ' amplitude of the molar MDCC-PBP-P i response changed in proportion to the concentration of y complex This result demonstrates that the same number of ATP molecules were hydrolyzed in the first turnover of the clamp loading I reaction when the relative concentration of y complex to MDCC-PBP was varied Figure 4-4, inset shows that approximately three molecules of ATP were hydrolyzed per molecule of y complex at a similar rate during the first turnover in each assay This result is consistent with the clamp loader structure containing three AAA + y (ATPase) subunits (Jeruzalmi et al ., 2001a), suggesting that each y subunit hydrolyzed one molecule of ATP during these assays. ATPyS-Chase of Pre-Steady-State ATP Hydrolysis Activity by y Complex ATPyS '' chase '' experiments were perfo1med to deterrnine the fate of ATP bound by y complex on addition of DNA These chase assays were done under identical sequential mixing conditions to pre-steady-state ATPase assays except that ATPyS ( 1 mM final concentration ) was added to the solution of DNA and 11DCC-PBP ATPyS is hydrolyzed 4 orders of magnitude more slowly than ATP by y complex (Hingorani and O'Donnell, 1998 ) and therefore is effectively non-hydrolyzable on the time scale of these experiments In these assays y complex is initially bound to ATP and addition of an excess ( IO-fold) of ATPyS to assays will limit y complex to a single round of ATP hydrolysis If securely bound ~ ATP is hydrolyzed more rapidly than it dissociates, then the pre-steady-state kinetics of ATP hydrolysis s hould be unaffected

PAGE 147

Figure 4-5 : Pre-steadys tate kinetics of ATP hydrolysis by y complex in the pre s ence and absence of the clamp in assays with and without an A TPyS cha s e Sequential mixing '' three-syringe ' MDCC-PBP A TPase assays were performed where y complex was incubated with ATP in the A ) absence ( black trace) or B ) presence (black trace) of~ .. for I s prior to the addition of solution containing DNA and MDCC PBP ATPyS chase experiments were done under identical conditions except that A TPyS was included in the solution of DNA and :tv1DCC PBP (red labeled + A TPyS ). The increase in fluorescence ofl\4:DCC PBP when it bound the P i product of ATP hydrolysis was measured in real time Fluorescence intensities were converted to the concentration ofMDCC-PBP bound to P i using the equation described in figure 4-3 and are plotted as a function of time Control experiments done in the absence of ATP are also shown (gray traces, labeled no ATP ). Final reaction concentrations were 0 27 My complex 100 M ATP 1 0 M clamp ( when present) 1 0 M DNA and 2 7 M :MDCC-PBP in assay buffer containing 20 mM TrisHCI pH 7 5 50 mM NaCl 8 mM MgC1 2 5 mM DTT and 40 g/ ml BSA In A TPyS chase assays, ATPyS at a final concentration of 1 mM was added to the solution of DNA and MDCC-PBP

PAGE 148

.-. :i ::1. -,. vr 0. I 0. al 0. I 0 0 C :E .-. :i .. ;; a. I a. al Q. I 0 0 C ::E 1.0 0.8 0.6 0.4 0.2 0.0 1 4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 1 33 A. +ATPyS no ATP 0.0 0.2 0.4 0.6 0.8 1.0 1 2 1.4 1.6 1.8 2.0 time (s) B. + ATPyS no ATP 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 time (s)

PAGE 149

134 If ATP dissociation competes with hydrolysis then bound ATP can be exchanged for ATPyS and the amount of ATP hydrolyzed will reflect relative rates of hydrolysis and dissociation In preliminary steady-state A TPyS chase assays, ATP hydrolysis activity was completely abrogated following addition of a 10-fold excess of ATPyS to reactions both in the absence and presence of p The steady-state ATP hydrolysis activity of 'Y complex in the absence of P showed some ability to continue in the presence of lower ATPyS : ATP ratios however the ATP hydrolysis activity in the presence of~ was poisoned even at substoichiometric ATPyS : ATP ratios Further details of these steady state chase reactions are outlined and discussed in chapter 5 of this dissertation. In assays without (Figure 4 5A red trace, + A TPyS) addition of A TPyS reduces the amount of ATP hydrolyzed by 'Y complex in the frrst twnover The rapid phase of ATP hydrolysis remains but most of the ATP that is hydrolyzed in the slow phase is no longer hydrolyzed. This result shows that ATP hydrolyzed in the rapid phase is hydrolyzed more rapidly than it dissociates whereas ATP dissociation is more rapid than hydrolysis in the slow phase In assays with p (Figure 4-5B red trace + ATPyS), addition of ATPyS does not affect the amplitude of the burst of ATP hydrolysis indicating that bound ATP is hydrolyzed more rapidly than it dissociates. Together these ATPase assays demonstrate that the p clamp increases both the pre-steady-state and state-state rates of ATP hydrolysis by y complex The sliding clamp appears to help y complex bypass a slower reaction step that fo11r1s product more slowly than it exchanges substrate A key question is what gives rise to the biphasic kinetics of ATP hydrolysis in a ssays without p The two most likely possibilities are that individual

PAGE 150

135 y subunits within the y complex hydrolyze ATP at different rates or that two populations of y cQmplex exist with one binding DNA and hydrolyzing ATP more rapidly than the second Pre-Steady-State Kinetics of p Clamp Loading by 'Y Complex Initiated at Different Steps of the Reaction Cycle To distinguish between the two possible mechanisms resulting in biphasic ATP hydrolysis kinetics in assays without~ the kinetics of y complex loading ~ on DNA were measured in assays that were initiated at different steps in the loading cycle The rationale for this set of experiments is that if two species of y complex give rise to biphasic A TPase kinetics then different populations of these species may be present at different stages of the loading reaction A real time fluorescence anisotropy-based assay was used to measure clamp loading where the anisotropy of an X-rhodamine (RhX) probe covalently attached to DNA (RhX-pt DNA ) increases when the DNA is bound by protein (Bloom et al 1996 ; Otto et al ., 1994) Polarized intensities of the RhX probe were measured during the time course of loading reactions and used to calculate the anisotropy of the probe as a function of time ( Lakowicz 1999 ). Clamp loading reactions were perfo11ned using different mixing schemes to add y complex ATP and to DNA, but all reactions contained final concentrations of 0 25 My complex, 500 M ATP 0 6 My and 0 05 M RhX-DNA. Kinetics of Clamp Loading when 'Y Complex is Equilibrated with ATP In the frrst mixing scheme y complex was pre-incubated with ATP prior to adding~ and DNA Because DNA binding is rapid and a pre-requisite for the burst of ATP hydrolysis (Ason et al ., 2003 ), the DNA binding / clamp loading kinetics are likely to mirror the kinetics of ATP hydrolysis If biphasic kinetics of ATP hydrolysis were due to I

PAGE 151

I I 136 individual 'Y subunits within the y complex hydrolyzing ATP at different rates, then a single species of y complex should fo1m on equilibration with ATP and give rise to relatively rapid monophasic kinetics of clamp loading. On the other hand if two species of y complex were formed on equilibration with ATP one of which rapidly bound p and DNA relative to the other , then biphasic kinetics of clamp loading should occur Clamp loading reactions done using this mixing scheme, produce a biphasic increase in anisotropy consistent with a mechanism where two species load clamps on DNA at I different rates (Figure 4-6A) The increase in anisotropy reaches a maximum value of about 0 31 in 200 ms and is followed by a small decrease to a steady-state value of about 0 3. The small decrease in anisotropy at the transition from the pre-steady-state to steady-state regimes is most likely due to dissociation of they complex from the clamp and DNA (Ason et al ., 2000 ; Bertram et al. 1998) The experimental conditions used in this anisotropy DNA binding assay were mimicked in an analogous pre-steady-state MDCC-PBP ATPase assay for examination of the kinetics of ATP hydrolysis during this clamp loading reaction (see below Figure 4-7) It is possible that when reactions are initiated in this manner y complex can either bind p frrst or bind DNA first If this were the case then the two species that give rise to the biphasic DNA binding/clamp loading kinetics would be free y complex and a P-r complex To test this possibility the experiment was repeated at two additional concentrations of p 1 2 and 2.4 If free y complex were present, increasing the concentration of~ should increase the rate at which 'Y complex binds p relative to DNA This increase in binding rates would be reflected in increases in the rate and amplitude of a kinetic phase due to pry complex binding DNA and a decrease in the amplitude of a

PAGE 152

137 phase due to free y complex binding DNA However increasing the concentration of~ in these reactions does not affect the kinetics of clamp loading (Figure 4-6A). This result I demonstrates that the biphasic clamp loading kinetics may not be the result of free y complex ruid a ~y complex binding DNA Instead, two different populations of y complex may form on equilibration with ATP one of which loads clamps more rapidly than the other. Another important result of these experiments is that the rates of the clamp loading reaction were largely unaffected by increasing the concentration of~ indicating that loading kinetics are independent of the rate of a bimolecular association reaction between and y complex Kinetics of Clamp Loading when y Complex is Equilibrated with ATP and P In A TPase assays the slow phase of ATP hydrolysis is not present when ~, y complex, and ATP are pre-incubated prior to addition of DNA To deterrnine whether the slow phase can also be eliminated in clamp loading reactions a second experiment was done where y complex ATP and~ were present in one stopped -flow syringe where they could establish equilibril1m and RhX-DN A was in the second syringe This reaction was done using the same concentrations of proteins and DNA as in the first mixing scheme Initiating reactions after y complex binds ATP and produces a single phase of increasing anisotropy to a value of about 0.33 in the first 50-100 ms ( Figure 4-6B, black trace) of the time course This rapid increase in anisotropy was followed by a slow decrease over about 500 ms to a steady state value of about 0 3 Again the increase is most likely due to a complex of~ and y complex binding DNA and the decrease due to dissociation of y complex from the clamp that has been loaded on DNA (Ason et al ., 2000 ; Bertram et al ., 1998 ). The anisotropy (0 3) remains higher than free DNA (0 21) because a steady-state population of~ remains bound to DNA (Bloom et al ., 1996)

PAGE 153

. Figure 4-6 Kinetics of clamp loading measuted in reactions that were initiated at different stages of the loading cycle The increase in anisotropy of an RhX probe covalently bound to DNA is plotted as a function of time for clamp loading reactions The anisotropy for free DNA (black) is also plotted and shows no change Assays contain final concentrations of0 25 M 'Y complex 500 MATP 0 05 M DNA and several concentrations of~ as indicated, in assay buffer containing 20 mM TrisHCl pH 7 5 50 mMNaCl 8 mMMgCl 2, 5 mMDTT and 40 g/ml BSA A ) Reactions were initiated by adding a solution of y complex and ATP to a solution of~ and DNA and contained 0 6 (blue ), 1 2 ( red) and 2 4 ( green ) M ~ B ) Reactions were initiated by addition of a solution of 'Y complex, ATP and to a solution of DNA (black trace ) and contain 0 6 M ~ For comparison of kinetics data using the mixing scheme shown in A is also plotted (blue trace) C) Reactions were initiated by adding 'Y complex to a solution of ATP ~ and 'Y complex and contained 0 6 M (black ) or 2 4 M (red ). D ) The clamp loading reactions in C with 0.6 M ~ were also repeated with different final concentrations of ATP : 100 M ( blue ), 250 M ( green ), and 500 M ( red ; same plot as the black trace in C ). The plot in (D ) is on expanded scale of time and anisotropy

PAGE 154

0 .b 0 en C ca Q. g 0 _, C ca 0.32 0 30 0.28 0.26 0.24 0 22 0.20 0.32 0.30 0 28 0.26 0 24 0.22 0 20 139 A. c u vette 0 0 0.1 0 2 0 3 0 4 0.5 0 6 0.7 0 8 0 9 1 0 time (s) B. 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 0 time (s)

PAGE 155

140 0 ATP C. cuvett e o.34 ~ [ P 1 -------~ 0 32 0 30 g0.28 ... 0 0 26 th c cu 0 24 0.22 0 20 0.0 0.1 0.2 0.3 D. 0.4 0 5 0.6 time (s) 0 7 0.8 0 9 1 .0 0.32 ~-------------~ 0 30 0.28 Q. _g 0.26 0 th 2 0.24 ca 0 22 0.20 Li [ATP] o.o 0.1 0.2 0.3 0.4 0 5 0.6 0.7 time (s) Figure 4-6 Co n tinued

PAGE 156

141 Kinetics of Clamp Loading when y Complex is Added Directly to a Solution of ATP, P, and DNA l E quilibration of y complex with ATP produces a biphasic increa s e in anisotropy whereas equilibration with ATP and P produce s only a rapid phase (Figure 4-6B, compare blue and black traces respectively ) If the slow s tep in clamp loading reactions were due to some ATP-dependent confor1national changes then it is possible that slow kinetics would be observed if y complex were not incubated with ATP prior to initiating clamp-loading reactions In a third set of experiment s, reactions were initiated by adding 'Y complex to a solution of P RhX-DNA and ATP Reactions contained 0 25 M 'Y complex, 0 05 MRhX-DNA 0 6 M p and saturating ATP ( 500 see below) as before When reactions are initiated prior to ATP binding a substantial lag of about 30 ms in the kinetics of clamp loading is produced ( Figure 4-6 C) This lag is followed by an increase in anisotropy to a value of about 0 31 that is complete in about 250 ms This increase in anisotropy is complete in about the same time it takes for the slow phase of reaction s using the fir s t mixing scheme ( Figure 4-6A ) These kinetics do not change when the concentration of p i s increased to 2 4 M and thu s are not limited by the rate of 'Y complex binding B The reactions described above initiated by adding 'Y complex to a solution of P RhX-DNA and ATP were repeated at three different concentrations of ATP to examine if ATP binding contributed to the slow step in these clamp loading reactions There was no apparent change in clamp loading kinetics observed when the ATP concentration was lowered from the amount used in the original assay ( Figure 4-6D ) The lag preceding slow clamp loading kinetics remained at approximately 30 ms at each concentration of ATP and was followed by an increase in anisotropy to a value of about 0 31 that was I

PAGE 157

/ 142 complete in about 250 ms Taken together these results demonstrate that the clamp loading reaction initiated by adding y complex to ATP p and DNA is limited by the rate of an intramolecular reaction, possibly ATP-induced confotmational changes in the y complex Kinetics of ATP Hydrolysis During Clamp Loading when y Complex is Equilibrated with ATP A pres teady-state MDCC-PBP A TPase assay was perfotmed under mixing conditions that mimicked those u se d in the pre-steady-state clamp loading anisotropy assay shown in figure 4-6A In that assay biphasic DNA binding/clamp loading kinetics were observed The biphasic clamp loading kinetics related to the biphasic ATP hydrolysis kinetics originally seen in the MDCC-PBP A TPase assay where y complex was equilibrated with ATP, and then mixed with pt DNA (Figure 4-3, black trace) Together these results indicated that there were two specie s of y complex present upon equilibration with ATP prior to reacting with DNA and Pin the clamp loading assay To investigate the kinetics of ATP hydrolysis when y complex is equilibrated with ATP and mixed with a solution of pt DNA and p a sequential mix MDCC-PBP A TPase assay was performed In this assay one syringe containing a solution of y complex ( 0 27 M) was mixed with a solution containing ATP ( 400 M) from a secon d syringe and preincubated for a duration of 2 s before mixing with a solution from a third syringe containing pt DNA ( 1 0 p ( 1 0 ), and MDCC-PBP ( 2 7 ). Final reaction concentrations are as indicated in parentheses Figure 47 shows a direct comparison of the ATP hydrol ysis kinetics for this clamp loading reaction with the biphasic hydrolysis kinetics originally observed with pt DNA only.

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143 1.4 --------r---------------~ 1.2 .-. 1.0 :i ::1. cuvette ._. 0. 0.8 I 0. al 0. 0.6 I 0 0 C 0.4 :i 0.2 0 .0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 time (s) Figure 4-7 Kinetics of ATP hydrolysis during the clamp loading reaction when 'Y complex is equilibrated with ATP Using a sequential-mix '' three-syringe '' MDCC-PBP ATPase assay with a mixing scheme ( inset ) analogous to that used in the anisotropy DNA binding assay ( figure 4-6A ), the ATP hydrolysi s kinetics of the clamp loading reaction where measured ( red trace ). One syringe loaded with a solution containing 'Y complex was mixed with the contents of a second syringe loaded with ATP The resulting mixture wa s pre incubated for a period of 2 s and then mixed with the contents of a third syringe loaded with a solution-containing pt DNA, p and 1v1DCC-PBP. The mixing scheme shown in the inset represents the contents of the second and third syringes All solution s were prepared in P i -mopped assay buffer : 20 mM Tris-HCI 50 mM NaCl 5 mM DTT 40 / mL BSA and 8 mM MgC1 2 Final reactant concentrations were 270 nM minimal complex or y complex 1 0 M ~ 400 M ATP 1 0 M pt DNA and 2 7 M MDCC-PBP The reaction ofy complex with pt DNA only was performed under identical conditions, except that there wa s no p present in the third syringe (black trace ). The flat trace (gray ) at the bottom of the plot showing no change was a negative control as s a y perfonned in the absence of ATP The s olid curve (blue ) is a fit of the clamp loading A TPase data ( red ) to an empirical expression regresenting a s ingle exponential rise followed by a linear rise : y = A 0 [l-exp (-ct) ] + C ( t ). Raw fluorescence data were transformed into the concentration of P i -bound lvID C C-PBP u s ing the equation shown in figure 4-3

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144 The kinetics for the clamp loading reaction ( red trace ) showed a rapid s ingle exponential rise thfit led directly into a s teady-state linear ri s e ( 0 74 Ms 1 ). The steady-state phase of the clamp loading reaction wa s identical to that previously ob s erved for reactions when 'Y ' complex was equilibrated with ATP and before mixing with DNA ( compare to Figure 4T5B black trace ). The single rapid phase matched s imilarly in amplitude and rate with the pre-steady-state rapid phase of the biphasic reaction with pt DNA only (Figure 4-7 black trace ). These results suggest that the predicted population o f y complex active specie s, (~ 40 o/ o ) in equilibrium with ATP rapidly bound to and loaded~ then entered a s teady-state clamp loading reaction as the smaller population of inactive species was converted to an active s pecie s, analogou s to the biphasic clamp loading anisotropy data above ( Figure 4-6A ). This result al s o suggests that the second phase observed in the biphasic anisotropy D N A binding assays is possibly equivalent to the rate of conformational chan g e ( s ) within 'Y complex, and that thi s rate limits the steady-state clamp loading reaction Kinetics of ATP Hydrolysis when y Complex is not Equilibrated with ATP Slow kinetic s of clamp loading were observed when 'Y complex was not preincubated with ATP prior to initiating reaction s. To determine whether this was also the case in A TPase as s a y s and whether the rate of ATP binding wa s contributing to the kinetic s of clamp loading s ingle mixin g A TPa s e a s says were done where 'Y complex was added directly to a s olution of ATP DN A, and MD CC -PBP Reactions contained 0. 2 7 My comple x, 1 M DNA 2. 7 M MD CC -PBP and 20 80 200 400 M ATP As in clamp loading assa ys, a s ub s tantial lag in product fo11nation preceded a burst of ATP h y droly s is ( Figure 4-8 )

PAGE 160

tl 0 >
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I I 146 result of these experiments i s that the bipha s ic nature of the burst of ATP hydrolysis persists even when 'Y complex i s not equilibrated with ATP The rate of the rapid phase of ATP hydrolysi s is reduced relative to reactions where 'Y complex wa s equilibrated with ATP making the biphasic nature of the burst more difficult to identify by visual I inspection alone Kinetics of Formation of the Two Populations of y Complex The results of the experiment s presented above demonstrate that two populations of y complex are forrned when y complex is eqilibrated with ATP and that an intramolecular reaction possibly ATP-induced conformational changes limits the observed rate of clamp loading reactions when y complex is not pre-incubated with ATP To more clearly define the kinetics of the confo11national transitions that occur when 'Y complex binds ATP a series of sequential mixing reactions were perfo11ned where the length of time that 'Y complex was incubated with ATP was varied If an ATP-induced conformational change limits the observed rates of clamp loading and ATP hydrolysis in assay s where 'Y complex is not pre-incubated with ATP then the populations of the conformational states should vary as a function of the incubation time with ATP After a defined preincubation time DNA was added to trigger ATP hydrolysis and probe the relative populations of y comple x species that have formed In these experiments y complex was incubated with ATP for 0 15 50 100 250 500 or 1000 ms prior to addition of DNA and MDCC-PBP Final concentrations were 0 27 My comple~ 400 M ATP 2 7 M 1\IDCC-PBP and 1 M DNA These data s how the evolution of the two major populations of 'Y complex species that are formed on equilibration with ATP. As the length of the incubation period increases the length of the lag phase decreases and

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Figure 4-9 Kinetics of ATP hydrolysis when y complex is incubated with ATP for a defined period of time prior to addition of DNA. Sequential mixing experiments were perfo11ned where y complex was incubated with ATP for 15 (red), 50 (blue) 100 (green), 250 (cyan), 500 (purple), or 1000 (black) ms prior to addition of DNA and MDCC PBP Kinetics of ATP hydrolysis were deter1nined by measuring the increase in fluorescence of:MDCC-PBP on binding inorganic phosphate and are plotted as the concentration of:MDCC-PBPPi complex as a function of time on time scales of A) 2 0 sand B) 0 4 s A control reaction with no ATP (gray) shows the signal for free MDCC-PBP as a function of time. The solid black curves through the data were calculated by fitting the data to the model shown in figure 410A. Final concentrations were 0 27 My complex, 400 M ATP, 1.0 M DNA, and 2.7 M MDCC-PBP in assay buffer containing 20 mM TrisHCl pH 7 5, 50 m.MNaCl 8 mMMgCl2 5 mMDTT, and 40 g/ml BSA. Raw fluorescence data were transformed into the j concentration of P i -bound MDCC-PBP using the equation shown in figure 4-3 I

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148 the rate of the initial phase of product formation i n c r eases (Figure 4 9 ). Incubation of y complex with ATP fo r 250 ins gives n early th e s ame ki n etics a s for 500 and 1000 ms indicating that the conformational transitions have reached equilibrium by this point in t i me A. 0 8 0 7 :E :i 0 6 0.. I 0 5 0.. m 0 4 0.. I 0 0 3 0 C 0 2 :z 0 1 0 0 0 0 0 2 0 4 0 6 0 8 1. 0 1.2 1 4 1 6 1 8 2 0 B. time (s) 0 55 0 50 0 45 ::1. ._, 0 4 0 0 35 0.. I 0 3 0 0.. m 0 25 0.. I 0 20 0 0 0 1 5 C 0 1 0 :E 0 05 0 00 0 0 0 0 05 0 1 0 0 1 5 0 20 0 25 0 3 0 0 35 0 4 0 time (s)

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149 Computer Modeling of ATP Hydrolysis Reaction Kinetics To determine the kinetic relationship between 'Y complex species that give rise to the biphasic kinetics of ATP hydroly s is and clamp loading data from sequential mixing experiments shown in Figure 4-9 were modeled using DynaFit ( Kuzmic 1996 ). Modeling demonstrated that at least three different s pecies of 'Y complex were required to ' I produce biphasic kinetics of ATP hydrol y sis and that these three species could not all exist on the linear pathway to products The minimal model that best fit the data is shown in Figure 4-1 OA ( see computer modeling methods in chapter 3 for details ). In this model, 'Y complex is initially present in a state, ETTI that can convert to two different species one that is active for DNA binding ATTT one that is inactive ITTT and does not directly form products The rate s of interconversion of these three states are relatively slow with rates in the range of 2 4 to 4 5 s1 In this minimal model it is assumed that each of these species is bound to three molecules of ATP because ATP hydrolysis rates were independent of the ATP concentration at this saturating concentration ( 400 M ATP ) However it is possible that the E and/or I states have fewer molecules of ATP bound and that very rapid ATP binding steps and conformational changes exist between the three states. The 'Y complex in the activated state ATTT rapidly binds DNA This DNAy complex then hydrolyzes each of its three molecules of ATP sequentially at the same rate The ATP hydrolysis steps are modeled using single forward rate constants because the presence ofMDCC-PBP limits the reverse reaction such that it cannot accurately be determined Previous work has shown that the DNA dissociation step i s likely to be very rapid relative to the steady-state recycling of the clamp loader and because these data do not directly measure this step DNA dissociation was set to be concurrent with hydrolysis

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I Figure 4-10 Kinetic modeling of ATP hydrolysis reactions A) The minimal kinetic model used to fit the ATP hydrolysis data in Figure 4-9 . Three states of the y complex, ETTI ATTT and ITTT are related by the kinetic constants shown ( cartoons are based on the structure of the clamp loader, see chapter2, figure 2-3 ). The ATTT form binds DNA (N) rapidly and hydrolyzes its three molecules of ATP (T ) in succession producing ADP (D) and inorganic phosphate (Pi) They complex dissociates from DNA on hydrolyzing its third molecule of ATP and is recycled back to the ETTT state Experimentally deterrnined rates of y complex binding DNA ( Ason et al 2000 ; Bertram et al ., 2000 ), ko nN, and MDCC-PBP ( PBP) binding phosphate (Brune et al ., 1994) ko n and ko ff were set constant in the fit All other rate constants were determined by fitting the data to the model using DynaFit (Kuzmic 1996) B) The concentrations of each of the three y complex species were simulated with KinTekSim (Barshop et al., 1983 ; Dang and Frieden 1997 ) and calculated based on the rate constants shown in A and are plotted as a function of time C) An additional step (PTTT -+ ETTT ) is required in the kinetic model shown in A to fit single mixing A TPase reactions as in Figure 4-8 where the pre-incubation time is effectively Oms Concentrations in this assay were 0 27 My complex 500 M ATP 1.0 M DNA and 2 7 M MDCC-PBP in assay buffer containing 20 mM TrisHCl pH 7 5 ., 50 mM NaCl 8 mM MgC1 2 5 mM DTT and 40 g/ml BSA All DynaFit program scripts and analysis for these models, and KinTekSim simulations are presented in the appendix

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151 A. k I ea 4.45s k -1 ei 2.37s ETTT k -1 ae 3.90s k -1 ie 4.23s ITTT ATTT+N J.< Cl> 0.10 Q. E 8 ?0.05 0.00 AT DN+P, l khyd 60S koff 13.6 s EI I I ATDDN+Pi -1 khyd 60s ADDD + N + P, B. ,: / : / I Alll --------------=i Elll I 1111 .,., I : ; ; j 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 simulation time (s)

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::E ::t a. I a. m a. I 0 0 C :i 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 152 C. PTTT k;,e = 53 s-i ETTT 0.5 :E ::1. 0.4 a. 0. 0.3 m 0. I 0 2 0 g 0.1 :E 0 0 0.1 0.2 0.3 0 4 time (s) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 time (s) Figure 4-10. Continued. of the third molecule of ATP Finally, the steady-state portion of the reaction was modeled as a pseudo-frrst order conversion of the ADP-bound 'Y complex (ADDD) back to the initial ATP-bound state (ETTT) This pseudo-first order approximation is reasonable because only a small fraction of the ATP substrate is consumed on the time scale of the reaction The three species of y com plex ETTT, ATTT and ITTT which are in equilibrium in the presence of ATP generate the two populations that give rise to the biphasic kinetics of ATP hydrolysis and clamp loading The rapid phase of ATP hydrolysis is produced largely from the population of 'Y complex in the ATTT state and the ETTT and ITTT states produce the slow phase. The populations of each of these species was simulated and calculated as a function of time using the kinetic constants in Figure 4-1 OA and are

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J 153 plotted in Figure 4-1 OB The concentration of each changes over the first 250 ms and then remains constant just as the kinetic data for ATP hydrolysis do not change at pre incubation times greater than 250 ms At equilibrium concentrations are 0 093 M ETTT 0 105 M ATTT and 0 052 M ITTT based on a total concentration of0 25 M 'Y complex calculated from ~e fit I In A TPase assays where 'Y complex was not pre-incubated with ATP (Figure 4-8 ), a pronounced lag (25 to 30 ms) in product formation is observed The minimal model in Figure 4-1 OA does not generate a lag that is lo~g enough to fit these data well An additional species of 'Y complex (PTTT) that converts relatively rapidly ( k pe = 53 s 1 ) to the ETTT state is required ; these data could be fit to the model using the rate constants shown in Figure 4-1 OA when this single step was included Figure 4-1 OC shows the data for an assay where 'Y complex was mixed with DNA MDCC-PBP and ATP The resulting fit from the minimal model (Figure 4-lOA) with the additional step PTTT + ETTT is shown over the experimental data Discussion The E. c oli 'Y complex functions as a molecular machine to assemble ring-shaped~ clamps on primers where DNA synthesis is slated to begin (reviewed in (Davey et al 2002 ). This mechanical task requires that the affinity of clamp loader for and DNA be modulated during the course of the clamp loading cycle Initially the 'Y complex must have a high affinity for both~ and DNA to efficiently bring them together ATP binding produces a high affinity binding state ( Ason et al ., 2000 ; Nak:tinis et al ., 1995 ; Turner et al ., 1999 ) where the 6 subunit can bind the clamp and '' crack '' it open ( Leu et al ., 2000 ; Stewart et al ., 2001 ; Turner et al ., 1999) Thus ATP binding allows the 'Y complex to fo1m a ternary clamp loaderclamp DNA complex (Bertram et al ., 1998 ) where an open

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154 clamp is most likely encircling DNA. At this point the affmity of the clamp loader for the clamp and DNA must be reduced so that the clamp can be closed around DNA and I the y complex can dissociate so as not to interfere with the polymerase binding the newly loaded clamp on DNA A primed DNA template provides the trigger to convert they c~mplex to a low affinity binding state (Ason et al ., 2000 ; Ason et al ., 2003) Upon binding a primer end, they complex hydrolyzes ATP and releases the clamp and DNA (Ason et al ., 2003 ). Binding and hydrolysis of ATP most likely induce conformational changes that produce the high and low affinity binding states respectively (Ason et al. 2000; Jenizalmi et al 2001a ; Naktinis et al ., 1995 ). Each clamp loader can hydrolyze up to three molecules of ATP as each y subunit has an ATP binding site These binding sites are located at the interface of two domains within a y subunit and also at the interface between two adjacent subunits within the complex (Jeruzalmi et al. 2001a ; Podobnik et al 2003) ATP binding by each y subunit is likely to promote a conformational change within that subunit that is then communicated to adjacent subunits. Thus, multiple conformational changes within the complex are likely to be associated with the transition to a high affinity binding state lTitimately this combination of confo11national changes exposes a region on the N-ter1ninal domain of the o subunit that binds the clamp A DNA binding domain is also created or exposed by these confor1national changes (Ason et al. 2000 ). Because there are three ATP binding sites within the clamp loader different conf 01 mational states are likely to exist depending on the occupancy of these sites In addition, the complex nature of the interactions between protomers and the conformational flexibility that must exist in the complex suggest that different I

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I 155 conformational states may exist even among complexes where the occupancy of the ATP binding sites is the same In this dissertation project the ATP-dependent conformational dynamics of the y complex have been investigated by measuring the kinetics of ATP hydrolysis and clamp loading under conditions where these changes contributed to the observed kinetics When y complex is completely equilibrated with ATP (pre-incubation times of ~ 250 ms) the first turnover of ATP is biphasic (Figure 4-3 black trace) Clamp loading I assays done under parallel conditions initiated by adding an equilibrated solution of y complex and ATP to and DNA, a biphasic increase in anisotropy is also observed indicating that two species were loading clamps at different rates ( Figure 4-6A ) If the biphasic kinetics of ATP hydrolysis were due to individual y subunits within the y complex hydrolyzing ATP at different rates, then a single phase of clamp loading resulting from a single population of y complex loading clamps would have been observed The ATPyS-chase assay of ATPase activity also showed different populations of 'Y complex when equilibrated with ATP before adding DNA, and showed that ATP is securely bound to one of these populations when directly compared to the original biphasic hydrolysis kinetics (Figure 4-5A) An ATP-bound population hydrolyzed ATP faster than it dissociates in a pre-steady-state rapid step that directly related to the rapid phase of the originally observed biphasic hydrolysis kinetics A pre-steady-state ATP hydrolysis assay mimicking the mixing conditions of the pre-steady-state clamp loading assay (Figure 47) showed that when 'Y complex was equilibrated with ATP before mixing with DNA and~ a single population rapidly hydrolyzed ATP then appeared to enter a steady-state clamp loading reaction The population of clamp loader that rapidly

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156 hydrolyzed ATP in this assay directly relates to the initial rapid phase observed for clamp loading kinetics as well as the population of 'Y complex which rapidly hydrolyzed ATP in I the biphasic reaction with DNA only Therefore this rapid phase may be due to some 'Y ' complex alone binding to pt DNA and hydrolyzing ATP The rate of the slower second phase of the biphasic hydrolysis kinetics ( i e some inactive species population) was increased to a rate identical to the steady-state rate of ATP hydrolysis for clamp loading in this assay designed to mimic the clamp loading assay (Figure 4-7) These ATP hydrolysis data for the clamp loading reaction in comparison to the reaction with DNA only are consistent with the ability of p to enhance the steady-state ATP hydrolysis activity of the clamp loader In the mimicked pre-steady-state clamp loading reaction (Figure 4-6A) the slower second phase may relate to confot ,national changes required for 'Y complex to gain affinity for p and load it on DNA A steady-state rate of ATP hydrolysis for clamp loading was observed during this time in the clamp loading ATP hydrolysis assay suggesting that the rate of the ATP-dependent confo11national changes giving the clamp loader affinity for P and DNA is a rate limiting step in the clamp loading reaction In A TPase assays where 'Y complex is incubated with p and ATP a single presteady-state phase of ATP hydrolysis is produced at the same rate as the rapid phase in assays without p ( Figure 4-3 red trace ). The amplitude of the single phase of ATP hydrolysis in assays with pis equal to the sum of the amplitudes of the two phases in assays without p and indicates that about 2 7 molecules of ATP are hydrolyzed per 'Y complex This number is interpreted as one molecule of ATP hydrolyzed by each 'Y subunit This experimental value is less than the theoretical value 3 most likely because

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I 157 of differences between the concentrations of active enzyme and total protein in the assay. ATPyS-chase of the pre-steady state hydrolysis activity in the presence of~ showed that the molecules bound to they subunits were hydrolyzed faster than they dissociate indicating that the clamp has an effect on the stability of the clamp loader Specifically in the presence of~ the bound-A TPs appear to be secure or '' trapped ' in the complex until DNA triggers hydrolysis upon clamp loading The observation that the sum of the amplitudes of the rapid and slow phases of ATP hydrolysis in assays without is also I equivalent to 2 7 molecules of ATP indicates that both populations of y complex are hydrolyzing all their ATP but at different rates The pre-steady state ATP hydrolysis assays performed in the presence of~ at three different y complex concentrations ( Figure 4 4) demonstrate further that the clamp loader hydrolyzed approximately three ATP molecules at nearly the same observed rate in the single rapid phase of each assay, and importantly that the molar response to hydrolysis by MDCC-PBP was adequately measured. This result has significance for any study of an energase enzyme ( i e ., ATPase, GTPase), in which the investigator is using the MDCC-PBP NTP hydrolysis assay to quantify the number of nucleotide molecules hydrolyzed under pre-steady-state ''burst ' conditions To determine the kinetic relationship between y complex species that give rise to the two phases of product formation in A TPase and clamp loading assays the kinetics of formation of these species was measured in pre steady state ATPase assays In these assays r complex was pre incubated with ATP for a defined period of time, 15 to 1000 ms, during which different populations of the various species formed. Kinetic data for ATP hydrolysis were dependent on the relative concentrations of these species

PAGE 173

158 Modeling of these data showed that the biphasic kinetics of ATP hydrolysis can be generated by three different 'Y complex species that we have denoted as ATTT ETTI I and ITTT (Figure 4-lOA and B) In other words three different states of the 'Y complex produce ~o different kinetic populations that give rise to two phases of product fo1mation In these reactions 'Y complex is initially present in the ETIT state and this state produces two new states A TTT and ITTT Modeling further demonstrated that one of these species ITTT, does not fortn products directly It is believed that these three species represent three different conf orrnational states of the 'Y complex ATP hydrolysis was also measured in assays where 'Y complex was not preincubated with ATP effectively a Oms time point (Figure 4-8 and Figure 4-lOC) These reaction time courses showed an extended lag of about 25 to 30 ms that was not present in assays where the pre-incubation period was 15 ms or greater This extended lag was also reflected in time courses for clamp loading where reactions were initiated by the addition of 'Y complex to ATP ~ and DNA (Figure 4-6C ). The model used to fit A TPase data for pre-incubation times of 15 1000 ms does not generate a lag that is long enough to adequately fit the ''Oms' preincubation data However, these data can be fit by the inclusion of an early step PTTT + ETTT which generates the ETIT state where the model in Figure 4-lOA begins A simulation of the model including the PTTT + ETTT step shows that formation ofETTT peaks at approximately 30 to 40 ms consistent with the extended lag observed in the assays where there was an effective O ms preincubation time (see Appendix A figure A-1 ). The rate of this early step is relatively rapid 53 s -1 Data for preincubation times of 15-1000 ms do not adequately define this kinetic step because it is rapid a half-life of about 11 ms relative to the shortest delay time To I

PAGE 174

159 define this step in these assays shorter pre-incubation times would be required which are not experimentally possible with our instrumentation Although an extra step was required to generate the lag in the Oms pre-incubation data the rest of the time course was fit very well by the model and rate constants shown in Figure 4-lOA The quality of I this fit indicates that the model adequately represents both data sets for the O ms and 151000 ms pre-incubation reactions What does this early step that generates the ETTI state represent? One likely I possibility is that this step reflects the rate of ATP binding to the complex These experiments were done under conditions where ATP binding was not rate-limiting and that are effectively ' V ma x ,, conditions. Therefore an ATP binding step could be represented as a pseudo-first order reaction Although it is not rate limiting, it would still take some finite amount of time for three ATP molecules to bind the complex and this time may be reflected in the lag Note that the length of the lag phase did increase substantially to about 70 ms in an A TPase reaction at a lower sub-saturating concentration, 20 of ATP ( Figure 4-8 blue trace) This observation is consistent with an ATP binding step contributing to the lag in product f 01 mation Another possibility is that this lag represents local conformational changes within a single 'Y subunit that occur when a single molecule of ATP binds the complex What is the physical nature of the three kinetic states ATTT ETTI and ITTT that give rise to the biphasic kinetic s of ATP hydrolysis? The model (Figure 4-lOA) describes the A TTT state as one that is activated for DNA binding suggesting that the ETTI to ATTT transition reflects confo1mational changes that either expose or create a DNA binding site This transition may also reflect conformational changes that expose

PAGE 175

160 the o subunit and activate the complex for P binding The pre-steady-state rate of ATP hydro,ysis in assays with p was identical to the rapid phase of hydrolysis in assays without p If p selecti v ely bound this activated specie s of 'Y complex then during the 1 000 ms equilibration period (Figure 4-3 red trace ) p could ultimately convert all 'Y complex to an activated P A TTT complex This would then produce a single phase of ATP hydrolysis that occurred at the same rate as the rapid phase in assays without p The high affinity of ATP-bound 'Y comple x for p Kd of 3-4 nM (Naktinis et al ., 1995) ( see also chapter 5 Figure 53), suggest s that p would be able to drive the equilibrium in this direction under our assay conditions Previous work has shown that when 'Y complex is pre-incubated with p and ATP for only 80 ms the pre-steady-state for1nation of ADP is biphasic (Hingorani et al ., 1999 ) as we have shown in assays without p This observation is consistent with selective binding of~ to an activated ATTT state although more prei ncubation time points need to be taken to provide stronger support for this model An alternative explanation for the effect of P on A TPase kinetics is that P could bind the E TTT state and this interaction could promote the formation of the A TTT state at the expense of the ITTT state This type of mechanism would require that P change the kinetics of this step rather than simply shifting the equilibrium of 'Y complex states by s electively binding one of the three specie s. These two possibilities could be differentiated in A TPase experiments where 'Y complex was pre incubated with ATP in the presence of p for different lengths of time A key feature of our kinetic model ( or any model that would adequately describe these data ) is that a nonproductive branch in the pathway to products must be present to generate the kinetic data s hown in Figures 4-8 and 4-9 ). In other words one s pecies of 'Y

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I 161 complex must form that does not produce products directly These kinetic data could not be adequately described by a series of state s that exist on the linear pathway to products (e g ElTTT + E2TTT + ATTT + products) In our minimal model, we have represented this branch as an interconversion of ETTT and I l I T We do not yet know what the physical nature of this ITTT .state is One possibility is that the ITTT state simply represents an alternative ATP bound for1n of the 'Y complex that is thermodynamically stable but not kinetically active Another interesting possibility is I that the ITTT state is not initially formed form the ETTT state but instead ATP loading into binding sites in a different order forrns the ETTI and ITTT states independently It is possible that ordered binding of ATP is required to generate the most rapid and efficient series of conf or1national changes to produce the high affinity binding state of 'Y complex ( Johnson and O'Donnell 2003) Structural data for the y 3 00' minimal complex suggests that two of the three ATP binding sites may be open in the nucleotide free complex so that two different orders of loading ATP may be possible ( Jeruzalmi et al 2001a ). Perhaps the ETTI state is fo11ned when ATP binds in the preferred order and the ITTT state is fo1med when ATP binds in an alternative order. The ITTT state then must convert to the ETTT state before it can go on to products It is possible that ITTT converts directly to ETTT or that ITTT converts to E TTT by dissociation and rebinding of ATP and the rates defined by our minimal model reflect the overall rate for the process Regardless of what the physical nature of these kinetic states is the data show that the rates of confo1mational changes within the 'Y complex are relatively s low and limit the rate of the pre-steady-state ATP hydrolysis and clamp loading reactions when 'Y complex

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162 is not pre-equilibrated with ATP The rates of transitions between ETIT and ATTT and ETTT and ITTT states are relatively slow on the order of2-4 s 1 with the rate of conversion ofETTT to ATTT being about 4 5 s 1 The magnitude of these values is similar to the steady-state rate constant or turnover number k catoomplex of 2 to 3 s 1 (Table 4 ... 1 ) (Bertram et al ., 2000; Hingorani et al ., 1999) for reactions with~ and may also reflect the rate-limiting step under the s e conditions Additionally kcat com p lex /Km values for steady-state reactions are relatively slow about 0 8 to 1 8 x 10 5 M 1 s1 ( Table 4-1) (Bertram et al ., 2000 ; Hingorani et al ., 1999 ), to repre s ent a bimolecular binding reaction between ATP and 'Y complex The magnitude of kc atcomplex /K m is likely to be reduced relative to the theoretical upper limit ( a diffusion controlled binding rate ) by a two-step binding reaction consisting of relatively rapid ATP binding followed by slow confo1mational changes induced by ATP binding

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I I CHAPTERS CHARACTERIZATION OF THE CLAMP LOADER CO:MPLEX AND COMPARISON TO GAMMA COMPLEX Introduction I The c oli sliding-clamp loader is a complex molecular machine made up of a heteropentameric core of AAA + subnits ('Y / t )3 00 along with two additional subunits ( x and 'V) (Davey et al 2002 ; Neuwald et al ., 1999 ; Onrust et al ., 1995) The core sub complex of the clamp loader has the necessary components for loading the sliding clamp onto DNA for promotion of processive synthesis (Onrust et al ., 1991) The X-ray crystal structure of this sub-complex (y 3 oo ') of the clamp loader was the first solved of any of the evolutionarily conserved clamp loaders (Jeruzalmi et al ., 2001a) and has had a broad impact across the entire replication field (Ellison and Stillman 2001 ; Jeruzalmi et al ., 2002 ; O'Donnell et al 2001 ). The mechanism and energetics of clamp loading has been extensively explored for 'Y complex ( y 3 00 X'V ) in both steady-state and pre-steady-state analyses (Bertram et al ., 2000 ; Hingorani et al ., 1999 ; Kelman and O'Donnell 1995 ). However the analysis of the sub-complex ( 'Y 3 oo ') has only been sparingly examined in steady-state assays only (Dong et al ., 1993 ; Onrust et al ., 1991 ), with the generalized conclusion that it has activity similar to 'Y complex ( Jeruzalmi et al ., 2001a ). The previous chapter ( 4 ) of this dissertation continues the detailed study of the clamp loading mechanism by 'Y complex and s hows the kinetic s of the ATP-dependent conformational transitions within the clamp loader using an array of ATP hydrolysis and DNA binding assays along with computer 163

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164 modeling and fitting the kinetic data Here many of the same analyses are presented for the '' n;iinimal complex '' clamp loader y 3 oo in direct comparison to those of y complex I with the goal of further understanding the activity of thi s conserved 'core' clamp loader, and additionally understanding the need for the x and \I' s ubunits in the E.coli clamp loader The work presented in this chapter encompasses the most detailed study of the y 3 oo clamp loader to date and includes steadys tate and pres teady-state analyses of DNA binding/clamp loading clamp binding and ATP hydrolysis activities of this minimal complex The results show that the activity of the minimal complex is nearly the same as y complex, as had been previously known ; however the data show that the missing x and \I' subunits are a necessity for y complex and confer confo11national stability to the clamp loader leading to increased optimal activity Another key result of the comparison of the y 3 00 minimal complex toy complex is that the clamp is responsible for the similarities observed for these clamp loaders It is well known that the sliding clamps enhance the ATP hydrolysis activity of their clamp loader and therefore the clamp loading activity of y complex (Onrust et al ., 1991 ; Stukenberg et al ., 1991), and also clamp loaders of other organisms (Ellison and Stillman, 2001). The comparison of y 3 oo and y complex steady-state and pre-steady-state kinetics in the absence and pre s ence of~ lead to a possible clamp loader enhancement mechanism wherein the clamp by binding the clamp loader converts it to a completely activated population and secures the bound molecules of ATP such that this ATP-bound -clamp loader-~ clamp complex is committed for the l oading reaction a.s soon as DNA is located I

PAGE 180

165 y 3 66' is the Minimal Clamp Loader Complex Which Can Bind DNA Analysis of DNA Binding Activity of the Individual Subunits or Sub-complexes of the Clamp Loader The steady-state anisotropy ofRhX-labeled ss DNA was analyzed to measure binding interactions between purified individual subunits of 'Y complex ( 'Y, o, o x-'I' ), as I well as several sub-complexes of subunits (yo po ', y"l)V, r'llo', roo ) The t subunit dimerizes the core polymerase in vivo and is also a constituent of the clamp loader in the holoenzyme Therefore the steady-state binding activity of the 't subunit alone as well as in a sub-complex with oo (too ') was also measured with the RhX-ss DNA The observed steady-state anisotropy of the RhX-probe on DNA is a population weighted average of the combined anisotropies for free DNA and protein-bound DNA. Therefore the observed anisotropy increases as the population of protein-bound DNA increases (Bloom et al ., 1996). The change in anisotropy of RhX covalently attached to a single stranded 50-mer DNA was measured from calculated anisotropy from titration assays with individual subunits or sub-complexes Increasing concentrations of the purified individual subunits sub-complexes or 'Y complex were titrated into a solution containing 50 nM RhX-ss DNA and O 5 mM ATP The ATP was added last in these assays such that the change in anisotropy in the absence then presence of ATP could be compared None of the individual subunits sub-complexes or 'Y complex bound ss DNA in the absence of ATP Figure 5-1 shows that none of the individual subunits and several of the combinations of subunits in sub-complexes have ss DNA binding activity under these conditions Only a specific combination of subunits the sub-complex y300 ', showed ATP-dependent ss DNA binding activity The corresponding too sub-complex also showed ss DNA binding activity

PAGE 181

166 0.06 ------------, 0 05 0. 0 b 0 04 0 ti) C 0.03 ca C Q) 0.02 C) C ca .r:. 0 0 01 Y4 6 6 X\V oS y6 = 0. 0. 0. 0. 't 1 eo eo Q. ttteo I I t,.t +ATP (.) + + + + + >0 c"eo eo eo eo Figure 5-1 Change in stea d y-state anlsotropy for RhX ss DNA in the p resence of individual s u bunits, s u b comp l exes and y complex with or withovt ATP Changes in t he steady-state anisotropy of a 50-nuc l eotide ss DNA labeled with RhX at the 5 '-end is p l otted against the va lu e for a single d ata point from titration expe r imen t s re p resenting saturated binding of the p rotein to Rhx-ss DNA (i e ., when there was binding) The 'Y4 o o X'V, t 2 oo ', yo subunits we r e all in the presence of ATP and y comp l ex or various s u b-complexes were in the absence o r p r esence of ATP as indicated The steady state polarized intensities from the RhX probe were measured upon the a d dition of a constant volume of a solution containing protein to a solution in the cuvette containing 50 nM RhX ss DNA A constant volume of ATP was s u bseq u ently a dd e d to the cuvette giving a fmal concentration of 0.5 mM, an d the po l arized intensities were again measured for calculation of anisotropy. All proteins were titrated over a range of 50 to 500 nM, and beyond 2 M in some cases The anisotropy value for free RhX ss DNA (r free) was subtrac t ed from the anisotropy for RhX-ss DNA in the presence of protein (rbound) to give the value of change in anisotropy p lotted Data from titration experiments for the 'Y4 o o X't' t 2, oo ', yo sub u nits were contributed by Xu Liu, Ph D I

PAGE 182

167 Analysis of the DNA Binding Activity of y 3 66' Minimal Complex in the Absence and Presence of p To further characterize the y 3 6o '' minimal complex '', titration assays were performed with RhX-pt DNA in the presence and absence of P clamp The change in steady-state anisotropy ofRhX-pt DNA was examined upon the addition of the minimal complex to determine the minimal complex-pt DNA binding activity as well as the P clamp loading activity when p was present in the as s ay The pt DNA substrate consisted of a 105-nucleotide template with a 30-nucleotide primer annealed approximately in the center of the template creating a 50-nucleotide-long ss DNA 5 -overhang of the template The sequence of the SO-nucleotide overhang was identical to the sequence of the 50-mer ss DNA used in the above titration assays ( see chapter 3 materials and methods for DNA sequences) It was previously shown that the 30 / 105-mer pt DNA substrate supports DNA synthesis by the polymerase ill core in the presence of y complex p and single-stranded DNA binding protein (SSB ) (Hingorani et al ., 1999 ). Increasing concentrations of the minimal complex ( 100 to 1200 nM ) were added to 50 nM RhX-pt DNA in the presence and absence of 500 nM P in a solution containing 8 mM MgCl 2 and 0 5 mM ATP In the absence of p the minimal complex appears to have low binding affinity for pt DNA similar toy complex ( Figure 5-2 ). Even though the sequence of the 50nucleotide ss DNA overhang of this pt DNA substrate is identical to the ss DNA 50-mer to which both the minimal complex and y complex clearly bind in figure 5-1 they do not appear to have much affinity for it within the context of the pt DNA substrate It was previously shown for y complex that there was a transient interaction with pt DNA and this interaction triggered rapid dissociation of y complex leaving it in a form that was

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1 68 0 14 0 12 > I 0. 0.10 0 .. .., i 0 08 C ca C 0 06 G) C) C 0 04 ca 0 .::: 0 (.) 8 D 0 02 8 0 8 8 C 0 00 I I I I I I I 0 200 400 600 800 1000 1200 1400 1600 1800 2000 y360 or y complex (nM) Figure 5-2 Steady-state binding of the minimal complex or 'Y complex with RhX-pt DNA with and without~ The steady-state polarized emission intensities of Rh.X-DNA were measured for calculation of anisotropy after solutions containing increasing concentrations of either the minimal complex alone ( open squares) or -y complex alone ( open circles) were added to a cuvette containing 50 nM RhX pt DNA 8 mM MgCl 2, 20 mM Tris-HCl pH 7 5, 50 mM NaCl 5 mM DTT 40glmL BSA and 0 5 mM ATP Titration assays were also performed for the minimal complex or 'Y comp l ex in the presence of 2 5 M ( filled squares or filled circles respectively ). The concentration of minimal complex or 'Y complex in the absence or presence of~ is plotted against the change in anisotropy from separate titration assays The anisotropy value for free RhX-pt DNA (r r r e e ) was subtracted from the anisotropy for RhX-pt DNA in the presence of protein (r bo und) to give the value of change in anisotropy plotted modeled to undergo a slow step to regain affinity for DNA resulting in the apparent low affinity for DNA in the steady state (Ason et al ., 2000) The comparable result here with the minimal complex, indicates that the ntinimal complex undergoes a similar slow step to regain affinity for DNA. I

PAGE 184

I I 169 For assays perfo1med in the presence of p the m i nimal complex gained high affinity for pt DNA and loaded p ( Figure 5-2) When y 3 80 wa s originally reconstituted and purified by other s ( Onrust et al ., 1991 ), it wa s shown that like y complex y 3 00 wa s capable of loading p and conferring processive DNA synthesis in assays with polymerase I III core p and SSB The minimal complex with p showed a slightly lower affinity for pt DNA than y complex however both appeared to saturate the pt DNA above a concentration of 1 Comparison of p Clamp Binding Affinity af the Minimal Complex and 'Y Complex Equilibrium ppyrene Binding Activity of the Minimal Complex and y Complex After determining that the steady-state DNA binding activity of the minimal complex is similar to that of y complex the equilibrium binding of y complex and the minimal complex to p clamp was examined in solution p pyrene binding assays were perf 01med by measuring the steadys tate polarized emission intensities of the pyrene probe covalently attached to p for calculation of anisotropy as a function of minimal complex or y complex concentration ( Figure 5-3 ). Like that for the RhX-labeled DNA substrates above the observed steady-state anisotropy of the pyrene-probe on~ is a population weighted average of the combined anisotropies for free-P and protein-bound P in s olution Therefore the ob s erved anisotropy increases a s the population of protein bound P increases Results show that minimal complex and y complex had es s entially identical affmity for p py r ene both in the presence or absence of ATP Dissociation constants for this binding interaction ( Ko ) were calculated ba s ed on a reversible second-order kinetic model : A + B!:;AB where A i s the ATP-bound clamp loader and Bis ~ pyrene_

PAGE 185

0.05 a. o 0.04 b 0 u, C 0 03 ca C Q) o, 0.02 C ca J:. 0 0 0 1 0 00 0 / ___.... h 7 / m 1 70 ____ ,..._ ____ __ o --.-~ ,,,.,--200 400 600 800 1000 1200 1400 1600 y380' or y complex (nM ) Figure 5-3 Steady-state anisotropy binding activity of r complex or the minimal complex with ppyr ene in the presence or absence of ATP The steady-state polarized emission intensitie s of p pyrene were measured and used to calculate anisotropy for solutions containing increasing concentrations of either the minimal complex in the presence of ATP ( closed squares) or absence of ATP ( open s quares ) or 'Y complex in the presence of ATP ( closed circles ), or absence of ATP ( open circles) Solutions containing the minimal complex or 'Y complex were added to a cuvette containing 50 nM p PYTene, 8 mM MgCl 2, 20 mM Tris-HCI pH 7 5 50 mM NaCl 5 mM DTT > and 40 g/mL BSA The ani s otropy value for free p pyrene ( r free) was subtracted from the anisotropy for p pyrene in the presence of different concentrations of y complex or minimal complex ( r oound) to give the value of cha nge in anisotropy plotted. Mean and standard deviation values are plotted for two independent measurements Nonlinear fits to the quadratic equation (s ee text) are plotted for minimal complex ( red solid) and r complex ( blue dashed ). The empirical s olution for the quantity AB was fitted to the experimental data using the following quadratic equation in terms of the change in anisotropy of p pyrene for e s timation of the Kt

PAGE 186

171 rAB1 =~ + [A] + [B] :'l~ +[A]+ (B] ) 2 4[A][B] ( r r \ + r J.bcund J.f:ree/ J.free 2[B) In the presence of ATP the minimal complex had a Kci of 3 nM, and y complex had a I<.(! of 4 nM foi~p yrene Both the minimal complex and y complex remained stably bound to ~~yr ene for a minimum of 10 minutes (not shown ). Surprisingly both the minimal complex and y complex bound p pyrene in the absence of ATP with dissociation constants of200 and 240 nM respectively Even though the dissociation constants for binding in the absence of ATP were ~ 50-fold higher than the Ki values measured in the presence of ATP they still reflect significant binding activity for the clamp loader with pp yrene in the absence of ATP A I<.(! of 3 nM was reported previously (Naktinis et al ., 1995) for 'Y complex and~ in the presence of ATP using surface plasmon resonance (SPR) methods The investigators also reported a Kt for interaction of y complex and P in the absence of ATP that was 1000-fold higher in the absence of ATP The dissociation constants measured using the steady-state anisotropy binding titration assays here with ~ pyrene, were estimated in solution equilibrium conditions (i.e., y complex was immobilized using the SPR method ) and the first measured for the y 3 66 minimal complex Apparent Dissociation Constant for ATP Binding the Minimal Complex or y Complex The dissociation constants for ATP binding the minimal complex or 'Y complex were deter1nined indirectly as a function of ATP-depen dent clamp loader binding to ~p yrene. ATP is required by the clamp loader to bind the clamp with high affinity and therefore the steady-state anisotropy assay was used to measure the minimal complex or 'Y complex binding activity with ~ pyrene as a function of ATP concentration I

PAGE 187

172 0.08 -----------------------, 0. 0.06 e ..., 0 u, C 0.04 as C (1) en C 0.02 as .r. (.) 0.00 0 5 10 15 20 25 30 35 40 [ATP] M Figure 5-4 Minimal complex or y complex binding p pyrene as a function of ATP concentration The steady-state polarized emission intensities of p.P yrene with the minimal complex or y complex were measured and used to calculate anisotropy following addition of a solution of a constant volume containing increasing concentrations of ATP in separate assays Concentrations were : pp yrene (65 nM) minimal complex or y complex ( 400 nM) in 8 mM MgCl 2, 20 mM Tris-HCl pH 7.5 50 mM NaCl 5 mM DTT, and 40 g/mL BSA The ch ange in anisotropy of p pyre ne in the presence of minimal complex ( squares) or y complex (circles) is plotted against the concentration of ATP The anisotropy value for free p pyrene ( r &ee ) was subtracted from the anisotropy value for ~ pyrene with minimal complex or y complex (r oo un d ) in the presence of varied concentrations of ATP to give the value of change in anisotropy plotted Mean and standard deviation values are plotted for two independent measurements A nonlinear fit to the quadratic equation (see text) (red curve ) is plotted for minimal complex only for clarity. Since this assay only measures the binding of clamp loader to p pyrene, and not ATP binding directly to the clamp loader it is an indirect measure of the affinity of ATP for the clamp loader The K dapparen t for A IP binding was estimated using the reversible second-order kinetic model : A + B !::. AB ( described above ), where A is the

PAGE 188

' 173 concentration of ATP-bound clamp loader at each concentration of ATP and Bis ~ pyrene. The result s showed that the minimal complex bound ATP with essentially identical affinity as y complex (:Ki appare n t = 23 ) This value is consistent with previous measures of ATP binding to the polymerase Ill holoenzyme ( ~ 2 ) in UV crosslinking I assays (Biswas and Komb~rg 1984 ), assay s with purified y subunit (~ 2 1 ) ( Tsuchihashi and Kornberg 1989 ), and with y complex ( ~ 2 M) ( Hingorani and O'Donnell 1998 ) Kinetics of ATP Hydrolysis by the Minimal Complex Measured Using the MDCC PBP ATPase Assay Steady-State ATP Hydrolysis Kinetics of y 3 66' Minimal Complex in the Absence and Presence of p Clamp It was previously determined for the minimal complex along with y complex that increases the steady-state rate of DNA dependent ATP hydrolysis ( Onrust et al ., 1991 ). To investigate the DNA-dependent ATP hydrolysis activity of the minimal complex further measurement of the initial velocities of ATP hydrolysis activity was performed in steady-state conditions with the MDCC-PBP ATPase assay Steady-state reactions were initiated b y addition of a range of ATP concentrations approximatel y 10-fold less than or greater than an apparent dissociation constant for ATP binding ( ~ 5 ), estimated from steady-state clamp loading a s says as a function of ATP concentration described in chapter 4 (F igure 42). Initial reaction velocities were mea s ured in three s eparate as s ays each for the minimal complex with pt DNA only and the minimal complex with pt DNA and ~ Initial velocities were averaged and plotted again s t ATP concentration and the re s ulting rectangular hyperbolic relationship wa s fitted with the Michaeli s -Menten equation : ( where vi s the initial velocity and [S] is ATP concentration ),

PAGE 189

I V [S] v = K.,,m: [S] 174 describing a system where the observed rate depends on the concentration of the enzymesubstrate complex The resulting steady-state kinetic parameters for ATP hydrolysis are presented in Table 5-1 Table 5-1 Steady-state ATP hydrolysis kinetic parameters for the minimal complex and com lex in the absence and resence of a Vma x r complex No 0.02 (0 003) 0 6 (0.07) 6 0 (3 0) 0.1 (0 04) + 0 07 (0 02) 2 0 (0 4) 11 (6 7) 0.2 (0 05) 300' minimal com lex 0 04 (0 01) 0 08 (0 01) 0 4 (0 13) 0 8 (0.14) 9.0 (3 0) 6 0 (4.0) 0.05 (0 02) 0.2 (0 09) a Steady-state MDCC-PBP A TPase assays were perfonned by addition of ATP ranging in concentrations from 0 6 to 77 M to a solution of 41 nM r complex with 50 nM ptDNA and 50 nM (when present) or 100 nM y 3 oo minimal complex with 100 nM ptDNA and 100 nM ( when present) All assays contained 2 4 M MDCC-PBP and Pi-mopped assay buffer containing 20 mM Tris -H Cl 50 mM NaCl, 5 mMDTT 40 g/mL BSA, and 8mMMgCl2 b Standard deviations were calculated from three separate steady-state MDCC-PBP A TPase assays c Since the clam~ loader has three subunits capable of hydrolyzing ATP, the turnover number (kca t com p e x) reflects the combined activity of the complex. The steady-state rate of ATP hydrolysis (kc atco m ple x) by the minimal complex increased 2-fold For 'Y complex,~ increased the rate of ATP hydrolysis activity approximately 3-fold. Changes in the K m values in the presence or absence of~ were not clear in this analysis. Previously a turnover rate of 1 8 s 1 and a Km of ~ 22 M for y complex in the presence of~ as reported (Hingorani et al ., 1999 ). The Km values derived from this analysis with the minimal complex and r complex are all higher than the apparent~ of 2 M dete1nlined for ATP with the ~p yr en e anisotropy binding assay suggesting that the K m value is not a simple binding affinity constant but some other

PAGE 190

I 175 combination of rates The enhancement of the turnover rate (kca tcompl e x) for both the minimal complex and 'Y complex by indicates the possibility that may partition a more active form of each, essentially increasing the clamp loaders concentrations towards the clamp loading pathway which requires ATP hydrolysis I The specificity constant for ATP ( kca tco m p le x /K m ) can be an apparent bimolecular binding rate constant This value increased 4-fold for the minimal complex and 2-fold for 'Y complex when P was present Interestingly these kca tcomplex /K. m values are all I roughly 1000-f old lower than would expected for diffusion-controlled bimolecular binding of a small nucleotide cofactor such as ATP This result indicates that rapid ATP binding was followed by a slow step leading to hydrolysis for both the minimal complex and 'Y complex Overall the results of the steady-state analysis show that in the absence or presence of p the minimal complex hydrolyzes ATP with slightly lower activity than 'Y complex The absence of sigmoidal curves in the analysis of initial velocity of ATP hydrolysis activity versus ATP concentration for the minimal complex or 'Y complex with pt DNA only, argues against p being an allosteric effector The mechanisms of positive cooperativity predict that without its allosteric effector an enzyme generally displays sigmoidal initial velocity kinetics (Fersht, 1999) Whether or not P was present, all plots of initial velocity against ATP concentration from the steady-state MDCC-PBP ATPase assays were rectangular hyperbolas that fit to the Michaelis-Menten equation not sigmoidal

PAGE 191

176 Pre-Steady-State Kinetics of ATP Hydrolysis by y 3 66' Minimal Complex in the Absence and Presence of P Clamp Pre-steady-state analysis was performed to characterize and determine if the DNAdependent ATP hydrolysis activity of the minimal complex required to load~ on pt DNA ' differs from the activity of y complex Does the minimal complex hydrolyze ATP in a different clamp loading reaction mechanism resulting in the small differences observed in the steady-state ATP hydrolysis parameters? The x and \JI subunits of y complex are not present in the minimal clamp loader complex Do the x and 'I' subunits change the way the clamp loader hydrolyzes ATP in the clamp loading reaction? For the pre-steady-state MDCC-PBP ATPase assay a sequential-mixing ''three syringe '' stopped-flow experiment was performed One stopped-flow syringe contained a solution of-y 3 00 minimal complex and~ (when present ), and a second syringe contained ATP The contents of these syringes were mixed and preincubated for 1 s After preincubation -y 3 oo ', ATP and~ (when present) were rapidly mixed with a solution from a third syringe containing pt DNA and MDCC-PBP Mixing with the third syringe triggered the detection system allowing real-time measurement of the fluorescence change due to inorganic phosphate binding MDCC-PBP upon pt DNA stimulated ATP hydrolysis activity of the minimal complex. The DNA substrate used in all experiments was the 30-nucleotide-primed/105-nucleotide template DNA described above The 1-s period of preincubation time was sufficient for equilibration of a complex of -y 3 00 and ATP or a complex of y 3 00 ', ATP and~ ( see simulations of eq uilibration in the appendix) The preincubation time was also increased up to 2 s and there was no further increase in ATP hydrolysis activity observed in the first turnover demonstrating that a 1-s period of preincubation is sufficient for for1nation of the y 3 oo ATP ,+/ -~ complex t

PAGE 192

177 The results of pre-steady-state MDCC-PBP ATPase assays for the minimal complex in the absence or presence of~ compared to identical assays for "f complex are shown in Figure 5-5 A and B The reaction time course for ATP hydrolysis for the minimal complex in the presence of~ showed a slight lag a single rapid phase of ~ 100 ms, another lag phase of ~ 1 150-200 ms constituting the fust turnover The single rapid phase in this assay is essentially an enzyme active-site titration depicting the amount of active minimal complex present in the assay Although the concentration of y complex or I the minimal complex used in these assays calculated to be the same the activity in the fust turnover was lower for the minimal complex than for y complex From the single pre-steady-state rapid phase amplitude an estimated ~ 1 5 molecules of ATP were hydrolyzed This result suggested that the concentration of '' live '' minimal complex was roughly half that calculated to be present in the assay This was not always the case during the course of this study In earlier assays the amplitudes of the rapid phase were in good agreement with the y complex activity Therefore, this difference may be due to error in calculation of clamp loader concentrations, or the minimal complex may be unstable in storage, and consequentially lose some activity The instability of the minimal complex has been noted previously (Olson et al ., 1995) However the mechanistic features of the pre-steady-state ATP hydrolysis reaction were preserved in relation to 'Y complex but the pause at the end of the fust turnover was less defined for the minimal complex In the assay for the minimal complex without~ (figure 5-5A, blue trace) there was a small lag ( ~ 20-30 ms) followed by two kinetic phases Fitting the data to a double exponential expression indicated that there were either two pre-steady-state phases

PAGE 193

Figure 5-5 Kinetics of ATP hydrolysis by the minimal complex or1 complex in the presence and absence of~ A) ATP hydrolysis kinetics for the minimal complex in the presence (black) and absence of~ (blue). B) ATP hydrolysis kinetic s for 'Y complex in the presence (black) and absence of~ ( blue) Using a sequential-mix ' three-syringe ' stopped, flow assay one syringe loaded with a solution containing the minimal complex or y complex and p ( when present ) was mixed with the contents of a second syringe loaded with ATP The resulting mixture was preincubated for a period of 1 s and then mixed with the contents of a third syringe loaded with a solution-containing pt DNA and MDCC-PBP All solutions were prepared in P i -mopped assay buffer : 20 mM Tris-HCl 50 mM NaCl 5 rnM DTT 40 g/mL BSA and 8 mM MgC1 2 Final reactant concentrations were 270 nM minimal complex or 'Y complex 1 0 Mp ( when present), 100 M ATP 1 0 M pt DNA and 2 7 M MDCC-PBP The flat trace (gray) at the bottom of each figure showing no change was a negative control assay performed in the absence of ATP Raw fluorescence data were transfonned into the concentration of P i -bound MDCC-PBP using the equation, X b (t) = Uob s (t )I r ] / [l b I r ] to solve for the fraction of Pi bound MDCC-PBP X b (t) then multiplying this value by the concentration ofMDCC-PBP present in the assay to get the value MDCC-PBP-P i (M) plotted I r was the fluorescence intensity of Pi free MDCC-PBP in assay buffer and lb was the saturated fluore scence intensity of completely Pi-bound MDCC-PBP in the presence of 200 M potassium phosphate (P i source ). I

PAGE 194

179 A. y300' minimal complex 1.0 ~-----------------, 0.9 i' 0.8 .,;; 0.7 0. I Q. m Q. I 0.6 0.5 0.4 0 o 0.3 C :e 0.2 0.1 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 B. y complex time (s) 1.8 -----------------. 1.6 .-. :E 1.4 ::1. 1.2 Q. 0. 1.0 m a.. 0.8 I 8 0.6 C 0.4 :E 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 time (s) preceding transition to the steady-state or that there was only a single rapid pre-steady state phase that transitioned into steady-state activity since the end of a putative second pre-steady-state phase was not well defined both visually or mathematically In either case the amplitude of the small pre-steady-state rapid phase was ~ 25% of the rapid phase activity observed for the reaction in the presence of~ ( i .e ., 25% of total active clamp loader ) For 'Y complex in the absence of~ (F igure 5-5B blue trace), pre-steady

PAGE 195

I I 180 state biphasic kinetics are clearly visible with the amplitude of the initial rapid phase approximately 40-50 o/o of the first turnover amplitude in the a ssay with (Figure 5-5B, black trace) Kinetics of ATP Hydrolysis when the Minimal Complex is not equilibrated with '.ATP I While investigating the dependence of the pre-steady-state ATP hydrolysis kinetics of y complex on the preincubation period with ATP assays were performed in which there was no preincubation period (Figure 4-8), In that study, the possibility that I ATP binding toy complex was limiting the hydrolysis kinetics was addressed. The results showed that the rate of ATP binding could be contributing to the slow confo1mational change kinetics in the clamp loader By performing the assay with different concentrations of ATP it was determined that ATP concentrations above 200 M ( ~ 100-fold Ki a pparen t ) were high enough such that ATP binding was not limiting the rate of hydrolysis but slow conformational changes may have been limiting For comparison of the ATP hydrolysis kinetics of the minimal complex to the kinetics by y complex when neither is equilibrated with ATP pre-steady-state MDCC PBP ATPase assays excluding equilibration with ATP were performed Figure 5-6 shows the resulting kinetics of ATP hydrolysis by the minimal complex compared toy complex with 500 M ATP Using a single-mix setup, a solution of the minimal complex or y complex was added directly to a solution containing ATP DNA and MDCC-PBP in the stopped-flow An extended lag phase preceded biphasic ATP hydrolysis activity The lag phase displayed by the minimal complex activity was approximately 50-60 ms at least twice the duration observed when the minimal complex was equilibrated with ATP for 1-s in the previous assays (Figure 5-5A, blue trace)

PAGE 196

0.8 0.7 :iE 0.6 0. 0.5 0. m 0.4 0. I 0 0.3 0 C 0.2 :E 0.1 0 .0 0.40 0.35 :iE ._a, 0.30 0. 0.25 I 0. m 0.20 t1.. I 0 0 15 0 C 0.10 0.06 0.00 A. y complex 0.0 0.2 0.4 0.6 B. y complex 181 y 3 '6'6' minimal complex 0.8 1.0 1 2 time (s) 1.4 1.6 1.8 2 0 0.00 0.06 0.12 0.18 0.24 0.30 0.36 0.42 0.48 0.54 time (s) Figure 5-6 Kinetic s of ATP hydrolysis of the minimal complex or 'Y complex directl y mixed with pt DNA and ATP A ) In the stopped-flow a simple ' twos yringe '' assay was performed where a solution from one syringe loaded with the minimal complex ( blue trace ) or 'Y complex (black trace) was mixed with the contents of a s econd syringe loaded with pt DNA ATP and MDCC-PBP B ) MDCC-PBP-P i is plotted against time on an expanded scale for better observation of the lag phase for y complex ( black ) and the minimal complex (blue ) Solid line s through the data ( red y complex and black y3 66 minimal complex ) are fits using the model described in chapter 4 figure 4-10 with DynaFit ( Kuzmic 1996 ) All solution s were prepared in Pi-mopped assay buff e r : 20 mM Tris-HCI 50 mM NaCl 5 mM OTT 40 g/ mL BSA and 8 mM Mg C 1 2 Final reactant concentrations were 270 nM minimal complex or 'Y complex 500 M ATP 1 0 M pt DNA and 2 7 M MDCC-PBP Raw fluorescence data were transformed into the concentration of P i -bound MDC C -PBP using the equation s hown in figure 5-5

PAGE 197

182 Notably, biphasic ATP hydrolysis kinetics following the initial lag persisted even witho\lt equilibration of the minimal complex with ATP This was a key result in the investigation of y complex in similar assays Raw fluorescence data for the minimal complex reaction were fitted with a double exponential expression, excluding the long lag phase revealing rates that were roughly 2 s 1 for the first phase and 0.2 s 1 for the second phase The minimal complex data were also fitted using the computer model presented in chapter 4 (Figure 4-10 ) (here figure-5-11 ) that includes the PTTT + ETTT step prior to the ATP equilibration steps The resulting fit to that model is shown in Figure 5-6 ( solid black trace ). In modeling the minimal complex data, the lowered concentration due to '' dead '' enzyme was accounted for, and the forward and reverse rates between the '' activated '' ATTT and '' inactivated '' ETTI states were determined from fitting experimental data from a 1-s equilibration with ATP (figure 5-5A blue) as well as the rates describing the ETTT !:+ ITTT transition ( appendix) The results of the experimental analysis and fitting suggest that the minimal complex has a slower forward I conformational change towards the ATTT species in the model The model estimate of this forward rate was 2 6 s 1 consistent with the empirical fit of the raw fluorescence data (2 s 1 ) This estimated rate was about 2-fold slower than the estimated rate for the fo11nation of '' activated '' y complex Qualitatively the comparison indicates that the conformational changes giving the clamp loaders affinity for DNA were much slower for the minimal complex than y complex It is possible that without the x and 'V subunits, the minimal complex has more inherent confotmational instability than y complex (Olson et al ., 1995 ), and cause s a slower transition towards an '' activated '' complex In the future I

PAGE 198

183 this assay should be repeated with different concentrations of ATP to study the rate of ATP binding to the minimal complex. A TPyS-Cbase of Pre-Steady-State ATP Hydrolysis Activity by the y366' Minimal Complex An analogue of ATP, adenosine 5 -0-(3-thlotriphosphate) ( ATP-yS ) was used in I MDCC-PBP A TPase assays to qualitatively study the nature of nucleotide binding to the minimal complex and 'Y complex A TPyS is essentially non-hydrolyzable by 'Y complex (kca t 1 X 10-4 at 37 C ) ( Hingorani artd O'Donnell 1998) However, it does bind 'Y complex with a similar Ki, cause confo11national change and allow binding of~ (Bertram et al ., 1998) To determine if the A TPs bound to the minimal complex can readily exchange with ATPyS pre-steady-state MDCC-PBP ATPa se assays were perfo1med where the hydrolysis of ATP bound to the clamp loader was chased upon the addition of excess ATPyS This A TPyS-chase assay was perfo11ned in the presence and absence of~ to determine if~ could in fact bind to and 'trap '' the minimal complex in an active confo11national state, 'locking in '' nucleotides To chase the ATP hydrolysi s activity with A TPyS the sequential-mix 'three syringe '' stopped-flow setup was used A syringe containing a solution of the minimal complex and ( when present) was mixed with a second syringe containing a solution of ATP This mixture was preincubated for 1 s and then mixed with the contents of the third syringe loaded with pt DNA, MDCC-PBP and A TPyS (i e at a 10-fold higher concentration than ATP) Figure 57 shows the results of ATPyS-chase assays in the presence and absence of~ for the minimal complex. For the chase in the presence of~ (Figure 57 A red trace), a single rapid phase of ATP hydro I ysis occurred which ended at an amplitude approximately equal to the original MDCC-PBP A TPase assay

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Figure 57 Pre-steady-state kinetics of ATP hydrolysis by the minimal complex when chased with non-hydrolyzable ATP-yS ATPyS-chase assays were performed A) for the minimal complex in the presence of P> and B) in the absence of~ ATP-yS-chase assays( + ATPyS, red) show that ATP originally bound to the minimal complex was hydrolyzed faster than it dissociates Using a sequential-mix ' three-syringe'' stopped, flow assay, one syringe loaded with a solution containing the minimal complex and p ( when present) was mixed with the contents of a second syringe loaded with ATP The resulting mixture was preincubated for a period of 1 s, and then mixed with the contents of a third syringe containing a solution of pt DNA, :MDCC-PBP and ATP-yS All solutions were prepared in Pi-mopped assay buffer : 20 mM Tris-HCl 50 mM NaCl, 5 mM DTT, 40 g/mL BSA, and 8 mM MgCl2 Final reactant concentrations were 270 nM minimal complex, 1.0 M (when present) 100 M ATP, 1 0 M pt DNA, 2.7 M MDCC-PBP and I mM ATP-yS. The flat trace (gray) at the bottom of each figure showing no change is a negative control assay performed in the absence of ATP Raw fluorescence data were transf or1r1ed into the concentration of Pi-bound MDCC-PBP using the equation shown in figure 5-5 I

PAGE 200

.,,.A :i :i .. u 0. I 0. DJ Q. I 0 0 C :E :i ..:!, 0. I 0. al 0. I 0 0 C :!!: 185 A y368' minimal complex with 1 0 ~------------------. 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0.2 0. 1 0 0 0 5 0.4 0 3 0.2 0 1 0 0 0 0 0 2 0 4 0 6 0.8 1.0 1 2 1 4 1.6 1 8 2.0 t ime ( s ) B. y306 minimal complex without 0.0 0 2 0 4 0 .6 0.8 1 ~ O 1.2 1 .4 1 6 1 8 2 0 time ( s ) This is es s entially an isolation of the first turnover of ATP hydrolysis in the clamp loading reaction and there was no s teady s tate ATP hydrolysis activity Increasing the preincubation period to 2 s in thi s assay resulted in no difference in activity ( not shown) These results are similar to tho se observed for y complex in the presence of~ (Figure 45 A ), and indicate that all ATP molecule s bound to the minimal complex active confo1mation are '' locked in '' and hydrolyzed faster than they di ss ociate

PAGE 201

186 In the chase assay without~ (Figure 5-7B, red trace), the addition ATPyS reduced the amount of ATP hydrolyzed by the minimal complex to a short biphasic increase in ATP hydrolysis preceded by a short lag The duration of the lag was similar to that observed in the original MDCC-PBP ATPase assay ( Figure 5-7B, black trace ). The total level of amplitude of thes~ two pre-steady-state phases was roughly equal to the amplitude of the small initial rapid phase observed in the original assay This result shows that ATP hydrolyzed in these two pre-steady-state phases by the minimal complex ' was hydrolyzed more rapidly than it dissociat~ and that the ATP hydrolyzed in the original assay slow phase or steady-state reaction readily exchanged with A TPyS ATPyS-Chase of Steady-State ATP Hydrolysis Activity by the y366' Minimal Complex or 'Y Complex The effect of addition of ATPyS in the steady state MDCC-PBP ATPase assay for the minimal complex and 'Y complex was studied along side the pre-steady-state A TPyS chase assays In the steady-state clamp loading cycle there must be a nucleotide exchange step following hydrolysis of ATP The A TPyS-chase of steady-state ATP hydrolysis activity was measured in assays containing the minimal complex or 'Y complex and pt DNA in the presence and absence of~ using concentrations of 50 nM minimal complex ory complex 150 nM pt DN~ and 150 nM ( when present ) ATP hydrolysis activity was initiated by addition of a solution of ATP giving a final concentration of 77 M. After approximately 10-s of ATP hydrolysis activity solution s containing varying concentrations of A TPyS were added in separate reactions providing a range of A TPyS to ATP ratios (Scheme 5-1 ). The ATPyS : ATP ratios for assays with~ were 0 25 : 1 0 5 : 1 I : 1 2 : 1 and 10 : 1 The ATPyS : ATP ratios for assays without~ were 0 25 : 1 1 : 1 2 : 1 and

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187 10 : 1 The assays where a 10 : 1 ATPyS : ATP ratio was present mirrored the ratio of ATPyS : ATP used in the pre-steady-state ATPyS-chase assays (T able 5-2 ) In the steady-state assay with ~ there ATP hydrolysi s activity was completely poisoned after addition of A TPyS at all A TPyS : A TP ratios tested with both the minimal complex and 'Y complex add add 10 s -Inhibition ? ATP ATPyS constant varied concentration concentration cuvette Scheme 5-1 Steady-state A TPyS-chase assay Table 5-2 Steady-state A TPyS-chase assay results : comparison of the minimal complex and 'Y complex in the presence or absence of B ATPyS : ATP minimal minimal 'Y complex + y complex ~ complex + B complex B 0 . 25:1 a 0.b + 0.5:1 n. n. 1:1 +c + 2:1 + 10:1 (I a Inhibition of ATP hydrolysis activity b Assay not perfor1ned at this ratio c ATP hydrolysis activity persists d Pre-steady-state ' mock-ratio ' When A TPyS was added to steady-state MDCC-PBP ATPase assays in the absence of~ a different effect was observed Over the range of A TPyS : A TP ratios from 0 .25 : l to 2 : 1 ATP hydrolysi s activity persisted for y complex although a ratio of 2 : 1 was enough to inhibit ATP hydrolysis activity of the minimal complex At a ratio of I 0 : 1 which mimics the pre-steady-state A TPyS-chase condition all ATP hydrolysis activity was

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188 abrogated for y complex and of course the minimal complex as well It appears that the pt DNA-stimulated hydroly s is of ATP continued until there was a large enough excess of I ATPyS to compete with ATP for all of the nucleotide binding sites in the clamp loader to stop the re action. These steady-state MDCC-PBP ATPase assays chased with ATPyS also show that the I 0-fold excess A TPyS used in pre-steady-state A TPyS-chase assays was sufficient to completely inhibit the steady-state ATP hydrolysis activity of both clamp loaders Clamp Loading Activity of the Minimal Complex is More Sensitive to ADP than 'Y Complex. A significant result of the A TPyS-chase analyses was the difference in sensitivity for nucleotide binding of the minimal complex compared toy complex To determine in more detail the nature of nucleotide binding to the minimal complex and 'Y complex, the effect of ADP on the steady-state clamp loading reaction was studied using fluorescence based anisotropy binding assays to measure clamp loading with RhX-labeled pt DNA Previously it was shown that ADP causes a change in confo1mation of 'Y complex similar to that caused by ATP in proteolytic digest assays (Hingorani and ODonnell 1998) This suggested that ADP could stably bind to the clamp loader However the conformation due to ADP binding must be different from that due to ATP because ADP cannot promote the clamp loading reaction To dete1mine if the ATP-bound clamp loader-~ clamp complex remained committed to clamp loading in the presence of excess amounts of ADP steady state DNA binding/clamp loading assays were perfo1med in the presence of increasing concentrations of ADP The steady-state clamp loading activity of the minimal complex or y complex was measured in several assays with RhX-pt DNA I

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/ 189 0.14 ~-----------------------, 0.12 0. 0 I 0.10 0 s 0.08 c,s C 0.06 G) a, C c,s s:. 0.04 0 0.02 0.00 -+--no ADP 0.2 mM 0.3 mM 0.6 mM 0.8 mM [ADP] Figure 5-8 Effect of increasing ADP concentration on the steady-state clamp loading reaction for the minimal complex and y complex In separate steady-state assays the polarized emission intensities ofRhX-pt DNA were measured for use in calculation of anisotropy upon the addition of solutions containing either the minimal complex ( 500 nM) (gray bars ) or y complex ( 500 nM) (black bars ) to cuvettes containing RhX-pt DNA ( 50 nM) (500 nM), ATP (0 3 mM) and a range of concentrations of ADP ( 0 0 0 2 0 3 0 6 0 8 mM) in assay buffer : 20 mM Tris-HCl pH 7 5 50 mM NaCl 5 mM DTT 40g/m.L BSA and 8 mM MgC1 2 Final concentrations are shown in parentheses The anisotropy value for free RhX-pt DNA (r&ee) in assay buffer with ATP and ADP was subtracted from the anisotropy for RhX-pt DNA in the presence of protein (roound) to give the value of change in anisotropy plotted The polarized emission intensities of the RhX-probe were measured for calculation of anisotropy upon the addition of a solution containing either the minimal complex or y complex to a solution of RhX-pt DNA, ~ ATP and varied concentrations of ADP in assay buffer The value of anisotropy reflects the steady-state clamp loading activity on RhX-DNA, and the constant concentration of ATP in these assays was high enough to promote the loading reaction for at least 200 s (see Figure 4-1 black trace)

PAGE 205

190 The calculated values of the change in anisotropy shown in Figure 5-8 were averaged for separate 30 s clamp loading reactions. Increasing ADP concentration hindered the minimal complex-catalyzed clamp loading activity The minimal complex I I clamp loading reaction was even inhibited at a concentration of ADP (0 2 mM), slightly lower than the constant concentration of ATP (0 3 mM ) The r complex-catalyzed clamp loading reaction remained unaffected until a large excess of ADP ( 0 8 mM) was present ADP does not completely stop the clamp loading reaction like ATPrS because, as a product of ATP hydrolysis ADP is part of the steady-state reaction cycle ADP does not become (' locked-in' to a clamp loader-P complex or even become part of a stable inactive complex with clamp loader because it is constantly exchanging with ATP Only when ADP was at a concentration above 0 6 mM did it begin to inhibit the clamp loading reaction by y complex This indicates that a product of ATP hydrolysis (ADP) inhibits clamp loading by the minimal complex to a greater extent than by y complex, and that the steady-state stages (i e ., conformational changes?) of the clamp loading reaction cycle for the minimal complex must differ from 'Y complex in some way Pre-Steady-State Kinetics of Clamp Loading by the Minimal Complex Initiated at Different Steps of the Reaction Cycle The kinetics of p clamp loading were studied in real time to dete1mine if similar populations of the minimal complex observed in pre-steady-state ATP hydrolysis assays could be observed in different stages of the loading reaction Fluorescence-based anisotropy binding assays were used to measure polarized emission intensities of RhX-pt DNA in real time using a stopped-flow apparatu s. The clamp loading reaction was initiated at different steps by changing the equilibration of and order of mixing the clamp loader with ATP and p, prior to rapidly mixing with RhX-pt DNA I

PAGE 206

I 191 Kinetics of Clamp Loading when the Minimal Complex is Equilibrated with ATP and P In the ATP hydrolysis assays there was only a single rapid phase observed when the minimal complex was preincubated with ATP and~ (Figure 5-5A) The monophasic reaction suggested that the minimal complex was completely in the '' activated '' state. Will similar rapid monophasic clamp loading kinetics be observed for the clamp loading reaction in the anisotropy binding assay ? A reaction was initiated after the ATP dependent conformational change arid assembly of the minimal complex with (Figure 5-9A scheme-I ). A solution containing the minimal complex, ATP and~ in one stopped-flow syringe was rapidly mixed with a solution containing RhX-pt DNA and ATP from a second syringe to initiate the reaction The schemeI results show a rapid single-exponential rise to an anisotropy value of roughly 0 31 in about 100 ms followed by a decay to steady-state anisotropy( ~ 0 25 ) over an additional 400 ms. The rapid rise in anisotropy most likely was the binding of the ~-bound minimal complex to RhX-pt DNA (see the correlation assays below ). After was loaded on DNA the anisotropy decayed as the clamp loader was released from DNA ending the first turnover of the clamp loading cycle The rate of the rapid rise in clamp loading activity for the minimal complex was similar to that observed for 'Y complex, but the decay into the steady-state activity was more rapid and more pronounced or '' deeper ''. The steady-state anisotropy ofRhX-pt DNA in the clamp loading reaction never decayed totally to the level of free RhX-pt DNA in all assays since the clamp loader and~ were in large molar excess over RhX-DNA This also shows that the end-point of the clamp loading reactions did not change with respect to when the reactions were initiated

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Figure 5-9 .. Pre-steady-state kinetics of the clamp loading reaction initiated at different steps The real-time change in polarized emission intensities of RhX-pt DNA was measured and used for calculation of anisotropy for clam p loading reactions by the minimal complex ( red ), compared to y complex (black) initiated at different steps by using the mixing schemes shown above the plots A ) ( Scheme 1 ), A solution from one syringe containing the minimal complex or y complex ~ and ATP was mixed with a s olution from a second sy ringe containing RhX-pt DNA and ATP. B) ( Scheme-2) One syringe, loaded with a solution containing the minimal complex or y complex and ATP was mixed with a solution from a s econd s yringe containing RhX-pt DNA, ~ and ATP C) ( Scheme-3 ), One sy ringe was loaded with a solution containing the minimal complex or y complex only then mixed with the contents of a second syringe loaded with a solution of RhX-pt DNA and ATP In all cases final reaction concentrations were 250 nM minimal complex or y complex, 600 nM ~ 0 5 mM ATP and 50 nM RhX-pt DNA in assay buffer containing 20 mM Tris-HCl pH 7 5 50 mM NaCl 5 mM DTT 40 g/mL BSA and 8 mM MgCl 2. The gray plots showing no change in anisotropy ( r rree ) was a DNA-only control where assay buffer was added to RhX-pt DNA All steady state clamp loading activity had matching anisotropy values respective to the clamp loader used, and the total intensities did not change with time indicating that initiating the clamp loading reaction at different steps had no effect on this s teady-state activity I

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>. 0. 0 .b 0 U) C ns >. a. 0 .::, 0 U) C l'G 0.34 0.32 0 30 0.28 0.26 0.24 0 22 0 20 A. scheme 1 0 0 0 2 0.4 193 c u vette 0 6 0 8 1 0 1 2 1 4 time (s) B. scheme-2 cu ette 0 34 ~---------==.i..=~-----------, 0 32 0 30 0 28 0.26 0 24 0 22 0 20 0 0 0 2 0.4 0 6 0 8 1 0 1 2 1 4 time (s)

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0.32 0.30 0 28 i o 0.26 fl) c C'G 0 24 0.22 0.20 0.0 0.2 0 4 0.6 0 8 time (s) Figure 5-9 Continued 194 0 ATP 1.0 1.2 1.4 Kinetics of Clamp Loading when the Minimal Complex is Equilibrated with ATP In a secon d assay (Figure 5-9B : scheme 2 ) the clamp loading reaction was initiated by adding an equilibrated solution of the minimal complex and ATP to a solution containing ~ RhX-pt DNA and ATP When the minimal complex was equilibrated with ATP in the corresponding pre-steady-state MDCC-PBP ATPase assay two kinetic phases were observed (Figure 5-5A) which suggested that a small '' activated '' population of the minimal complex was present and rapidly reacted with DNA followed by a slower hydrolysis reaction. T he amount of the '' activated 1 population of the minimal complex was estimated at the low value of ~ 25 o/ o of total enzyme

PAGE 210

I 195 Unlike that observed for r comple~ biphasic clamp loading kinetics were not seen for the minimal complex in this assay following equilibration with ATP (Figure 5-9B red trace) In fact a lag of ~ 70 80 ms in duration was present preceding a slow monophasic rise to an anisotropy value of roughly 0 26 that peaked at ~ 400 ms The anisotropy then I decayed into the same steady-state clamp loading reaction ( ~ 0 25) observed in the scheme-1 assay The minimal complex did not show any rapid clamp loading phase, suggesting that there was little or no '' activated ' species of minimal complex initially present This result contrasted with the pre-ste ady-state MDCC-PBP ATPase assay where a small initial rapid phase after equilibration with ATP was observed It is possible that the predicted concentration ( ::;63 nM) of the minimal complex present in the rapidly ATP hydrolyzing population was unable to produce a rapid DNA binding phase in this clamp loading assay In comparison to the DNA binding kinetics displayed by y complex (black trace) the minimal complex may be slower to form a DNA binding surface when it binds p Taken together these results could indicate that the minimal complex undergoes a significant slow step after equilibration with ATP before it can bind and load~ Kinetics of Clamp Loading When the Minimal Complex is Mixed Directly with a Solution of ATP, I}, and DNA To examine the kinetics of the slow phase the clamp loading reaction was initiated prior to the ATP binding confo1mational change(s ) and p binding steps (Figure 5-9C scheme-3 ) Anisotropy binding assays were performed where a solution containing the minimal complex alone in one stopped flow syringe was mixed with a solution from a second syringe containing ATP p and RhX-pt DNA lftbe minimal complex has a

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196 slower ATP-dependent conformational change compared to y eomplex, then will the obseryed clamp loading kinetic s by the minimal complex also be slower in this assay? In thi s scheme-3 assay ( Figure 5-9C red trace ), the minimal complex showed slow clamp loading kinetics rising to an anisotropy value of about 0.26 preceded by a 70-80 ms lag consistent with a reaction where a slow step forms prior to a fast phase This result was nearly identical to that observed in the scheme 2 assay ( Figure 5-9B red trace ), although in scheme-3 there was no equilibration of the minimal complex with ATP Similar sigmoidal kinetics were observed for y complex in the scheme 3 assay ( black trace ). The slower kinetics observed for the minimal complex in the scheme-3 assay do suggest that the minimal complex undergoes a slower rate-limiting transition than y complex does Direct Real Time Correlation of the Minimal Complex DNA Binding and ATP Hydrolysis Kinetics in the Presence and Absence of p Clamp To directly examine the timing off or111ation of the minimal complex clamp loader binding DNA with the kinetics of ATP hydrolysis in the presence and absence of p pre steady-state fluorescence anisotropy binding and MDCC-PBP ATPase assays were preformed under identical conditions These combined assays allow direct comparison of the kinetics of ATP hydrolysis activity ( Figure 5-5A ), and DNA binding activity (Figure 5-9A ) by the minimal complex in the presence of p These correlation assays were performed in a stopped-flow either outfitted for simple fluorescence emission detection ofMDCC-PBP for measurement of ATPase kinetics or for polarized emission detection for measurement of the RhX-pt DNA binding kinetics A sequential-mix 'three-syringe '' setup was utilized where one syringe was loaded with the minimal complex and P (when present ), and a second syringe was loaded with ATP The contents of these two syringes I

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197 were mixed and preincubated for 1 s then mixed with the contents of a third syringe containing RhX-pt DNA for RhX-anisotropy binding assays or unlabeled-pt DNA and MDCC-PBP for ATPase assays The concentrations of all reactants and buffer were the same in both assays Previously similar c 1 orrelated ~alyses were performed with 'Y complex for investigation of mutant p clamps (Bertram et al ., 2000) and recently for study of clamp loading activity on elongation proficient pt DNA substrates ( Ason et al 2003) Key results from these correlation assays with 'Y co~plex showed that a complex of the clamp loader, p (when present ), and DNA rapidly fo11nedjust prior to DNA-stimulated ATP hydrolysis activity which was then followed by release of the clamp loader from the complex as the first turnover of clamp loading was completed Figure 5-10 shows the results of correlation assays performed with the minimal complex When equilibrated with ATP in the presence of P (Figure 5-lOA) the anisotropy (DNA binding/clamp loading) data showed that a ternary complex rapidly peaked at an anisotropy value of about 0 25 in ~ 50-60 ms just prior to a '' burst '' of ATP hydrolysis ending at approximately 100 ms The anisotropy of the rapidly fo11ned ternary complex quickly decayed to the level of free RhX-pt DNA upon a single rapid phase of ATP hydrolysis In these assays the concentrations of the minimal complex and P were not saturating with respect to the concentration ofRhX-pt DNA and therefore the anisotropy decayed as expected to a level near that of free RhX-DNA During this anisotropy decay phase, the minimal complex was released from p loaded on DNA and the ATP hydrolysis activity showed a corresponding pause before the steady-state

PAGE 213

Figure 5-10 Direct correlation of the kinetics of DNA binding and ATP hydrolysis by the y 3 00 minimal complex in the absence and presence of~ clamp A ) Correlated RhX-pt DNA binding kinetics (red ) and ATP hydrolysis kinetics (black ) for the minimal complex in the presence of~ B ) Correlated RhX-DNA binding kinetics (red) and ATP hydrolysis kinetics ( black ) for the minimal complex in the absence of~ Using a sequential-mix '' threes yringe '' stopped-flow assay one syringe loaded with a solution containing the minimal complex and~ ( when present ) was mixed with the contents of a second syringe loaded with ATP The resulting mixture was preincubated for a period of I s, and then mixed with the contents of a third syringe loaded with a solution-containing RhX-pt DNA fot RhX anisotropy binding assay s, or unlabeled-pt DNA and MDCC-PBP for ATPase as s ays. All reactions were perfor1ned in assay buffer: 20 mM Tris-HCl 50 mM NaCl 5 mM DTT 40 g/ mL BSA and 8 mM MgCl 2 Final reactant concentrations were 225 nM minimal complex, 500 nM ( when present ), 2 00 MA TP 450 nM pt DNA, and 4 4 M MDCC-PBP The real-time change in polarized emission intensities of RhX-pt DNA was measured and used for calculation of anisotropy by the stopped-flow software Anisotropy binding data were fitted to an empirical double exponential function and plotted as solid curves (blue ). Raw MDCC-PBP fluorescence data were transformed into the concentration of P i -bound MD C C-PBP plotted using the equation shown in figure 5-5

PAGE 214

2.0 1.8 ........ 1.6 :E ..,:; 1.4 a.. 1.2 I a.. 1.0 m Q. 0.8 I 0 0.6 0 C 0.4 :E 0.2 0.0 199 A. min lmal complex with p -.-----------------.0.26 0.25 0.24 0.23 0. e 0.22 0 "' 0.21 C 0.20 0.19 '-r--"' -----'---''--r-'.__..__.__,_..__.__......._._~...__,_.....__,___.__,_......___._.....,__,..__,__,_----'--+ 0. 18 0 0 0.2 0.4 0.6 0.8 1 0 1.2 1.4 time (s) CG B. minimal complex without p 1.6 [ ~ ~ -~ =~====:::::::;: 7 0.215 1.4 0.210 ..... 1 2 1.0 I a.. 0.8 m a.. 0.6 I 0 0 0.4 C :e 0.2 0.0 0.205 Q. l e 0.200 .., 0.195 0.190 0.0 0.2 0.4 0.6 0.8 1 0 1.2 1.4 time (s) 0.185 0 "' C cu It is d uring this stage of t h e reaction where t h e n u c l eotide exchange steps are tho u ght to occur ending the firs t turnove r T he res u lts of t h ese correlated a ss ays are consistent with the p re stea d y s t ate kine t ic s of DNA bin d ing/c l am p l oading when the minimal comp l ex was equili b rated with ATP an d ~ (Figure 5 9A scheme 1 ), and with t he pre stea d y-state

PAGE 215

I 200 kinetics of ATP hydrolysis by the minimal co mplex when equilibrated with ATP and (Figure 5-5-A) In the absence of~ (Figure 5-lOB), the anisotropy (DNA binding ) data showed that a binary complex of the minimal comple x and DNA rapidly formed to an anisotropy value of roughly O 21 in ~ 70 ms j ust prior to stimulation of biphasic ATP hydrolysis I I activity. Unfortunately there was considerable noise in the calculated anisotropy in the DNA binding assay for the minimal complex in the absence of p under these correlation assay conditions The results albeit noisy do show that the minimal complex, after equilibration with ATP bound DNA just prior to the initial rapid phase of ATP hydrolysis activity The minimal complex then appeared to be releasing the DNA during the second phase of ATP hydrolysis before entering the steady-state reaction The MDCC-PBP ATPase assay (Figure 5-5A ) for the minimal complex was performed under more optimal conditions for examination of ATP hydrolysis kinetics ( i e ., when the concentration of pt DNA was saturating ) The previous assay showed much clearer results, to which the correlated assay appeared to be consistent Discussion Understanding y Complex Kinetics by Characterization of and Comparison with "(366' Minimal Complex The results described in this chapter address the function of the clamp loading machine by comprehensive study of a minimal complex clamp loader (y 3 00 ) and direct comparison to the activity of 'Y complex In the clamp loading reaction the rings haped p clamp must be opened placed at the correct location on DNA and released to allow attachment to DNA polymerase for processive synthesis To accomplish these well ordered tasks the clamp loader acts in a switch-like manner where ATP binding and

PAGE 216

201 hydrolysis modulate its affinities for and DNA The status of nucleotide binding to the three..ry motor subunits of the clamp loader coordinates intramolecular communication I between all subunits through a structural ''language'' of conformational transitions, 'switching '' between inactive and active specie s. ATP binding induces confo11national canges revealing sites for binding DNA and in the clamp loader DNA-stimulated ATP hydrolysis causes inactivation and release of the clamp loader from loaded ~' and ultimately, during nucleotide exchange, conformational changes occur that reset the clamp loader for continued activity The combination of y 3 0 1 0 1 subunits has been known to be the minimal subcomplex with clamp loading ability suitable for processive DNA synthesis in in vitro reactions (Onrust et al 1991 ; Stukenberg et al ., 1991) The y 3 00 minimal clamp loader structure has been solved is currently the most complete structure of any clamp loading machine ( Jeruzalmi et al ., 2001a ). By AAA + structural homology to the eukaryotic and archaeal clamp loaders, the y 3 00 structure has provided investigators with many fundamental deductions for conserved clamp loading functions (Davey et al ., 2002 ; Neuwald et al ., 1999 ; O'Donnell et al. 2001) Here steady-state and pre-steady-state ATP hydrolysis and DNA binding / clamp loading analytical methodologies were utilized with fluorescence-based techniques revealing the importance of the x and 'ti subunits for optimum ATP hydrolysis and clamp loading activity by 'Y complex This work extends the previous analysis of 'Y complex by mirroring and comparing results in identical biochemica l assays and addresses the theoretical model presented in chapter 4 for the clamp loader ATP-dependent conforrnational dynamics by providing additional insight into these conformational transitions that drive the E. c oli clamp loader I

PAGE 217

I 202 y 3 66' is the Minimal Complex with DNA Binding Ability, and Binds ATP and p with Affinity Similar toy Complex Examination of the r complex individual subunits and different combinations of these sub11nits initially revealed that only the combination of ( y / 't ) 3 o o X,'V (''r complex '' ) and(r / t) 3 0 o'(''minimal complex ''), had ATP-dependent DNA binding ability (Figure 51 ) Although the exact nature of the DNA binqing site within the clamp loader is currently undefined, this study shows that a DNA binding '' site ~ ( or surface ) was fonned in the presence of ATP A tetramer of the r subunit, for example cannot obtain an ATP' dependent confo11nation for for1nation of the putative DNA binding surface This indicates that the o and o' subunits are required and may physically contribute to formation of this DNA binding surface The ATPand clamp-binding constants, estimated using the ~ pyrene anisotropy binding assays were both nearly identical for y complex and the minimal complex Both bind ATP with an apparent dissociation constant of2 M (Figure 5-4 ). The ATP dependent affinity for ~p yrene was estimated at 3-4 nM for both clamp loaders (Figure 53 ). Thus under the assay conditions presented in this work, means that nearly all clamp loaders would be bound to when it is present The similar magnitude of these affinities for each clamp loader to ATP and suggest that the y-subunits nucleotide binding site accessibility was similar in both clamp loaders and the subsequent ATP-dependent changes inducing exposure of the 6-subunit ~-interaction element were also similar Pre-Steady-State ATP Hydrolysis and DNA Binding Kinetics: Analyses of the Active and Inactive Clamp Loader States The kinetics of ATP hydrolysis by the minimal complex are discussed in te11ns of the model presented in chapter 4 which was based in the computerized fitting of the pre steady-state ATP hydrolysis kinetic s of r complex (K 11z1nic 1996 )

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1r ? 1 'a s ETTT k ? 1 ae S ATTT+N ATTTN 203 l I I I kon l 36}vf s 1 60S 1 PBP+P, PPBP I koff 13.6 s hyd 60S ETTT AT DN+P, 1 khyd 60s ADDD + N + R I Figure 5-11 Kinetic modeling of ATP hydrolysis reactions This model is reproduced from chapter 4 (F igure 4-lOA ). Three states of the clamp loader ETTT ATTT and l l l T are shown ( cartoons are based on the structure of the clamp loader see chapter2 figure 2-3 ). The ATTT form binds DNA (N) rapidly and hydrolyzes its three molecules of ATP ( T ) in s ucce ssion producing ADP (D ) and inorganic phosphate (Pi). The clamp loader dis s ociates from DNA on hydrolyzing its third molecule of ATP and is recycled back to the ETTI state. Experimenta lly detertruned rates of 'Y complex binding DNA ( A s on et al 2000 ; Bertram et al., 2000), konN and MDCC-PBP (PBP ) binding phosphate (Brune et al ., 1994 ), kon and ko rr are presented All DynaFit program scripts and analysis for these models and KinTekSim simulations are presented in the appendix This model demonstrated that at least three different species of 'Y complex were requir ed to produce biphasic kinetics of ATP hydrolysis and that these three species could not all exist on the linear pathway to products The minimal model that be s t fit the data ha s been reproduced here as Figure 5-11 and is presented without the rates deterrnined for the 'Y complex conf or1national change because the assays perfor1ned to reliably estimate those

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204 rates have not been repeated for the minimal complex ( see computer modeling methods in chapter 3 for details ). I In this model the clamp loader is initially present in a state ETTI that can convert to two different species one that is ' activated '' for DNA binding A TIT, one that is ~active ITTT and does not directly fo11n products In this minimal model, it is asst1med that each of these species is bound to three molecules of ATP because ATP hydrolysis rates were independent of the ATP concentration at this saturating concentration ( 400 M ATP ) However it is possible that the E and/or I s tates have fewer molecules of ATP bound and that very rapid ATP binding steps and additional confo1mational changes exist between the three states. In this model the activated s tate ATTT rapidly binds DNA This DNA-clamp loader complex then hydrolyzes each of its three molecules of ATP sequentially at the same rate The ATP hydrolysis steps are modeled using single forward rate constants because the presence of:MDCC-PBP limits the rever s e reaction such that it cannot accurately be deter1nined Previous work ha s shown that the DNA dissociation step is likely to be very rapid relative to the steady-state recycling of the clamp loader and because these data do not directly measure this step DNA dissociation was set to be concurrent with hydroly s i s of the third molecule of ATP Finally, the steady-state portion of the reaction was modeled as a pseudo-first order conversion of the ADP-bound clamp loader ( ADDD ) back to the initial ATP-bound s tate (ETTT) This pseudo-frrst order approximation is reasonable because only a s mall fraction of the ATP s ubstrate is consumed on the time scale of the reaction

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I 205 Here examination of the pre-steady-state ATP hydrolysis and clamp loading activities reveal that the minimal complex also exists in a mixture of confo11national states in equilibrium with ATP However several notable differences were observed for the minimal complex mechanism suggesting a slower confotmational transition and less stable '' activated, ATTT c9nformatio~al state in the minimal complex The pre-steadyI state biphasic kinetics of ATP hydrolysis observed when the minimal complex alone was equilibrated with ATP and mixed with DNA ( Figure 5-5A, blue trace) or not equilibrated with ATP prior to mixing with D~A and ATP (Figure 5-6 ) suggested that two species of the minimal complex were present but the estimated population of the rapidly acting ATTT species was significantly smaller ( ~ 25 o/ o of total enzyme) in comparison to 'Y complex (Figure 4-3) ( -40 % of total enzyme) present under similar conditions The two kinetic phases observed in the minimal complex assay may not represent the same biphasic kinetics described for 'Y complex in chapter 4. For the minimal complex, the short pre-steady-state rapid phase is followed by a second pre-steady-state phase that is likely indistinguishable from steady-state activity, or the pre-steady-state rapid phase is directly followed by steady-state hydrolysis kinetics In assays with the minimal complex alone (figure 5-5A blue trace) several possible mechanisms could produce the two kinetic phases 1 ) A single population of the clamp loader was present in which the individual y subunits hydrolyze ATP a different rates 2 ) like 'Y complex, two distinct species of the clamp loader were present that differ in DNA-stimulated ATP hydrolysis activity or 3 ) two different species of clamp loader interact with pt DNA in separate parallel reactions For example pt DNA binding may not be coupled with the

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206 pre-steady,.state rapid hydrolysis phase due to a 1 15 molar excess of ss-primer DNA over pt DNA likely to be present in these assays After the small population of t 'activated ' ATTT species reacted with ss DNA, then the steady -state formation of ATTT species from ETTI would be the separate reaction with pt DNA In the later two cases, tl;le two species could differ by the ATP-dependent confo1mational change(s) required for binding DNA described by the model One confor1national species ATTT has high affinity for DNA and therefore binds pt DNA ( or ss DNA ) and hydrolyzes ATP at a faster rate than the second The second phase of ATP hydrolysis could arise from the ETTT species slow ly changing conformation forming the ATTT species, and therefore slowly gaining affinity for DNA The equilibriwn mixture of the minimal complex species with ATP resulting in a small '' activated ' ATTT population ( ~ 25 %) could provide a mechanistic explanation for the slower steady state ATP hydrolysis activity observed without ~ In the pre-steady-state DNA binding/clamp loading assay (Figure 5-9B scheme-2), when the minimal complex was equilibrated with ATP prior to mixing with a solution containing~ RhX-pt DNA and ATP biphasic clamp loading kinetics were not exhibited This was in contrast to the expected biphasic activity based on the model that predicts the mixture of species in equilibration with ATP dominated by the ETTT / l l l "' f and A TTT states A lag of ~ 70-80 ms followed by slow clamp loading kinetics were observed It is interesting to note that the lag phase directly related to the duration of the rapid phase of the biphasic kinetics displayed by y complex (Figure 5-9B) showing that there is some significant disparity in the clamp loading reaction when x and 'I' subunits are missing from the clamp load er I

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207 The differences between the DNA binding / clamp loading kinetics by the minimal clamp loader from 'Y complex could have been due to a difference in equilibrium between activated (ATTT) and inactive states (ETTT !:. ITTT ) predicted modeling the pre-steady state ATP hydrolysis analysis Another likely possibility is that the model based on the ATP hydrolysis kinetics dqes not adequately describe the kinetics of DNA binding/clamp loading Although similar populations of activated and inactive clamp loader species could be expected during equilibration with ATP based on the model when mixed with I DNA and ~ a different reaction may have beet). measured in the DNA binding/clamp loading assays For 'Y complex the pre-steady state biphasic DNA binding/clamp loading kinetics did show that distinct species were present but it is possible that they were not the same as those described in the model It is currently unknown which of the ATP-dependent conformational states~ can bind to The data shown in figure 5-3 even shows that~ binds the clamp loader in the absence of ATP albeit with 50-fold less affinity than in the presence of ATP The model based on the pre-steady-state ATP hydrolysis experiments satisfactorily describes the mixture of clamp loader ATTT, E TIT and ITTT conformational states in equilibrium with ATP but whether p binds any one or all of these species is unclear For example ( with 'Y complex ), the activated ATTT clamp loader species may rapidly bind DNA before binding ~ and the E TTT or ITTT states could bind ~ and then convert into an activated '' ATTT~ '' -complex that loads the clamp in the second slower pre-steady-state phase For the minimal complex equilibrated with ATP the pre-steady-state ATP hydrolysis kinetics predicted that a small population of activated A TTT species was

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208 present that hydrolyzed ATP in a rapid pre-steady-state phase . Why then was there an initial lag observed in the DNA binding/clamp loading assay when the minimal complex 1 was equilibrated with ATP? It is possible that the small-activated population did load~ on DNA initially but the resulting change in anisotropy was too low to observe The minimal complex DNA binding/clamp loading kinetics suggested that the~ clamp loading reaction was preceded by a slow phase Following the above example (for y complex) may have bound to any one or all of the minimal complex species in equilibrium with ATP ( ATTT ETTT ITTT) and a slow conversion ofETTT and/or ITTT to an activated '' ATTT~ '~ -complex occurred while the small original activated ATTT population rapidly bound DNA and hydrolyzed ATP When DNA binding kinetics (in the absence of~) were correlated with ATP hydrolysis for the minimal complex equilibrated with ATP (Figure 5-1 OB ), a small amount of DNA binding activity just prior to ATP hydrolysis was observe~ further suggesting that only a small activated population of the minimal complex was present Although this experiment was not a clamp loading assay, it did show that a small amount of the active minimal complex reacted rapidly with DNA instead of displaying an initial lag phase The minimal model (Figure 5-11) describes intramolecular confo11national transitions that are likely to separate the mixture of states in equilibrium with ATP for y complex and probably the minimal complex as well However without knowing to which ATP-dependent confo1mational state or states~ binds it is not yet possible to reliably model the DNA binding/clamp loading kinetics displayed in these assays when either clamp loader was initially equilibrated with ATP

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209 The monophasic ATP hydrolysis kinetics for the minimal complex in the presence of~ indicate that there was only a single species of minimal complex present that was active for DNA binding, and therefore clamp loading. These results clearly show that the x and 'V subunits missing from the minimal complex are required by y complex to maintain a higher population of ATTT activated conformational species in equilibrium I I with ATP For example, they may facilitate the conformational changes separating the ATTT and ETTT species of the clamp loader The~ clamp appears to help the minimal complex increase its pre-steady-state activity perhaps by bypassing a slower reaction step (i e the [ITIT !::; ETTT] !::; ATTT transition) that occurs in the first turnover of ATP In pre-steady-state ATP hydrolysis experiments in the presence of~' reactions were initiated following a preincubation period allowing equilibration of the minimal clamp loader with ATP and ~ The minimal complex and y complex displayed similar rapid monophasic kinetics in the first turnover (Figure 5-5A and B) Pre-steady-state ATPyS chase assays perfo1rr1ed in the presence of~ (Figure 57 A) were also similar toy complex (Figure 4-5B), showing rapid monophasic kinetics of a single turnover of ATP hydrolysis These results indicate that equilibration with ATP and converted or ' trapped'' all species of the minimal complex into an '' activated' ATTT-~ bound conformational state effectively increasing its population before being mixed with DNA to initiate measurement of ATP hydrolysis activity for the clamp loading reactions The correspo nding pre-steady-state DNA binding/clamp loading assays where the minimal complex was equilibrated with ATP in the presence of~ to initiate the reaction after the ATP-dependent confo11national change also showed a single rapid phase of activity (Figure 5-9A, scheme-I). The decay in anisotropy as the clamp loader released

PAGE 225

210 from DNA"7~ occurred at a faster rate and was '' deeper' for the minimal complex compared to y complex as the reaction entered the steady-state The decay to a lower anisotropy value for steady-state activity indicates that there was little minimal complex rebinding 'to DNA during this phase A possibility is that the steady-state reaction was ~peded as a consequence of slower conforn1ational change ( s) (i.e. [ITTT ETTI] ATTT) required for conversion to the reactivated '' ATTT~ '' state Slower conversion of the minimal complex and less clamp loading activity could explain the quicker rate of the decay compared to y complex as well as the reduced steady-state anisotropy observed in this and other assays with the minimal complex Further the correlation of pre-steady state ATP h y drolysis and DNA binding/clamp loading activity (Figure 5-IOA) demonstrated that the rapid monophasic DNA binding/clamp loading reaction ( analogous to scheme-I in Figure 5-9A ) directly preceded a monophasic '' burst '' of ATP hydrolysis by the minimal complex Together the pre-steady-state ATP hydrolysis and DNA binding assays in the presence of~ clearly show that the clamp has the ability to convert the mixture of ATP-dependent confo1mational states of the clamp loader into a single activated population '' ATTT~ '' capable of rapid loading of~ on DNA followed by rapid ATP hydrolysis in the first turnover Again whether binds the A TIT state only and '' traps '' it or binds to the E TIT or ITTT states and increases the rate of their conversion to the activated '' ATTT~' species is unclear The x and 'I' Subunits, Missing from the Minimal Complex, May Facilitate the Conformational Dynamics of 'Y Complex The only difference between y complex and the minimal complex in these experiments is the presence of the x and 'l' subunits in y complex Based on the model for equilibration with ATP and pre-steady-state ATP hydrolysis activity ( Figure 5-11 ), the

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I 211 absence of the x, and 'V subunits from the minimal complex may cause conformational instability or some other deficiency in ATP-dependent conformational communication between subunits within this clamp loader, resulting in a low quantity of activated A TTT versus inactive [ETTT !+ ITTT] confo1mational states A possibility is that the overall forward rate of confo1matipnal change:s ( ... ETTT ATTT .. ) producing the minimal complex activated species may be slower than the corresponding rate for y complex Also an increased reverse rate of conformational transitions ( ... ETTT A TIT .. ) could potentially result in an elevated population of tpe '' inactive '' minimal complex species The opposite conformational dynamics could exist for y complex resulting in the difference in estimated activated species population observed between y complex ( ~ 40 0 /o) and the minimal complex (~ 25 /o ) when equilibrated with ATP For example 'Y complex could be optimized for an overall faster forward rate of confo1mational changes (i.e ., facilitated by the x, and 'V subunits) towards an activated species and a slower reverse rate towards an inactive population p Clamp Enhances the Switch from Inactive to Active Clamp Loader Populations It has been known for some time that the sliding clamp enhances the ATP hydrolysis activity of the clamp loader and therefore the clamp loading activity of y complex ( Onrust et al ., 1991 ; Stuk:enberg et al ., 1991) and also clamp loaders of other organisms (Ellison and Stillman 2001 ). Here steady-state and pre-steady-state ATP hydrolysis assays show that the activity of the minimal complex is nearly identical to 'Y complex in the presence of~ consistent with previous analyses enhanced the steady state ATP hydroly s i s turnover rate approximately 2-fold for the minimal complex and characteristically 3-fold for y complex ( Table 5-1 ). An even more significant result was the enhancement of an apparent s econd-order binding constant (~ 1 complex /K.m ) in the

PAGE 227

212 presence of~ This steady-state parameter is roughly 1000-fold slower than would be expected for diffusion-controlled binding of a small nucleotide cofactor such as ATP to I an enzyme and thus indicates that binding of ATP is followed by a slow step leading to hydrolysis such as the proposed confo1mational changes modulating clamp and DNA b~nding affinities The increase in the specificity constant for y complex by the presence of~ was about 2-fold whereas the minimal complex showed a much higher increase in the specificity constant ( 4-f old) demonstrating that did more '' work '' in conversion of this putative slow phase following ATP binding Based on these steady-state data, along with the similarities observed for the minimal complex and y complex in pre-steady-state ATP hydrolysis data, it is concluded that clamp allows the clamp loader to overcome the slow phase, most likely by conversion of the population of inactive clamp loader into a population of all completely activated conformational state ' ATTT~ ''. Further the enhancement of the minimal complex specificity constant supports that clamp had a much greater effect in --ETTT ___.~ Al"l"I + ~ATTI~ 4 4 Scheme 5-2 clamp enhancement of '' activated '' clamp loader conversion of the large estimated population of minimal complex '' inactive ' species (95 % of total enzyme) Enhancement by effectively increases the concentration of the active species most likely by selectively binding to ATTT and '' trapping '' it into an active ATTT~-complex poised for clamp loading Alternately could conceivably bind to an 'inactive '' species such as ETIT and increase the rate of conversion to ATTT~ ( see scheme 5-2 ). I

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r 213 Experiments when the Minimal Complex was not Equilibrated with ATP Reveal Slower Conformational Change Kinetics than 'Y Complex In the pre-steady-state DNA binding/clamp loading assay where the minimal complex was not equilibrated with ATP slow sigmoid-like clamp loading kinetics were seen in the first turnover (Figure 5-9C scheme-3 ) Interestingly this initial lag was of the same duration ( ~ 70-80 1 ms) observed when pie minimal complex was equilibrated with ATP before mixing with DNA and~ (compare with Figur~ 5-9B) These kinetics were similar to with those observed with y complex in the same assay conditions and I show that the DNA binding/clamp loading kinetics were preceded by a slow step The same step responsible for the lag phase most likely limited the slow clamp loading kinetics as well and it is hypothesized here that this slow step was an intramolecular confo1n1ational change(s ) following ATP binding Previously for y complex it was observed that the ATP-dependent conformational change was limiting the clamp loading reaction progress Pre-steady-state kinetics of ATP hydrolysis were also measured when the minimal complex (Figure 5-6 ) (or y complex figure 4-8) was not equilibrated with ATP The observed sigmoid-like kinetics were analogous to those observed in the DNA binding/clamp loading assay (Figure 5-9C scheme-3) The minimal complex when directly mixed with DNA and ATP displayed a lag of r--00 ms that was then followed by slow hydrolysis kinetics These analogous clamp loading and ATP hydrolysis kinetics indicate that a rate limiting complex-confo1mational transition switching an inactive species into an active species occurred when the clamp loader was mixed with ATP However it remains possible that the kinetics represent a mechanism different than when the clamp loader is equilibrated with ATP before mixing with DNA ( +/ -~) It is entirely

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214 possible and likely that the clamp loader is fluctuating between confor1national states in the absence of any nucleotide that are different than the conformational states observed in equilibrium with ATP Biphasic kinetics in either case are still consistent with the domination of two states overall including a '' branch' pathway towards no product fq1mation, modulating activity of the clamp loaders Compu ter modeling of the data shown in Figure 5-6 was done using the model presented in chapter 4 (F igure 4-10 here figure 5-11) The computer modeling results for the minimal complex estimate that the forward confo11national change towards an active complex is ~ 2-fold slower than for y complex and the reverse conformational change is roughly 3-fold faster than for y complex Fitting these single data sets for the minimal complex doe s not provide enough data for a completely reliable fit however the fit shown in figure 5-6 as well as simulations shown in the appendix (Figures A-3-sim, A-4-fit ) are consistent with slower forward conformational changes producing a small active population of the minimal complex species The small change in MDCC-PBP fluorescence due to the small active population limited the ability to further examine the A TPase kinetics as a function of equilibration time with ATP The Nature of Nucleotide Binding to the Minimal Complex and 'Y Complex The pre-steady-state ATP hydrolysis activity of the minimal complex in the presence of~ was cha s ed by the addition of a 10-fold exce s s ofnon-hydrolyzable ATPyS to examine the nature of the nucleotide binding The minimal complex was preincubated with ATP and before mixing with DNA to trigger ATP hydrolysis in the presence of A TPyS ( Figure 57 A red trace ). It is known that still binds the clamp loader in the presence of ATPyS but cannot load~ onto DNA without ATP hydrolysis (Bertram et al 1998 ). E xperiments for the minimal complex were perfor1ned under conditions identical

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I 215 to assays with 'Y complex (Figure 4-5B ) For both clamp loaders in the presence of p a single turnover of ATP hydrolysis occurred and there was no further steady-state activity. The results demonstrated that in the presence of p the ATPs bound to the clamp loader were hydrolyzed faster than they exchanged with A TP'YS in the pre-steady-state suggesting that they were s~bilized or trapped '' in an activated confo11nation clamp loader-P complex '' ATTT~ ''. In the steady-state the excess amount of ATP'YS easily out competed ATP for binding to the minimal complex or 'Y complex poisoning further hydrolysis activity For steady-state ATP'YS-chase of the minimal complex or 'Y complex activity in the presence of~ A TPyS was added directly to the steady-state ATP hydrolysis reaction approximately 10-s after reaction initiation (see Scheme 5-1) Several ATPyS : ATP ratios were tested in separate assays, including a 10 : 1 pre-steady-state mock-ratio Under all ATPyS : ATP ratios tested, there was no further hydrolysis activity after chasing the reaction with ATPyS (Table 5-2) Due to the presence of~ the 'activated '' confo11national state resulting from A TPyS binding was trapped in a state that could not load on DNA, and therefore there was no further ATP hydrolysis or nucleotide exchange Even a substoichiometric ATPyS : ATP ratio of 0 25 : 1 was sufficient to stop the reaction in these assays indicating the possibility that A TPyS may share one or two of the three nucleotide binding sites with ATP in the clamp loader and still prevent ATP hydrolysis-dependent loading of~ These results support the idea that clamp traps the clamp loader in an active conformation where the nucleotides (ATP or A TP'YS) are stably bound. This provides additional evidence for a mechanism of clamp loader enhancement

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216 by the sliding-clamp, showing that an activated clamp loader ~ complex '' ATTT~'' is stable and committed to clamp loading upon pt DNA triggered ATP hydrolysis Pre-steady-state ATPyS-chase assays in the absence of~ were also performed The minimal complex was preincubated with ATP and then mixed with DNA in the presence of a 10-fold excess of ATPyS (Figure 5-7B red trace). A small biphasic level of ATP hydrolysis activity resulted that was roughly equal to the level of the small rapid phase seen in original assay (Figure 57B, black trace) This result could indicate that there was considerable conforrnational instability leading to more competition between ATP and ATPyS even in the active species population Another possibility is that these two pre steady-state phases observed in the A TPyS-chase assay represent the same two kinetic phases seen in the original assay however they are significantly reduced in amplitude due to competition between ATP and ATPyS in binding the '' unstable'' minimal complex. When the preincubation time was increased to 2 s for this assay there was no difference in the resulting kinetics of ATP hydrolysis activity (not shown) This A TPyS-chase assay did isolate the small population of the activated conformational species (A TIT) of the minimal complex that was present when equilibrated with ATP Under the same assay conditions with y complex (Figure 4-5A) a single rapid phase of ATP hydrolysis followed by a second low-level slow phase was observed prior to complete loss of steady state activity. Both assays show that ATP hydrolyzed in the rapid pre-steady-state phase is hydrolyzed more rapidly than it dissociates and that the ATP hydrolyzed in the second-slow phase of the original assay readily exchanged with ATPyS When the steady-state ATP hydrolysis activity of the minimal complex in the absence of~ clamp was chased with ATPyS it was inhibited at a lower ATPyS : ATP ratio

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I I 217 than 'Y complex was (Table 5-2 ). Since the ATPyS-chase wa s initiated during a steadystate reaction, the nucleotide exchange s tep following DNA-stimulated ATP hydrolysis was vulnerable During this step the clamp loader would be in equilibrium with ATP if only for a short period of time ( i e ., the ATP concentration was saturating in these assays) and therefore only 1 a small population of the minimal complex compared to y complex would in be in the ATP-dependent confo11national activated state A pos s ible reason for the elevated sensitivity of the minimal complex to ATPyS in these steady-state I I chase assays is that the estimated high population of '' inactive '' minimal complex conformational states would easily bind A TPyS and become unavailable for further ATP binding and hydrolysis stopping the reaction In contrast at similar ATPyS : ATP ratios less y complex '' inactive '' species is present during nucleotide exchange, and maintained continued ATP hydrolysis activity at nearly a normal steady-state rate When chased with stoichiometric ( or even slightly higher i e 2 : 1 ) ATPyS : ATP ratios 'Y complex may either be able to maintain a population of an all ATP-bound active state or sustain the ability to bind DNA and hydrolyze ATP even when there is mixed A TPyS and ATP binding during the steady-state reaction It is known that A TPyS can induce conformational changes in y complex allowing DNA binding If the nucleotide binding site s were mixed with ATP and ATPyS the results show that DNA binding is s ufficient to trigger hydrolysis of a minimum of one ATP in 'Y complex Thi s indicates that when y complex is in equilibrium with both ATP and ATPyS additional confor1national states could be identified in pre-steady-state ATP hydrolysis reactions that represent species where only one or two hydrolyzable A TPs are present

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218 Additional analysis of the nature of nucleotide binding during the steady-state clamp loading reaction by the minimal complex and 'Y complex was performed It was I t deter1nined that increasing relative concentrations of ADP could inhibit the DNA binding/clamp loading activity of the minimal complex but not 'Y complex ( Figure 5-8 gray bars ). ADP alone is insufficient for DNA binding and clamp loading activity In steady-state DNA binding/clamp loading as s ays the concentration of ATP was held constant at a level that could sustain s teady-state clamp loading activity for at least 200 s ( see chapter 4 figure 4-1 black curve ). When ADP was added at increasing concentrations 'Y complex continued loading p cycling through the steady-state reaction Only when the ADP concentration was raised to a level ~ 2 7-fold above ATP was the clamp loading cycle of 'Y complex somewhat hindered The clamp loading activity of the minimal complex however was diminished beginning at a substoichiometric concentration of ADP relative to ATP and decreased further with increasing ADP. It was previously shown that ADP could stably bind 'Y complex and cause a confonnational change (Hingorani and O'Donnell 1998 ; Naktinis et al ., 1995 ). The p clamp must do more work to convert and '' trap '' the minimal complex in an activated ATP-dependent conformational state. Therefore in these steady-state assays there is predicted to be a significant level of '' inactive-state '' minimal complex during nucleotide exchange after ATP hydrolysis It is believed that ADP can readily compete with the 'inactive confo1mational s tates of the minimal complex and inhibit steady-state clamp loading activity These results add to the ATPyS-chase analyses and support a mechanism where the sliding cJamp enhances its clamp loader by affecting the conformational dynamics I

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I I 219 > that modulate DNA binding by trapping the clamp loader in ah activated clamp loader-~ clamp complex with A TPs secured 'ATTT~ ''. Investigation of the minimal clamp loader complex has proven to be invaluable for understanding the activity of 'Y complex in clamp loading This comprehensive analysis of the minimal complex sqows that it does have activity similar to 'Y complex, however in the absence of the~ clamp it is less stable and has reduced DNA-stimulated ATP hydrolysis activity Together these kinetic analyses show that in the presence of ATP I I both the minimal complex and 'Y complex clamp loaders exist in some dynamic conformational equilibrium dominated by two states with distinct DNA binding abilities. The ATP-dependent confonnational dynamics of the minimal complex appear to be dominated by a species inactive for DNA binding that must go through a '' forward '' conformational change to become activated which may occur at a slower rate compared to the related confor1national change for -y complex. A key to the clamp loading mechanism is that when either of the clamp loaders is equilibrated with ATP in the presence of~ a slow step is bypassed, and their activities achieve considerable similarity under identical experimental conditions clamp binding enhances the clamp loader specificity for ATP converts the mixture of ATP-dependent confo1mational species into an activated conformational state, essentially increasing its concentration and affects the stability of the nucleotide binding sites In comparison to -y complex, the minimal complex is missing the x and 'I' subunits These subunits are unique to the E. coli clamp loader and probably other gram-negative prokaryotic clamp loaders as well (Xiao et al ., 1993a ; Xiao et al ., 1993b) Therefore it is proposed that the missing x and 'I' subunits are responsible for the differences in the kinetics observed here This chapter encompasses

PAGE 235

220 the most detailed study of the y 3 66 minimal complex to date and therefore provides additi,onal significant biochemical data for understanding the mechanism of the clamp I loading reaction catalyzed by the y complex molecular machine I

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I CHAPTER6 CONCLUSIONS AND RECOMMENDATIONS Introduction Within the DNA polymerase ill h oloenzyme the principal enzyme responsible for E. c oli chromosomal replication, are processivity proteins Processivity proteins confer I the ability of DNA polymerase III to replicate stretches of DNA thousands of nucleotides long at a rate approaching 750 nucleotides per second without dissociation These speed and processivity characteristics are required by the polymerase for synthesis of the leading and lagging strands of the entire E. c oli chromosome in a period of about 40-60 minutes (Kelman and O'Donnell 1995 ; Kornberg and Baker 1992) The processivity proteins fo1m a ring-shaped DNA sliding-clamp and a complex molecular machine, the clamp loader. These processivity proteins have been functionally conserved in all branches of life and are involved in all forms of DNA metabolism including recombination and repair, underscoring their importance in the cell and the importance of investigating their functions The E c oli sliding-clamp is a dimer of crescent-shaped~ subunits that topologically links DNA polymerase to the template DNA (Kong et al ., 1992 ). The clamp loader is an energase enzyme consisting of seven sub11nits (1: 2 y, o o ', x., and 'lf) ATP binding and hydrolysis by the -c / y-AAA + ATPase motor subunits drives conformational changes in the clamp loader that modulate affinities of the clamp loader for the sliding clamp and DNA (Davey et al., 2002) ATP-dependent conformational changes cause exposure of the o subunit from occlusion by o ', and cause fo11nation of a DNA binding surface on the clamp loader The o subunit binds the p 2 2 1

PAGE 237

222 clamp and induces confor1national changes within the clamp that open it at a single dimer interf~ce allowing threading onto DNA (Jeruzalmi et al ., 2001b ). As a fmal step in clamp loading DNA stimulates ATP hydrolysis b y the clamp loader that leads to its detachment ' from the loaded clamp ( Ason et al ., 2000 ; Bertram et al. 2000 ). DNA polymerase then binds the clamp for processive replication Although the basic clamp loading reaction mechanism is understood several key questions remain to be answered The kinetics of ATP-dependent confor1national changes driving the clamp loader DNA and binding interactions are unclear. X-ray crystal structures of the clamp loader and a truncated y subunit have exposed details of the nucleotide binding sites within the clamp loader and steady-state ATP binding constants have been estimated (Hingorani and O'Donnell 1998 ; Jeruzalmi et al ., 2001a ; Podobnik et al ., 2003 ). However the nature of nucleotide binding to the clamp loader during the real-time clamp loading reaction has not yet been explored Sliding clamps of all organisms are known to enhance the activity of their respective clamp loader but the mechanism of enhancement remains poorly characterized (E llison and Stillman, 1998 ; Onrust et al ., 1991 ). A heteropentameric minimal clamp loader complex (y 3 66 ') can be reconstituted, which is missing the x and 'I' subunits This '' minimal '' clamp loader is fully functional in clamp loading promoting processive DNA synthesis and consists of the evolutionarily con s erved AAA + motor and clamp interaction subunits (Neuwald et al ., 1999 ; Onrust and O'Donnell 1993 ). The x and 'I' subunits are unique to many prokaryotic clamp loaders ; therefore what distinctive properties do they contribute to the clamp loading mechanism ? I

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223 > Each of these poorly understood facets have been addressed in this dissertation by two fluorescence-based methodologies designed for investigation of steady-state and pre steady-state ATP hydrolysis and DNA binding kinetic analyses of the clamp loader. The kinetics of DNA and clamp binding were addressed directly in solution-based anisotropy binding assays with fluorescent-labeled~ and DNA substrates The calculated changes in anisotropy in these assays directly reflect protein-protein or protein-nucleic acid binding by the clamp loader with labeled-~ or DNA respectively (Ason et al., 2003) ; I The ATP hydrolysis activity of the clamp loader in the presence or absence of~ was measured with fluorescent-labeled E. coli phosphate binding protein assays (Ason et al. 2003 ; Brune et al., 1994) These ATP hydrolysis assays were also used to address the nature of nucleotide binding to, and the kinetics of the conf onnational changes within the clamp loader By comparison of the activities of 'Y complex (y 3 00 X\II) to the minimal complex (y 3 66 ) using both methodologies the functions of the 'X and 'V proteins in clamp loading were addressed. The studies outlined in this dissertation show that the clamp loader exists in a mixture of conformations in equilibrium with ATP There were two dominant populations of conf orinational states, one of species activated for DNA binding and clamp loading and one of 'inactive '' species The possibility of two major populations of the clamp loader was originally proposed based on observation of pre-steady-state biphasic ATPase kinetics in the absence of~ then confirmed based on relating pre steady-state biphasic clamp loading kinetics observed in DNA binding assays The kinetics of the confot 1national changes separating these species have been dete1mined by investigating their evolution in increasing equilibration times with ATP Computer

PAGE 239

224 modeling of the experimental data from these ATP hydrolysis experiments gave reliable estimates of the rates of the conformational changes occurring between a mixture of I I ATP-dependent conformational states of the clamp loader Further ATPase and DNA binding as~ays when the clamp loader was not equilibrated with ATP showed that the conformational changes were likely to be rate limiting in the clamp loading reaction. Together ATP hydrolysis and DNA binding/clamp loading kinetics describe a possible mechanism of enhancement by the p clamp where the clamp essentially increases the concentration of the activated clamp loader species 'trapping '' ATP and committing this ATP-bound clamp loader-P clamp complex to the loading reaction upon binding primed-template DNA. Comparison of the minimal complex (y300') toy complex ( y 3 00 xv) revealed that the x and 'tf subunits most likely facilitate the confo11national changes towards the active clamp loader species As a result an inactive population considerably dominated the mixture of conf 01 mational states of the minimal complex in equilibrium with ATP The p clamp, through the enhancement mechanism converted the '' unstable '' minimal complex into a nearly complete active species population with ATP hydrolysis and clamp loading properties similar toy complex A non-hypothesized aspect of this comparison of clamp loaders is the idea that x and 'V are AAA + adaptor proteins that provide the clamp loader with increased stability of the activated conformation by facilitation of conformational dynamics and communication between the AAA + subunits of the clamp loader Steady-State Kinetics of the Clamp Loader in the Absence or Presence of p Originally under study in this research project was how the p clamp affected the kinetics of DNA binding and ATP hydrolysis by y complex. Among the aims of the research was to determine how p increases the DNA-stimulated ATP hydrolysis activity I

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I I 225 of y complex and how affected the stability of nucleotide binding in y complex The goal was to gain a better understanding of these aspects of the clamp loading reaction The early findings showed that~ clamp conferred upon the clamp loader an apparent higher affinity for DNA that could be sustained for hundreds of seconds depending on the concentration of ATP in steady-state DNA binding/clamp loading assays The apparent higher affinity for pt DNA in the presence of~ was due to an increase in y complex affinity for ATP A series of steady-state clamp loading reactions were performed at I I several different concentrations of ATP Analysis of the resulting reactions gave an estimate for an apparent dissociation constant for ATP of ~ 5 Along with the steady state ~p yr en e binding a ssays it is concluded that y complex and the minimal complex have a similar apparent Ki for ATP of ~ 2 M ( see Figure 5-4) The DNA binding assays for y complex in the presence or absence of~ indicated that decreases the dissociation constant for ATP binding giving the clamp loader more affinity for ATP Additional analysis of the steady-state clamp loading reaction where the relative starting concentration of ADP was increased in separate DNA binding assays showed that excess amounts ADP could not inhibit the clamp loading reaction This result is consistent with the conclusion that increases the binding constant for ATP specifically Steady-state ATP hydrolysis assays were perfo1med to directly assess how the clamp affects the binding and hydrolysis of ATP by y complex The '' Michaelis Menten '' kinetic parameters were determined for y complex in the presence and absence of~ The maximum velocity (V m a x ) and ATP specificity constant (kca tcomp le x /K m ) for y complex increased in assays with ~ and thus V max, which is dependent on enzyme concentration is increased about three-fold over y complex in the absence of~ to ~ 0 07 1 s 1

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226 Steady-state analysis of ATP hydrolysis also showed that~ increased both the V max and kca tcom~ l ex /K m values detetmined for the minimal complex to levels similar to 'Y complex ( see Table 5-1 ). These results are consistent with the early studies that showed the ' minimal complex had ATP hydrolysis and clamp loading activity similar to y complex Concluded here however is that this similar activity by the minimal complex is produced only in the presence of~ clamp The kca tcomp l ex /K m values in the absence or presence of~ are relatively slow about 0 8 to 2 0 x 10 5 M 1 s 1 too slow to represent a bimolecular binding reaction occurring at diffusion controlled limit between a small nucleotide cofactor ( ATP ) and the clamp loader The magnitude of kca tco m plex /Km is likely to be reduced relative to the theoretical upper limit ( i e a diffusion controlled binding rate 1 x 10 9 M 1 s 1 ) by a two-step binding reaction consisting of relatively rapid ATP binding followed by slow step induced by ATP binding The slow second step of this putative two-step binding reaction is concluded to be ATP-dependent confotmational changes within the clamp loader Kinetics of ATP-Dependent Conformational Changes within the Clamp Loader Binding and hydrolysis of ATP molecules by the y subunits of the clamp loader modulate the activity of the clamp loader The energy derived from ATP binding and hydrolysis is transduced mechanically into conformational changes that give the clamp loader differential ( switch-able) affmities for~ and DNA In the clamp loading mechanism binding of ATP to this '' energase '' enzyme drives all of the steps necessary for placement of the clamp at the correct position on primed DNA (Turner et al ., 1999 ). DNA stimulated ATP hydrolysis drives further conformational transitions within the clamp loader that cause s it to release on DNA Nucleotide exchange following hydrolysis and clamp loader release allows another round of ATP binding and continued I

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I I 227 steady-state clamp loading activity There exists both biochemical (Hingorani and O'Donnell, 1998) and structural (Jeruzalmi et al ., 2001a ; Podobnik et al 2003) evidence for a conformational change in the clamp loader upon nucleotide binding to the three y subunits The nucleotide binding sites lie within interfaces between the o -Y1 Y1-Y 2 and I Y2-Y3 subtinits of the clamp loader. At least one and possibly two of these interfacial nucleotide binding sites is thought to be constitutively open whereas the third is deeply buried in an interface This lead to a model describing a sequential series of individual y1 I subunlt conformational changes whereupon ATP binding to an open interface caused a conformational change opening the adjacent interface and so on (Jeruzalmi et al ., 2001a) These conclusions drawn respective to ATP-dependent conformational changes in this study of the clamp loader structure were strongly limited by the simple fact that the structure was solved in the absence of nucleotide, and its conformational state was thought to be largely due to a crystal packing artifact The structure does predict that ATP binding to any one of the y subunits will directly affect adjacent subunits by undergoing a conformational change Along this line, it could easily be imagined that the y subunits are not static, and could constantly be undergoing confo1 mational transitions in the absence of ATP that result in random opening and closing of the interfacial binding sites allowing molecules of ATP to bind and stabilize the clamp loader in several different forms with varying affinities for the clamp and DNA All together, these subjective remarks point out that the clamp loader most likely exists as a mixture of confo1mational states both in the absence and presence of ATP The previously undefined kinetics of ATP-dependent conformational changes separating these states were measured in ATP hydrolysis assays and DNA binding/clamp

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228 loading assays in this research project The overall conclusion regarding ATP-dependent conforiational dynamics is that the clamp loader exists in a mixture of conformational states in equilibrium with ATP dominated by two populations one activated for DNA I binding and one incapable of DNA binding The rate of the conformational change separating the two major species of 'Y complex was directly addressed in pre-steady-state ATP hydrolysis reactions when 'Y complex was equilibrated with ATP for defined periods of time prior to mixing with DNA to initiate biphasic ATP hydroly s is reactions Simultaneous global fitting of the experimental data for all equilibration times using the kinetic model ( Figure 4-10~ reproduced here a s Figure 6-1 ) ( see also appendix ) was performed with the computer program DynaFit (Kuz1nic 1996) and simulations of equilibration were performed with the program KinTekSim (Barshop et al ., 1983 ; Dang and Frieden, 1997 ). This minimal model of three conformational species adequately fitted the experimental biphasic ATP hydrolysis kinetics The model showed that the mixture of confo1rnational species in equilibrium with ATP is dominated by two of these species ATTT and ETTT where ATTT is the activated conformational state and ETTT is an inactive conformational state defmed by the slow branch transition to an inactive ITTT state that does not produce product The ph y siological relevance of the non-productive ITTT species is unknown, however a '' branch-pathway '' producing a slow s tep in the reaction was necessary for modeling of the slow pres teadys tate pha s e of the experimentally observed biphasic kinetic s. A significant limitation of this kinetic model is that it is not sufficient for describing the DNA binding/clamp loading kinetics from the pre-steady-state anisotropy

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229 assays in the presence of~ When y complex was equilibrated with ATP then mixed with DNA ~ and ATP biphasic DNA binding/clamp loading kinetics were observed These biphasic kinetics did show that two species of the clamp loader were present after equilibration with ATP but the exact nature of these two species cannot be deter1nined without understanding how~ affects the pre-steady-state kinetics in this a ss ay 1r -1 A.ea 4.45s k -1 ei 2.37 s ETTT 1, -1 e 3.90s ATTT+ N ~:A t 23 5.Ml s l Al I IN k 6 os 1 hyd kie 4.23s kon136M 1 s 1 PBP + P, 4 PPBP 1 ~tr t3.6 s A DN+P, -1 kb yd 60S E f I I AT DN +P, .J k hyd 60s ADDO+ N + P, Figure 6-1 Kinetic modeling of ATP hydrolysis reactions The minimal kinetic model is reproduced from Figure 4-10 used to fit the ATP hydrolysis data in Figure 49 Three states of they complex ETTT ATTT and ITTT are related by the kinetic constants shown ( cartoons are based on the structure of the clamp loader see chapter2 figure 2-3). The ATTT form binds DNA (N) rapidly and hydrolyzes its three molecules of ATP ( T ) in succession producing ADP (D) and inorganic phosphate (P i ) The y complex dissociate s from DNA on hydrolyzing its third molecule of ATP and is recycled back to the ETTT state Ex perimentally dete11nined rates of y complex binding DNA (As on et al ., 2000 ; Bertram et al ., 2000) ko nN, and MDCC-PBP (PBP ) binding phosphate ( Brune et al ., 1994 ) kon and ko rr, were set constant in the fit All other rate constants were determined by fitting the data to the model u s ing DynaFit (K uzmic 1996 ). This model doe s not describe the only possible way to explain the experimental kinetics of ATP hydrolysis and other possible mechanisms can and should be considered in the future As an example an alternate minimal model that contains a slow step following hydrolysis of two molecules of ATP and release of DNA fits the data well and is presented in the appendix.

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I 230 Although the above model was satisfactory in describing the conformational changes separating the mixture of clamp loader states in equilibrium with ATP it remains an open question to which of these states clamp binds Therefore the biphasic DNA binding/clamp loading kinetics observed after equilibration with ATP may represent a different mechanism of clamp loader activity in the presence of~ Consistent with this idea is the result of a pre-steady-state ATP hydrolysis assay where y complex was equilibrated with ATP prior to rapid mixing with DNA ~ and ATP (see Figure 4-7) In that assay only a single pre-steady-state rapid phase followed by steady-state ATP hydrolysis kinetics were observed This assay is analogous to the DNA binding/clamp loading assay were pre-steady-state biphasic kinetics were observed ( above, Figure 46A), and indicated that some amount of activated y complex alone binds DNA and rapidly hydrolyzes ATP then loads the clamp in a slower pre-steady-state step that occurs at a rate which limits the continuing steady-state activity. Based on comparison of these two analogous ATP hydrolysis and DNA binding/clamp loading assays it is concluded that the clamp loader entered the pre steady-state reaction in the mixture of ATP-dependent conformational species described by the computer model, and then clamp converted this mixture by binding to one or more of the conformational species Conversion of the mixture of confor1national species by~ to a single new species activated for clamp loading (ATTT~ ) would then relate to the rate-dete11nining step of the clamp loading mechanism In the pre-steady-state ATP hydrolysis and DNA binding/clamp loading assays where y complex was equilibrated with ATP and~ prior to rapid mixing with DNA only a single pre-steady-state rapid phase of activity was observed followed by transition into the steady-state reaction.

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231 From these assays it was concluded that converted the equilibrium of ATP-dependent confo~n1ational states of the clamp loader into a single activated species before reacting with DNA. Mod~ling the ATP hydrolysis kinetics for the reaction of y complex with DNA gave an estimate of the rate of the conformational change f orrning ATTT from ETTT (4.45 s1 ) This estimated ''forward' conformational change rate is consistent with the complex conformational changes this multi-subunit clamp loader must make to become activated, perhaps by binding the ATTT species or binding and converting the ETTT species (scheme 6-1) This estimated confo1mational change rate is in consistent with the ~ 2 s 1 turnover rate (kca tco mpl ex ) calculated in the steady state ATP hydrolysis analysis. EITT + ll ATTTfi Scheme 6-1 The turnover rate measured by steady-state analysis is generally smaller than the actual rate constant limiting the fortnation of product, therefore, the rate of the conformational change estimated from pre-steady-state assays and computer modeling is likely to represent a closer approximation of the rate-limiting step in the clamp loading reaction The contribution of ATP binding to the rate of the conformational change was addressed in assays when y complex was not equilibrated with ATP then mixed with DNA and ATP The assay was repeated at several concentrations of ATP in an attempt to determine the ATP -binding rate toy complex. It was concl uded that when the ATP concentration was < 200 Min the assay conditions ATP binding contributed to the rate of the conf 01 mational changes, but the complexity in modeling three possibly I

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I I 232 independent ATP-binding steps hampered estimation of the ATP binding rate. The experimental data from an assay where the ATP was above 200 mM was fitted to the minimal model shown in Figure 6-1 An additional species of y complex (PTTT) that converts relatively rapidly (53 s 1 ) to the ETTT state is required to fit to the lag phase in the experimental data When this single initial step was included the minimal model using the rate constants shown in Figure 6-1 gave an adequate fit to the data (see Figure 4-lOC ). It was concluded from this fitting that y complex went through the same reaction I pathway producing biphasic kinetics when not equilibrated with ATP as when it was equilibrated with ATP A simulation of the equilibration with ATP for y complex including the PTTT + ETTT step is shown in the appendix ( A-1 ). For the minimal complex, the above assays wherein equilibration times with ATP were changed to deterrnine the rate of the conformational change for y complex were not plausible due to the reduced amplitudes of biphasic ATP hydrolysis by the minimal complex in the absence of~ and therefore the reduced fluorescence change during the assay Although the research was limited by this experimental factor the minimal model was applied to a single data set where the minimal complex was equilibrated with ATP for a period of 1 second (Figure 57B appendix ) The estimated the rate of the minimal complex forward confo1111ational change from this reasonable fitting was ~ 2 6 s 1 Although this approximation is limited by the absence of additional data sets for increa s ing equilibration times it i s in agreement with the lower calculated steady-state turnover rate for ATP hydrolysis limiting the clamp loading reaction compared to y complex A large reverse conformational change ( ATTT + ETTT ) rate ( 12 6 s1 ) was also estimated by this fitting analysis and is consistent with the greatly reduced activated

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233 species population of the minimal complex Simulation of the minimal complex equili bration with ATP using rates dete11nined from the fitting of the single data set from I Figure 5-7B gave estimate of ~ 10 % ATTT species at 1 second (appendix, A-2) A Po~sible Mechanism for p Clamp Enhancement of Clamp Loader Activity The majority of experiments presented in this dissertation were perfonned in the absence and presence of~ to understand how this clamp affects 'Y complex A comprehensive comparison of the kinetics of ATP hydrolysis and DNA binding/clamp loading has produced a possible answer to the question : How does sliding-clamp enhance the activity of its clamp loader? Due to the functional conservation between clamps and clamp loaders of all f orrns of life the possibilities presented here may be applicable across evolution as well The steady-state ATP hydrolysis kinetics of the clamp loader are affected by the clamp (Hingorani and ODonnell, 1998 ; Onrust et al. 1991). The Michaelis-Menten analysis presented in this dissertation produced parameters consistent with the previous studies and additionally provided novel analysis of the minimal complex. An important conclusion is that the increase of the ATP specificity constant for the minimal complex was of greater magnitude than for 'Y complex The predicted active population of the minimal complex in equilibrium with ATP in the absence of~ is considerably small in comparison to 'Y complex and the presence of ~ confers upon this population enhanced activity similar to y complex This reflects on the overall effect the clamp has on the clamp loader The clamp appears to partition the clamp loader to an activated fo1m kinetically more robust than in the absence of the clamp The combined results of the kinetic assays presented here point to a possible mechanism where~ clamp selectively binds the activated species of the clamp loader I

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234 during equilibration with ATP and~' converting all clamp loaders into an activated ATP clamp loader-~ complex (ATTT + !:+ ATTT~) poised for rapid clamp loading Both the I minimal complex and 'Y complex have similar affinity for ATP, Kci ~ 2 M (Figure 5-4) I (Hingorani and O'Donnell 1998), and the ATP-bound clamp loaders have low nanomolar affmity for~ Ket appar e n t ~ 3-4 nM (Figure 5-3) (Naktinis et al ., 1995 ), therefore during the equilibration period in the assays would be able to pull the equilibrium between the mixture of ATP-dependent conformational clamp loader states from the inactive species towards a completely activated species. An alternative to this mechanism is a possible situation where could bind to an inactive species as well as the activated species, promoting conversion to the activated species ( see scheme 6-1 ) This possibility would require a change in the conformational change kinetics rather than simply changing the equilibrium between the states by selectively binding to the activated species Future work will be required to differentiate between these two possible mechanisms Pre steady-state ATP hydrolysis assays should be repeated when the clamp loader is I equilibrated with ATP and for several different periods of time before mixing with DNA to initiate the reaction Analysis of the resulting kinetics from these assays could be done in a manner analogous to the analysis of the series of assays when 'Y complex was equilibrated with ATP for different periods of time If~ selectively binds to the activated species only then a decrease in rapid hydrolysis pres teady-state kinetics would be observed with decrea s ing equilibration time s. The resulting pre-steady-state kinetics may also be biphasic as the inactive specie s is intrinsically converted to the activated species similar to the pre-steady-state ATP hydrolysis kinetic s observed in assays without clamp (ETTT ATTT + ATTT~ ). I I

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' ' 235 Preliminary pre-steady-state experiments when the equilibration time for the clamp loader, ATP, and~ was decreased to 250 ms have already been performed in this dissertation project (not shown) The resulting pre-steady-state hydrolysis kinetics were biphasic in nature Previous pre-steady-state analysis using radio-labeled ADP release as a measure of ATP hydroly~is kinetics was performed when 'Y complex was incubated with ATP and for a duration of 80 ms The published results showed that the pre steady-state formation of ADP was in fact biphasic (Hingorani et al 1999). Although I the possible mechanism explained by the equilibrium shift by selective binding to the activated clamp loader is supported by these data further analysis like that proposed above is required perhaps along with computer modeling to fully understand how converts the clamp loader into a completely activated form within a 2:: 1-second incubation period These experiments would also help the future investigator acquire more kinetic data concerning which clamp loader conformational species binds. Conclusions from the A TPyS-chase assays shed additional light on the putative clamp enhancement mechanism. An essentially non-hydrolyzable ATP analogue, A TP"(S was used in pre-steady-state and steady-state ATP hydrolysis assays to dete11nine the nature of nucleotide binding by the 'Y complex or minimal complex clamp loaders. Pre-steady-state A TP"(S-chase assays in the presence of~ revealed that the molecules of ATP bound to either clamp loader were hydrolyzed faster than they dissociate (see 'Y comple~ Figure 4-5 and the minimal complex Figure 5-7) Pre-steady-state ATP"(S chase assays were also perfo11ned in the absence of~, and showed that only the molecules of ATP bound to the activated clamp loader species were hydrolyzed faster than they dissociate and that ATP molecules bound to any inactive species exchange

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236 more rapidly with A TPyS than they were hydrolyzed It is thus concluded that molecules of A zy bound to the activated clamp loader are more secure or stably bound than A TPs I bound to any inactive species In the presence of~ all of the clamp loader is in the ATPI bound active state with the molecules of ATP e ss entially '' trapped '' in the activated complex with ~ The ATPs remain securely bound until the activated clamp loader-~ complex forms a ternary complex with DNA DNA then triggers hydrolysis of the bound ATP molecules causing the clamp loader to lose affinity for the clamp and DNA finishing the clamp loading reaction It is concluded that the clamp enhances the clamp loader by a mechanism wherein it selectively binds to the activated species of the clamp loader Selective binding to the activated clamp loader effectively increases its concentration by driving conformational equilibrium between inactive and active species towards the activated state (ETTT + A TTT~ ). The sliding-clamp secures the molecules of ATP bound to this active state and commits it to a productive clamp loading reaction on primed-template DNA for processive replication The Missing x and 'I' Subunits are Responsible for the Kinetic Differences Between the Minimal Complex and y Complex As stated above the x and w subunits are mis s ing from the minimal complex, and are the likely causes of the differences described here for the DNA binding clamp loading and ATP hydrolysis acti v ities by the minimal comple x and r complex The most notable dissimilarities between the minimal comple x and r complex appeared in assays without the clamp Without ~ the clamp loader is dynamically changing between a range of conforn1ations that acquire some new equilibrium mixture when molecules of ATP bind essentially defmed or separated in majority by inactive and activated species I

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I I 237 populations The ATP-dependent conforrnational dynamics in the clamp loader are the driving force of this energase molecular machine Without the x and 'V proteins, the minimal complex conf orn1ational dynamics appear to be hampered in such a way that this sub-complex clamp loading machine had diminished intersubunit communication The land 'If Subunits are E.coli Clamp Loader AAA+ Adaptor Proteins I The minimal complex has activities similar to 'Y complex in the presence of~This should be expected given that the five-subunit assembly of AAA + proteins in the minimal complex appears to be conserved acrqss evolution (Davey et al ., 2002 ; Ellison and Stillman 2001 ). It should be noted that the E. coli clamp loader differs from clamp loaders of other organisms There is the need for the additional x and 'V clamp loader subunits at the replication fork for direct interaction with single-stranded DNA binding protein (SSB), and for indirect interaction with primase The x and 'JI subunits based on the work presented in this dissertation are also required for optimum clamp loading activity They confer additional conf ortnational stability to the clamp loader when in equilibrium with ATP essentially '' tightening ' it up. This is accomplished possibly by facilitation of the ATP-dependent confonnational changes towards the activated clamp loading species or by slowing the rate of conformational change towards the inactive population It is likely that x and 'V confer both of these functions to the 'Y complex clamp loader, giving it a true advantage over the evolutionarily conserved five-subunit clamp loader architecture found elsewhere It is proposed in this dissertation that the x and 'V subunits are members of a growing group of AAA + adaptor proteins (Dougan et al. 2002) Among this group are the small subunits of Clp proteases (Zeth et al ., 2002) in E. c oli and the general function of these adaptor proteins appears to be in conferring substrate specificity for the AAA +

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238 motor proteins they assist T