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The Kinetics of Gamma Complex, the Escherichia Coli Clamp Loader, Binding the Beta-Clamp Before Dna During the Clamp Loa...

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Title: The Kinetics of Gamma Complex, the Escherichia Coli Clamp Loader, Binding the Beta-Clamp Before Dna During the Clamp Loading Reaction
Physical Description: 1 online resource (88 p.)
Language: english
Creator: Thompson, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: clamp, dna
Biochemistry and Molecular Biology -- Dissertations, Academic -- UF
Genre: Biochemistry and Molecular Biology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: THE KINETICS OF GAMMA COMPLEX, THE ESCHERICHIA COLI CLAMP LOADER, BINDING THE BETA-CLAMP BEFORE DNA DURING THE CLAMP LOADING REACTION By Jennifer A. Thompson May 2010 Chair: Linda Bloom Major: Biochemistry and Molecular Biology In Escherichia coli, the gamma complex clamp loader loads the beta-sliding clamp onto DNA. The beta clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, gamma complex binds beta and DNA to form a ternary complex. Binding to primed template DNA triggers gamma complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of gamma complex binding beta and DNA. A fluorescence intensity-based beta binding assay was developed in which the fluorescence of pyrene covalently attached to beta increases when bound by gamma complex. Using this assay, an association rate constant of 2.3 x 107 M-1s-1 for gamma complex binding beta was determined. The rate of beta binding was the same in experiments in which gamma complex was pre-incubated with ATP before adding beta or added directly to beta and ATP. In contrast when gamma complex is pre-incubated with ATP, DNA binding is faster than when gamma complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP pre-incubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote beta binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for gamma complex to bind beta before DNA during the clamp loading reaction cycle.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jennifer Thompson.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Bloom, Linda B.

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
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Permanent Link: http://ufdc.ufl.edu/UFE0041677/00001

Material Information

Title: The Kinetics of Gamma Complex, the Escherichia Coli Clamp Loader, Binding the Beta-Clamp Before Dna During the Clamp Loading Reaction
Physical Description: 1 online resource (88 p.)
Language: english
Creator: Thompson, Jennifer
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: clamp, dna
Biochemistry and Molecular Biology -- Dissertations, Academic -- UF
Genre: Biochemistry and Molecular Biology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: THE KINETICS OF GAMMA COMPLEX, THE ESCHERICHIA COLI CLAMP LOADER, BINDING THE BETA-CLAMP BEFORE DNA DURING THE CLAMP LOADING REACTION By Jennifer A. Thompson May 2010 Chair: Linda Bloom Major: Biochemistry and Molecular Biology In Escherichia coli, the gamma complex clamp loader loads the beta-sliding clamp onto DNA. The beta clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, gamma complex binds beta and DNA to form a ternary complex. Binding to primed template DNA triggers gamma complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of gamma complex binding beta and DNA. A fluorescence intensity-based beta binding assay was developed in which the fluorescence of pyrene covalently attached to beta increases when bound by gamma complex. Using this assay, an association rate constant of 2.3 x 107 M-1s-1 for gamma complex binding beta was determined. The rate of beta binding was the same in experiments in which gamma complex was pre-incubated with ATP before adding beta or added directly to beta and ATP. In contrast when gamma complex is pre-incubated with ATP, DNA binding is faster than when gamma complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP pre-incubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote beta binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for gamma complex to bind beta before DNA during the clamp loading reaction cycle.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jennifer Thompson.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Bloom, Linda B.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041677:00001


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1 THE THE ESCHERICHIA COLI CLAMP LOADER CLAMP BEFORE DNA DURING THE CLAMP LOADING REACTION By JENNIFER A. THOMPSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Jennifer A. Thompson

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3 To my family and friends

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4 ACKNOWLEDGMENTS I thank Dr. Linda Bloom, my mentor, for her helpful guidance, advice, and support as well as Dr. Laipis and Dr. Cain of my supervisory committee for their mentoring. I thank Chris Paschall, Ankita Chiraniya, Jaclyn Hayner, Dr. Melissa Marzahn, and the other members of the Bloom lab over the past three years for their research assistance, thoughtful discussions, and friendship. I also thank the National Institutes of Health for its generous support. I thank my parents and my family for their loving support through all the years and to Chris Meyer for being a constant source of encouragement motivation, and love.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF FIGURES .......................................................................................................... 7 LIST OF ABBREVIATIONS ............................................................................................. 9 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION .................................................................................................... 13 Escheri chia coli DNA Replication ............................................................................ 13 Eukaryotic DNA Replication .................................................................................... 14 Processivity Factors ................................................................................................ 16 Structure of Sliding Clamps .............................................................................. 16 Structure of Clamp Loaders .............................................................................. 17 Clamp Loading Mechanism .................................................................................... 20 2 MATERIALS AND METHODS ................................................................................ 27 Materials ................................................................................................................. 27 Nucleotides and Oligonucleotides .................................................................... 27 RFC and Rad24RFC Purification .................................................................... 27 Buffers .............................................................................................................. 27 Methods .................................................................................................................. 28 Purification of DNA Polymerase III Proteins ..................................................... 28 Sliding Clamp Mutagenesis ........................................................................... 28 Sliding Clamp Transformation ....................................................................... 28 Sliding Clamp Purification ............................................................................. 29 Covalent Labeling of the clamp with Pyrene ................................................. 31 PCNA Sliding Clamp Mutagenesis ................................................................... 31 PCNA Sliding Clamp Transformation ............................................................... 32 PCNA Sliding Clamp Purification ...................................................................... 33 Covalent Labeling of the PCNA clamp with AlexaFluor 488 ............................. 35 Protein Concentrations ..................................................................................... 36 Labeling Primer DNA with DCC ........................................................................ 37 Experimental Procedures ........................................................................................ 37 Steady State Fluorescence Ass ays .................................................................. 37 Pre Steady State Fluorescence Assays ........................................................... 38 Data Analysis .......................................................................................................... 38 Steady State Fluorescence Assays .................................................................. 38 Pre Steady State Fluorescence Assays ........................................................... 39 Kinetic Modeling ............................................................................................... 39

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6 3 RESULTS ............................................................................................................... 41 Introduction ............................................................................................................. 41 ..................................................................... 42 omplex binding ............................................................................... 43 .................................................................................. 44 ATP ...................................................................................................................... 45 DNA Binding with and without ATP Preincubation .................................................. 46 Effect DNA Binding Kinetics ......................... 47 Kinetic Modeling of DNA Binding Reactions ........................................................... 48 Discussion .............................................................................................................. 50 Two Sets of ATP induced Conformational Changes ........................................ 51 ................... 52 4 DISCUSSION AND FUTURE STUDIES ................................................................. 67 A PPENDIX: DEVELOPMENT OF A NEW FLUORESCENCE BASED ASSAY: MEASURING PCNA OPENING IN THE SACCHAROMYCES CEREVISIAE DNA REPLICATION SYSTEM ................................................................................ 70 Introduction ............................................................................................................. 7 0 A fluorescence intensity based assay to measure opening and closing of an RFC PCNA complex ........................................................................................... 71 Measuring Rad24RFC PCNA Interactions using the PCNA AF488 Opening Assay ................................................................................................................... 73 Discussion and Fu ture Studies ............................................................................... 74 REFERENCE LIST ........................................................................................................ 81 BIOGRAPHICAL SKETCH ............................................................................................ 88

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7 LIST OF FIGURES Figure page 1 1 A Model of the Escherichia coli Replisome. ........................................................ 22 1 2 Structure of sliding clamps. ................................................................................ 23 1 3 Structure of the E. coli ................................................ 24 1 4 Structure of the S. cerevisiae clamp loader, RFC. .............................................. 25 1 6 Th e ATP dependent steps of the clamp loading reaction. .................................. 26 3 1 Fluorescence intensity clamp binding assay. ...................................... 55 3 2 Emissio n Spectra of pyrene fluorescence. ........................................................ 56 3 3 ................................................................ 57 3 4 PY. ............................................................... 58 3 5 ............................................................................. 59 3 6 binding to DNA. ............................................................... 60 3 7 with ATP. ............................................................................................................ 61 3 8 ATP. ................................................................................................................... 62 3 9 DNA DCC. ................................................................................................................... 63 3 10 x DNA binding kinetics. ...................... 64 3 11 Kinetic model fit to p/t DNA binding data. ........................................................... 65 3 12 Model for formation of a ternary clamp loader clamp DNAcomplex ................. 66 A 1 Crystal Structural Model of PCNA highlighting the residues mutated for the PCNA opening assay. ........................................................................................ 76 A 2 Relief of the AF488 Fluorescence quench in the presence of SDS. ................... 77 A 3 Emission spectra of PCNA AF488 were taken at excitation wavelength of 495 nm. .............................................................................................................. 78

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8 A 4 Equilibrium binding and opening of RFC PCNA. ................................................ 79 A 5 Equilibrium binding and opening of Rad24RFC PCNA. .................................... 80

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9 LIST OF ABBREVIATION S AAA+ ATPases associated with cellular activities AF488 AlexaFluor 488, C5 maleimide ATP adenosine triphosphate adenosine 5 O (thiotriphosphate) PY BSA bovine serum albumin DCC 7 diethylaminocoumarin3 carboxylic acid, succinimidyl ester DMSO dimethyl sulfoxide DNA deoxyribonucleic acid E. coli Escherichia coli GINS complex of Sld5Psf1 Psf2 Psf3 (Go Ichi NiSan or 51 2 3)) IDCL interdomain connecting loop LB Luria Broth MCM mini chromosome maintenance PAGE polyacrylamide gel electrophoresis PCNA proliferating cell nuclear antigen PCNA PCNA covalently labeled on residues I111C and I181C with AlexaFluor 488 AF488 PIP box PCNA interacting peptide Pol III DNA polymerase III p/tDNA primer/template DNA p/tDNA primed template DNA with an amino linker covalently labeled with DCC DCC PY N (1 pyrene) maleimide

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10 RFC replication factor C RNA ribonucleic acid SRC serine arginine cysteine SSB single stranded DNA binding protein TCEP tris (2 carboxyethyl) phosphine

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11 Abstract of Thesi s Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE THE ESCHERICHIA COLI CLAMP LOADER CLAMP BEFORE DNA DURING THE CLAMP LOADING REACTION By Jennifer A. Thompson May 2010 Chair: Linda Bloom Major : Biochemistry and Molecular Biology In Escherichia coli, sliding clamp onto ry complex. Binding to primed template DNA ba complex. Using this assay, an association rate constant of 2.3 x 107 M1s1 ing was the same in experiments incubated with ATP, DNA binding is faster me time. Slow DNA binding in the absence of ATP preincubation is the result of a ratelimiting ATP induced conformational change. Our results strongly suggest that the ATP induced e slow ATP -

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12 induced conformational change that precedes DNA binding may provide a kinetic

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13 CHAPTER 1 INTRODUCTION Escherichia coli DNA Replication DNA replication occu rs semiconservatively such that each parental DNA strand is replicated in a 5 to 3 direction to create two new daughter strands (reviewed in (1)). At the replication fork, a large multicomponent holoenzyme compl ex simultaneously organizes and coordinates the continuous and discontinuous synthesis of the leading and lagging strands, respectively, of the double helix (reviewed in (2)) (Fig. 1 1). DNA synthesis is initiated when the replicative DNA polymerase extends the 3 end of an RNA primer. Primase (DnaG), an RNA polymerase, synthesizes these short (1012 nt) RNA primers (3,4) DnaB, the ring shaped replicative helicase, unwinds the duplex DNA with 5 3 polarity ahead of the replication fork (reviewed in (5,6) ) The helicase is bound to both the clamp loader and to primase to stimulate helicase activity at the fork (7,8) On the leading strand, only one RNA primer is required because synthesis is continuous but on the lagging strand mul tiple primed sites are required for each 12 kb DNA segment created called Okazaki fragments. During Okazaki fragment maturation, the 5 RNA ends are replaced with DNA and the DNA fragments are joined by ligase. The DNA polymerase III (Pol III) holoenzyme consists of ten subunits that are divided into three separate subassemblies clamp; and 3) DnaX complex clamp loader. Two Pol III cores, one for each DNA strand, are required at the replication fork (9 11) 5

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14 core clamp loader. Without the help of accessory proteins, the Pol III core incorporates only approximately 20 nucleotides in a single DNA binding event and can easily dissociat e from the template. Sliding clamps and clamp loaders enhance the processivity of DNA synthesis (12) Clamps encircle DNA while tethering DNA polymerases to the DNA template and freely slide along with the polymeras e in either direction (13) The addition of clamps increases the processivity of replicative DNA polymerases from tens to thousands of nucleotides per DNA binding event (14) ATP dependent clamp loaders are required to actively open and close the sliding clamps onto DNA. Only one clamp is required on the leading strand where DNA replication i s continuous but the lagging strand synthesizes DNA discontinuously and requires a new clamp to be loaded per Okazaki fragment made. Clamp loading on the lagging strand must be fast since typically 1 kb of DNA is synthesized per second and must be coordinated with the leading strand synthesis. Eukaryotic DNA Replication In eukaryotes, the process of DNA replication is much more complex and many of the mechanisms are not well known. There are many more components in cluding over 15 DNA polymerases, involved in the replication and repair pathways (10) Although eukaryotic replication may be more complex than bacterial replication, the core proteins in the replisome are more analogous than different in both structure and function. Leading strand synthesis begins with the primase activity of DNA polymerase /primase laying down an 810 nt RNA primer. The DNA polymerase subunit of DNA pol

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15 /primase extends the RNA primer with DNA creati ng an RNA DNA hybrid. The eukaryotic helicase is thought to be a heterooligomeric complex consisting of a Cdc45/MCM/GINS (CMG) complex, although the exact composition of the active helicase is still unclear (15 18) The CMG complex unwinds with 3 to 5 polarity, which is the opposite of the bacterial DnaB helicase Replication factor C (RFC), the eukaryotic clamp loader, loads the sliding clamp, proliferating cell nuclear antigen (PCNA), onto the 3 end of the RNA DNA primer, T he leading strand polymerase, which is thought to be once RFC dissociates and processive synthesis can continue (19) Eukaryotic Okazaki fragments are about 10fold smaller than the bacterial counterparts and are made at a rate 10fold slower at the replication fork. Synthesi s of /primase creating RNA DNA hybrid primers but a new primer is needed every 100200 nt for each new Okazaki fragment. RFC loads a new PCNA sliding clamp per DNA fragment being created. In comparis on with th e leading strand, DNA pol thought to be on the lagging strand that associates with the clamp and allows for processive synthesis (20,21) Okazaki fragment maturation occurs when a ribon uclease, typically RNaseHI removes all of the RNA in the primer except for the last RNA nucleotide, which is removed by flap endonuclease 1 (FEN 1) (22) Other pathways to degrade the primer, depending on the structure of the 5 end of the Okazaki fragment, can occur including t hose that involve Dna2 (23) Okazaki fragments together.

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16 Processivity Factors Structure of Sliding C lamps The overall structure and function of sliding clamps are conserved from bacteria to humans (reviewed in (10,24) ) Most clamps are ring shaped complexes composed of identical protein subunits (13,25,26) The E. coli sliding clamp is a dimer composed of two identical protein monomers while both th e Saccharomyces cerevisiae and human PCNA sliding clamps are trimers (Fig. 1 2A C). Although there is little sequence globular domains with a comparable fold that are linked by interdomain connecting loops (IDCL) clamp has three domains per subunit creating two interfaces while PCNA has two domains per subunit creating three interfaces. Due to the uniform clamp structure, the clamp loader can in principle open any inter face pair on the clamp to allow DNA to pass through the center of the ring. The subunits of sliding clamps are arranged in a headto tail fashion giving clamps a rotational axis of symmetry through the center of the ring, and two distinct faces. Theoreti cally, proteins can bind either face of the clamp but it has been found that most proteins bind the same face, the face i n which the C termini protrude (27,28) helices, which encircles cla mp has a central opening of 35 in diameter and is approximately 34 thick allowing the ring to cover one helical turn of DNA when bound. PCNA has a similar diameter of about 34 for the central opening and a thickness of about 30. DNA sliding clamps h ave been found to be involved in almost all processes related with DNA metabolism including replication, repair, and modification. Clamps

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17 have been found to interact with a variety of proteins and protein complexes during these processes. Many proteins that bind clamps interact through a conserved peptide sequence motif. Based on sequence alignments and binding studies, this motif is clamp and Qxx (I/L/M) xxF (F/Y) for PCNA, termed the PCNA Interacting Peptide box (PIP box) (29 31) PCNA has been found to bind cell cycle regulators, repair enzymes, and protein kinases among others. The PIP box folds into a 310 x, which has 3.6 residues per turn. An important feature of this binding motif is the presence of hydrophobic amino acid residues including phenylalanine and tyrosine, which come in contact with the hydrophobic pocket on the clamp under an IDCL (32 34) Sliding clamps have one hydrophobic binding pocket per monomer therefore PCNA has three clamp. Recent work has shown that more than one protein can be bound at a time (reviewed in (35) ) The sliding clamp tool belt model sugges clamp at once to facilitate polymerase switching (36) The clamp loader and the replicative DNA polymerase are not able to bind the clamp at the same time even though other proteins may be able to bind a different pocket Steric effects require the clam p loader to dissociate from the clamp before the polymerase can bind and proceed with replication (27,28,34,37) Structure of Clamp L oaders Clamp loaders are multi subunit, spiral shaped complexes of similarly str uctured proteins that are part of the AAA+ family of ATPases (38 42) The ATPase regions of the clamp loader use ATP binding and hydrolysis to promote molecular interactions that catalyze the different steps of the clamp loading mechanism. Each subunit of the clamp

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18 loader contains three domains joined by flexible linkers. Domains I and II located at the N terminal region are structurally conserved and form the AAA+ family of ATPase region while domain III at the C te rminal end is unique to clamp loaders (43) (Figure 1 3A). An active clamp loader consists of five subunits and is held tightly together at the C terminal domains to form a collar while the N terminal domains are more loosely suspended with the A and E subunits not touching at all creating an open area (33,44,45) (Figure 1 3B). This gap could potentially allow DNA to enter the central chamber in the clamp loader, which positions DNA into the open clamp. The clamp is opened into a right handed helix and fits the spiral of the clamp loader (33,46) Electron microscopy studies of archaeal RFC PCNA DNA open lock washer appearance that allows the clamp to dock on the surface o f the helical RFC (47) At the replication fork, the E. coli clamp loader is comprised of seven subunits, including three copies of the dnaX (45,48,49) The dnaX gene identical sequence except for the last amino acid (50 52) The C terminal extension on well as to the DnaB helicase (11,53) In cells, the clamp loader with the composition 2 has been isolated and was found tethered to two copies of the core polymerase. The clamp loader with this specific co mposition is thought to be the main clamp loader in the holoenzyme complex. Clamp loaders with various in clamp loading

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19 but not in processive replication in vitro (48) The five subunit core, DnaX3 minimal complex, is an active clamp loader but less efficient than the seven subunit complex (54 56) In the clamp loader, the three DnaX subunits bind and hydrolyze ATP and are often thought of as t he motor due to the ATPase activity (56 58) clamp and is called the wrench because this subunit alone can open the clamp to unload from DNA (59,60) stabilizes the clamp loader stranded DNA binding protein (SSB) and increases the efficiency of clamp loading (55,61,62) RFC, the eukaryotic clamp loader, consists of five subunits that are found in a similar arrangement to that of the bacterial clamp loader. The yeast and human cl amp loaders are composed of four small subunits and one large subunit, which has additional N terminal and C terminal extensions that are not present in the small subunits (63 65) (Figure 14A B). The exact function of these two additional regions is not yet known. The N terminal region (removed from structure in Fig. 14) binds to DNA but is not directly required for clamp loading and deletion of this region increases clamp loading activity in vitro and cells are st ill viable in vivo (66 68) All five subunits are part of the AAA+ family of ATPases but only subunits 14 (A D) are functional. RFC1 (A) is E. coli clamp loader but is a functional ATPase unlike its bacterial counterpart (69,70) RFC5 (E) hydrolyze AT P but the subunit does have an arg inine finger that can interact with the

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20 ATP site of the adjacent subunit RFC2. The remaining subunits, RFC24 (B D) are In all species of clamp loaders, t he ATP sit es are located at the interfaces of the clamp loader subunits and contain conserved Walker A and Walker B sequence motifs. An arginine finger residue located in a conserved Ser Arg Cys (SRC) motif reaches into the interface of the ATP site of the adjacent subunit and senses when ATP is bound (71,72) This location may enable the subunits to undergo conformational changes in response to ATP binding and hydrolysis, which would allow the clamp loader into interact with the other molecules. Different conformational states formed by the clamp loader may drive different steps in the clamp loading mechanism. There are slight differences between these systems in how the ATP sites fill and when hydrolysis occurs. Binding studi es with yeast RFC show that the ATP sites fill sequentially Using ATP two molecules of ATP S promotes binding of either PCNA or DNA, and binding of PCNA or DNA promotes binding of a third molecule of ATP formation of a ternary RFC PCNA DNA complex promotes bi nding a fourth ATP S molecule. In contrast to yeast all three sites in the E. coli complex bind ATP in the absence of the clamp or DNA Clamp Loading Mechanism Clamp loading is a dynamic process, which includes many different steps that lead to the form ation of a ternary complex and ultimately the assembly of the clamp onto DNA (Figure 15). The clamp loader must first be primed with ATP and undergo a conformational change that increases its affinity for binding the clamp (27,73,74) T he ATP primed clamp loader can bind to either the clamp or to DNA first and ultimately form a ternary clamp loader clamp DNA complex. The binding of the clamp loader to

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21 DNA triggers hydrolysis of ATP in one or more of the ATP sites (75,76) Hydrolysis of ATP most likely causes another conformational change in the clamp loader prompting it to lose affinity for the clamp and DNA. The sliding clamp is closed around DNA and the clamp loader dissocia tes from the complex. All these steps must occur rapidly and in a defined order to allow the processive DNA polymerase to then bind and proceed with elongation. Although there have been many recent developments in this complex mechanism, there are still many questions left to be answered.

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22 Figure 11. A Model of the Escherichia coli Replisome. The E. coli replisome uses two Pol III core enzymes (purple) to copy leading and lagging strands. The replicative helicase (light blue) unwinds duplex DNA ahead o f the replication fork and stimulates primase activity. Primase ( green) synthesizes s hort RNA primers for each Okazaki fragment. The gray ) give processivity to the Pol III core by tethering the polymerase to the DNA template The clamp loader, (blue) uses the energy of ATP hydrolysis to assemble sliding clamps on DNA at primed sites. The clamp loader c onnects the leading and lagging str and polymerases. SSB (orange) prevents secondary structure formation of singlestranded DNA This figure was based on (2).

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23 Figure 12. Structure of sliding clamps. Ribbon diagrams of the E. coli S. cer evisiae PCNA (B), and human PCNA (C) sliding clamps, with each subunit represented in a different color. All clamps are composed of six globular domains with comparable fold that are linked by interdomain connecting loops. Structures were generated using P DB files 2POL (13) 1SXJ (33) and 1AXC (32)

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24 Figure 13. S tructure of the E. coli with the subunit domains labeled starting with domain III at the C terminal region and domain I at the N terminal region and (B) a cartoon representation of the minimal E. coli 3 (blue) (yellow), (red) with the subunits labeled A E based on their position. The clamp loader is a spiral shaped complex with its subunits held tightly together at the C terminal domains to form a collar while the N terminal domai ns are more l oosely suspended. The structure was generated using PDB file 1XXH (45)

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25 Figure 14. Structure of the S. cerevisiae clamp loader, RFC. (A) A ribbon diagram and (B) a cartoon representation of RFC with the subunits numbered and labeled A E based on their positions The subunits corresponding for the human RFC clamp loader are p140 (A), p40 (B), p36 (C), p37 (D), and p38 (E). RFC has similar structure and function to the E. coli clamp loader. The structure was generated using PDB file 1SXJ (33)

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26 Figure 1 6 The ATP dependent steps of the clamp loading reaction. In the first phase of clamp loading, formation of a ternary clamp loader clamp DNA complex is promoted by ATP binding. In the second phase, DNA binding triggers hydrolysis of ATP, the release of the clamp on DNA, and the dissociation of the clamp loader from the complex.

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27 CHAPTER 2 MATERIALS AND METHOD S Materials Nucleotides and Oligonucleotides Concentrations of ATP (Amer sham Biosciences/GE Healthcare) diluted with 20 mM Tris Diagnostics) were determined by measuring the absorbance at 259 nm and using an extinction coefficient of 15,400 M1cm1. Syntheti c oligonucleotides were obtained from Integrated DNA Technologies ( Coralville, IA) and purified by 12% denaturing polyacrylamide gel electrophoresis (PAGE) The sequences of the 60nucleotide template and the complementary 30nucleotide primer are as foll ows: 5 TTC AGG TCA GAA GGG TTC TAT CTC TGT TGG CCA GAA TGT CCC TTT TAT TAC TGG TCG TGT 3 and 5 ACA CGA CCA GTA ATA AAA GGG ACA TTC (C6dT) GG 3 where C6dT is a T with a C6 amino linker which was covalently labeled with 7diethylaminocoumarin3 carboxyli c acid succinimidyl ester (Invitrogen) as described (61,73) Primed templates were annealed by incubating the 30nucleotide primer with the 60nucleotide template in 20 mM Tris HCl (pH 7.5) and 50 mM NaCl at 85 r 5 minutes and then allowing the solution to slowly cool to room temperature. For all assays, the molar ratios of primer to template were 1:1.2 in annealing reactions. RFC and Rad24 RFC Purification These proteins were provided by the ODonnell lab. Buf fers Assay buffer used to study the E. coli DNA replication system contained 20 mM Tris HCl (pH 7.5), 50 mM NaCl, and 8 mM MgCl2 with the addition o f 4% glycerol where

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28 indicated. Assay buffer used to study the S. cerevisiae DNA replication system contained 30 mM HEPESNaOH (pH 7.5), 150 mM NaCl, and 8 mM MgCl2. E. coli p roteins were stored in Buffer A ( 20 mM Tris HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT and 10% glycerol ) The S. cerevisiae proteins were stor ed in 30 mM HEPESNaOH (pH 7.5), 0.5 mM EDTA, 150 mM NaCl, 2 mM DTT, and 10% glycerol. Methods Purification of DNA Polymerase III Proteins (77) (78) (78) (55) 3 (48) as previously described with minor modifications as described (supplementary online material (79) ) Sliding Clamp Mutagenesis Site directed mutagenesis was performed to change the Glu 299 to Cys construct in which surface cysteines 260 and 333 were replaced with serine so that Cys 299 could be selectively labeled. The QuikChange mutagenesis ki t (Stratagene) was used as directed by the manufacturer for site directed mutagenesis with the following primers (and the complementary strands) : Q299C, 5 CAC CGC CAA CAA CCC GGA ATG CGA AGA AGC GGA AGA GAT C; C260S, 5 CAT CTG GAA GCT GGC TCA GAT CTG CTC AAG CAG GCG; C333S, 5 CTG AAC GCG CTG AAA CTG AGA ACG TCC GCA TGA TGC. Sliding Clamp Transformation Transformation was done by t haw ing of E. coli BL21 (DE3) competent cells on ice and add ing 10 ng of the Q299C mutant plasmid to the competent cells The cells were chilled on ice for 5 min followed by heating at 42 C for 45 s and finally chilled

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29 on ice again for 2 min The transformed mixture was then add ed to 1 mL LB media and then allowed to recover in the shaker at 37 C, 250 rpm, for 1 h. A 1:10 dilution of the recovered culture was prepared by adding of LB media. Cells were plate d ( diluted culture and 1:10 diluted culture) on separate LB 37 C overnight for no more than 16 h. A starter culture was created by inoculating 2, 50 mL flasks containing 10 mL with one colony (per culture flask) from the overnight transformation. The cultures were i ncubated in the shaker at 37 C, 250 rpm, for 4 to 5 h and the flasks were stored at 4 C overnight. The starter culture was aliquoted into 4, 2 mL tubes and spun at 6 000 RCF for 10 min. The supernatant was d ecant ed and each pellet was r esuspended of LB 2.8 L flasks were inoc ulated (with 500 mL of LB ) with the resuspended pellets Cultures were i ncubated in the shaker at 37 C, 250 rpm, until the OD600 reached around 0.6 to 0.7 (3 to 4 h) Expression was induced by the addition of IPTG to 1 m M which is equivalent to flask The incubation was continued in the shaker at 37C for 4 h. C ultures were chilled on ice to slow expression and then spun at 5,000 RCF at 4 C for 30 min. Pellets were decant ed from the supernat ant w eigh ed, and stored at 80 C S amples of pre and post induction at different time points were saved to run SDS PAGE later. Sliding Clamp Purification (80) as previously described with the additional modifications as described (79) The pellets were t haw ed on ice and resuspended in 30 to 40 mL lysis buffer (20 mM Tris HCl ( pH 7.5) 0.5mM EDTA, 50

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30 mM NaCl, and 2 mM DTT) The resuspended pel lets were pressed two times in a French Press at 1 00 0 psi on the gauge and approximately 17,000 psi in the cell. The lysate was cleared by centrifugation at 10,000 RCF, 4 C for 45 min The cleared he pellet. Samples of the pellet and lysate were saved to run SDS PAGE later. All remaining purification steps are carried out at 4C unless otherwise noted. Ammonium sulfate (0.194 g/mL, 35% saturation) was added to the clarified cell lysate. The precipi tate was removed by centrifugation at 31,000 RCF for 30 min and discarded. Additional ammonium sulfate (0.218 g/mL, 70% saturation) was added to the supernatant, and the precipitate was recovered by centrifugation at 9, 800 RCF for 20 min. The pellet was re suspended in Buffer A and dialyzed against Buffer A. The dialyzed protein was loaded onto two 5mL HiTrap Q Sepharose columns (GE Healthcare) equilibrated in Buffer A and joined in tandem. After loading, the columns were washed with 3 column volumes (30 mL ) of Buffer A, and elut ed with a linear gradient of 0 to 500 mM NaCl. Fractions containing a peak eluting at about 225 mM NaCl were pooled and dialyzed against 10 mM sodium acetate (pH 7.5) and 0.5 mM EDTA. Dialyzed protein was loaded onto two 5mL HiTrap HeparinSepharose columns (GE Healthcare) equilibrated in 10 mM sodium acetate (pH 7.5) and 0.5 mM EDTA and joined in tandem. The material that was not retained on the column was collected and adjusted to pH 6.0 with acetic acid before loading onto the sam e two 5 mL HiTrap HeparinSepharose columns joined in tandem and equilibrated in 10 mM sodium acetate (pH 6.0) and 0.5 mM EDTA. The column was washed with 3 column volumes (30 mL) of the equilibration buffer and eluted with a linear gradient of 0 500 mM NaCl

PAGE 31

31 pooled, dialyzed against Buffer A that did not contain DTT, aliquoted, and stored at 80 C. The total protein yield was 73.5 mg/ L and had a final concentration of 4.76 m g/mL clamp with Pyrene Q299C mutant was labeled as described (81) The mutant protein was dialyzed against 1 L of 50 mM K2PO4 (pH 7.4) overnight at 4 C and for another 5 h after was transferred into 1 L of fresh 50 mM K2PO4 (pH 7.4). The N (1 pyrene) maleimide (Molecular Probes) was dissolved in the final concentration of DMSO was 8% in solution. clamp was incubat ed with a 10 fold excess of pyrene ( and 560 nmol pyrene) Q299C mutant and the pyrene in D MSO solution were warmed at 37 C temperature in the dark and then stored overnight at 4C. Q299C mutant was purified as described (72,82) using a BioRad P6DG desalting column with buffer containing 20 mM Tris HCl (pH 7.5) and 0.5 mM EDTA to remove excess free dye PY PY were then loaded onto a 1 mL HiTrap Q Sepharose anion exchange column (GE Healthcare) equilibrated with Buffer A and washed for 10 column volumes (10 mL) to remove excess pyrene noncovalently bound. The protein was eluted with Buffer A containing 0.5 M PY was dialyzed against Buffer A that did not contain DTT, aliquoted, and stored at 80 C. PCNA Sliding Clamp Mutagenesis Site directed mutagenesis was performed to change the Ile 111 and Il e 181 to Cys in a PCNA construc t in which surface cysteines 22, 62, and 81 were replaced with

PAGE 32

32 serine so that Cys 111 and Cys 181 could be selectively labeled. The QuikChange mutagenesis kit (Stratagene) was used for sitedirected mutagenesis as per the man ufacturers instructions with the following primers (and the complementary strands) : I111C, 5 GGA TAC CAG AAA GAC CGT TGT GCC GAA TAC TCT CTG ; I181C, 5 CGG ATC AGG TTC AGT CTG TAT AAA ACC ATT CGT GG ; C22S, 5 GGA AAT TGA CCA ACT GGA CGG AAT CTT TGA AAC CA T C ; C62S, 5 CCT AAC GTA ACA GGA TGG TCG GAT CTA TAT TCT TGG AAG GC; C81S, 5 GTA TCG GTG TTG TTA CCG GAA CGT AGG ATT TTA CTT AG PCNA Sliding Clamp Transformation The mutant PCNA was purified based on a protocol previously described (83) with the additional modifications as described. Transformation was done by t haw ing 50 of E. coli BL21 (DE3) competent cells on ice and a dding 10 ng of PCNA I111C/I181C mutant plasmid to the competent cells The cells were chilled on ice for 5 min followed by heating at 42 C for 45 s and finally chilled on ice again for 2 min The tra nsformed mixture was then add ed to 1 mL LB media and then allowed to recover in the shaker at 37 C, 250 rpm, for 1 h. A 1:10 dilution of the recovered culture was prepared by adding of LB media. Cells were p late d ( of 1:1 d iluted culture and 1:10 diluted culture) on separate LB agar 37 C, overnight for no more than 16 h. A starter culture was created by inoculating 2, 50 mL flasks containing 10 mL with one colony (per culture flask) from the overnight transformation. The cultures were i ncubated in the shaker at 37 C, 250 rpm, for 4 to 5 h and the flasks were stored at 4 C overnight.

PAGE 33

33 The starter culture was aliquoted into 4, 2 mL tubes and spun at 6 000 RCF for 10 min. The supernatant was d ecant ed and each pellet was r esuspended of LB 2.8 L flasks were inoculated (with 500 mL of LB icillin ) with the resuspended pellets Cultures were i ncubated in the shaker at 37 C, 250 rpm, until the OD600 reached around 0.6 to 0.7 (3 to 4 h) Expression was induced by the addition of IPTG to 1 mM which is equivalent to er flask The incubation was continued in the shaker at 37C for 4 h. C ultures were then chilled on ice to slow expression and then spun at 5,000 RCF at 4 C for 30 min Cell pellets were d ecant ed from the supernatant w eigh ed, and stored at 80 C S amples of pre and post induction at different time points were saved to run SDS PAGE later. PCNA Sliding Clamp Purification The cell pellets were t haw ed on ice and resuspended in 30 to 40 mL lysis buffer (20 mM Tris HCl ( pH 7.5) 0.5mM EDTA, 50 mM NaCl, and 2 mM DTT) The resuspended pellets were pressed two times in a French Press at 1 000 psi on the gauge and approximately 17,000 psi in the cell. The lysate was cleared by centrifugation at 10,000 RCF 4 C for 45 min The cleared lysate/supernatant, where PCNA is found, was decanted from the pellet. Samples of the pellet and lysate were saved to run SDS PAGE later. All remaining purification steps are carried out at 4C unless otherwise noted. The volume of the cleared lysate was adjusted to 50 mL with Buffer A c ontaining 50mM NaCl and ammonium sulfate (0.2 g/mL, 40% saturation) was slowly added to the clarified lysate over 30 min with gentle stirring The p recipitation continue d fo r about 1.5 h after the final addition of ammonium sulfate. The precipitate was removed by

PAGE 34

34 centrifugation at 31,000 RCF at 4 C for 30 min and discar ded The recovered supernatant volume was determined and then multiplied by 0.15 g/mL (60% saturation). Ammonium sulfate was s lowly added over 1 h and allowed to continue precipitation for 1.5 h after the final addition The precipitate was removed by centrifugation at 31,000 RCF at 4 C for 30 min and discar ded The recovered supernatant volume was determined and then multiplied by 0.15 g/mL (85% saturation). Ammonium sulfate was slowly added over 1.5 h and allowed to continue precipitation for 1 h after the final addition. The precipitate was recovered by centrifugation at 9,800 RCF for 25 min at 4 C PCNA is not in the supernatant but in the pellet. The pellet was resuspended in about 10 mL Buffer A containing 50 mM NaCl and then the re suspension was di alyzed against 2 L Buffer A containing 50 mM NaCl overnight using SpectraPor 2 (Spectrum Laboratories) dialysis membrane with a molecular weight cutoff of 12,000 to 14,000 g/mol. PCNA was placed into fresh dialysis buffer (2 L Buffer A containing 50 mM NaCl) and dialyzed for another 5 to 6 h. Two 5 mL HiTrap Q Sepharose columns (GE Healthcare) were equilibrate d with Buffer A containing 50 mM NaCl. The r ecover ed PCNA solution from dialysis wa s loaded onto the column via a peristaltic pump at a rate of 2 mL/min and the flow through was collected. The column was washed with 50 mL Buffer A containing 50 mM NaCl A linear gradient was run from 50 mM to 700 mM N aCl over 150 mL at 2.5 mL/min and 1.8 mL fractions were collected. PCNA is expected to elute at approximately 450 mM NaCl. To confirm presence of PCNA, SDS PAGE was run on the fractions to be pooled. T he pooled fractions were dialyzed overnight against 2 L of 25 mM KPO4 ( pH 7.0 ), 2 mM DTT, an d 10% g lycerol in a SpectraPor 2 membrane with a molecular weight cutoff

PAGE 35

3 5 of 12 000 14 000 g/mol. PCNA was put into fresh dialysis buffer at least one time and dialyzed for another 5 to 6 h. A 5 mL HiTrap SPSepharose column (GE Healthcare) was equilibrat ed with 25 mM KPO4 ( pH 7.0), 2 mM DTT, and 10% glycerol. The recovered PCNA solution loaded onto the column via a peristaltic pump at 2 mL/min. The PCNA will flow through this column. An 8 mL MonoQ column (GE Healthcare) equilibrated with 25 mM KPO4 ( pH 7. 0 ), 2 mM DTT, and 10% glycerol. Inject the PCNA collected from the SP Sehparose column flow through via a large injecting loop. Wash the column with 40 mL of 25 mM KPO4 ( pH 7.0 ), 2 mM DTT, and 10% glycerol. Run a linear gradient from 0 to 700 mM KCl over 8 0 mL at 2 mL/min and collect 1 mL fractions. PCNA is expected to peak at approximately 420 mM KCl. To confirm presence of PCNA, run SDS PAGE of the fractions to be pooled. Dialyze the pooled fractions overnight against 2 L of 30 mM HEPESNaOH (pH 7.5), 150 mM NaCl, 2 mM DTT, and 10% g lycerol in a SpectraPor 2 membrane with a molecular weight cutoff of 12 000 14, 000 g/mol. Change to fresh dialysis buffer at least one time and dialyze for another 5 to 6 h. Aliquot the PCNA and store at 80C. The total protei n yield was 20.5 mg/ L and had a final concentration of 5.94 mg/mL determined by measuring the absorbance at 280 nm in 6 M guanidine hydrochloride and using the calculated extinction coefficient 6,170 M1 cm1 for PCNA. Covalent Labeling of the PCNA clamp w ith AlexaFluor 488 For the labeling reaction, a solution of 50 mM TCEP in 0.2 M Tris base (pH 8.0) is added to PCNA and allowed to sit for 5 min. TCEP would reduce any disulfide bond formation that may occur between the cysteine residues. The AlexaFluor 488 C5 maleimide (AF488) (Molecular Probes) was dissolved in DMSO. After the addition of the PCNA I111C/I181C solution, the final concentration of DMSO was 8% The PCNA

PAGE 36

36 solution is incubated with a 30fold excess of AF488, (65 nmol AF488). The AF488 in DMSO solution was added to PCNA and incubated for 4 h at room temperature in the dark The AF488 labeled PCNA was purified using a BioRad P6DG desalting column with buffer containing 30 mM HEPES NaOH (pH 7.5) 150 mM NaCl, and 0.5 mM EDTA to remove excess free dye. One mL fractions were collected and pooled. Fractions containing PCNA AF488 which eluted in the void volume, were combined and loaded onto a 1 mL HiTrap Q Sepharose anion exchange column (GE Heathca re) equilibrated with 30 mM HEPESNaOH (pH 7.5) 150 mM NaCl, 0.5 mM EDTA and 10% glycerol. The loaded protein was washed for 10 mL with 30 mM HEPES NaOH (pH 7.5) 150 mM NaCl, 0.5 mM EDTA and 10% glycerol to remove excess AF488 and then eluted with 30 m M HEPESNaOH (pH 7.5) 150 mM NaCl, 0.5 mM EDTA and 10% glycerol containing 0.5 M NaCl. The PCNA AF488 was dialyzed against 30 mM HEPESNaOH (pH 7.5) 150 mM NaCl, 0.5 mM EDTA 2 mM DTT, and 10% glycerol aliquoted, and stored at 80 C. Protein Concentra tions Concentra were determined by measuring the absorbance at 280 nm in 6 M guanidine hydrochloride and using the calculated extinction coefficient (220,050 M 1cm1 PY was determined using the Modified Lowry Protein A ssay using bovine serum albumin (BSA) as standards (Pierce). Based on the concentration of protein and the concentration of pyrene determined by its absorbance at 338nm (extinction coefficient of 40,000 M1 cm1), the typical labeling efficiency was 60%. The concentrations of RFC and PCNA were determined my measuring the protein absorbance at 280 nm in 6 M guanidine hydrochloride and using

PAGE 37

37 the calculated extinction coefficient 162,120 M1 cm1and 6,170 M1 cm1, for RFC and PCNA, respectively. Labeling Pri mer DNA with DCC The 12% PAGE purified 30nucleotide primer was covalently labeled with a 20fold excess of DCC overnight at room temperature. The DCC labeled primer DNA was extracted using chloroform and concentrated BuOH then run on a desalting column containing rehydrated BioRad P6DG resin with 20 mM Tris HCl (pH7.5) and 0.5 mM EDTA to remove excess free dye. To remove unlabeled oligonucleotides from the labeled oligonucleotides, the DCC labeled DNA was purified by 12 % PAGE, eluted in a sterile buffer of NaCl, Tris, and EDTA and stored at 20 C. Experimental Procedures Steady State Fluorescence Assays Fluorescence emission spectra were measured using a QuantaMaster QM 1 fluorometer (Photon Technology International) or an Edinburgh Analytical Instruments FS900 fluorometer. DCC labeled DNA was excited at 440 nm and emission scanned from 450 to 550 nm using a 3.6nm bandpass on the Edinburgh SF900 fluorometer. PY m and emission scanned from 355 to 455 nm using a 5 nm bandpass on the Photon Technology International fluorometer. Equilibrium binding P Y was done by adding reagents sequentially to the cuvette starting PY PY or DNA DCC wa s done by adding reagents sequentially to the cuvette starting with PY or p/t DNA

PAGE 38

38 equilibrium an PY binding assays, emission spectra were measur ed following each addition, and intensities relative to free PY at 375 nm were plotted as a PCNA AF488 was excited at 495 nm and emission scanned fro m 505 to 605 nm using a 2.5nm bandpass on the Photon Technology International fluorometer. Equilibrium binding of RFC or Rad24RFC to PCNA AF488 was done by adding followed PCNA AF488 RFC or Rad24RFC for a total reaction volume of 80 L. E mission spectra were measured following each addition and relative intensities at 518 nm were plotted as a function of RFC concentration. Pre Steady State Fluorescence Assays Assays were done using an Applied Photophysics SX20MV stoppedflow apparatus at 20 reagents immediately before they entered the cuvette. Data were collected for a total of 5 s at intervals of 1 ms. Final concentrations for each experiment in assay buffer with 4% glycerol are indicated in the Figure Legends and/or Results Measurements of DCC fluorescence were made using a 455nm cut on filter to collect emission while exciting at 430 nm with a 3.72nm bandpass. A 365nm cut on filter was used to collect PY emission when exciting at 345nm using a 3.72PY binding experiments. Data Analysis Steady State Fluorescence Assays Emission spectra were corrected for background by subtracting the signal for free PY, DCC, or AF488 signal during analysis. The dissociation constant (KD PY

PAGE 39

39 complex was calculated by fitting the observed intensity data (Iobs) to equation 1 in c is the conce PY Imin is the PY and Imin complex PY The intensity of complex PY (Imax) and KD were fit as adjustable parameters by nonlinear regression using KaleidaGraph. Iobs ( KDc) ( KDc)2 4c 2 ( Imax Imin) Imin (2 1) Equation 2 1 was also used to fit the quench in DCC fluorescence as a function of complex in the DNA binding assay to calculate the relative intensity of a clamp loaderDNA complex and a KD value. In this case, the c oncentration of DNA was substituted for Imax represents the intensity of free DNA, and Imin represents the intensity of bound DNA. Equation 2 1 was also used to fit the quench in AF488 fluorescence as a function of RFC in the PCNA opening assay to calc ulate the relative intensity of a clamp loader clamp complex and a KD value. In this case, the concentration of PCNA was substituted for Imin represents the intensity of free PCNA AF488 and Imax represe nts the intensity of bound RFC PCNA AF488. Pre St eady State Fluorescence Assays PY binding were empirically fit to a single exponential rise, y = a (1 ekobst) + c, using KaleidaGraph to determine values for observed rate constants, kobs, Ki netic Modeling DNA binding data in Fig. 3 6, 3 8, and 310 were globally fit to t he model illustrated in Fig. 3 11 using DynaFit to calculate the rate constants given in the figure. All of the

PAGE 40

40 rate constants were treated as adjustable parameters; none w ere set at fixed values. The relative intensity for free DNA and for the initial clamp loaderDNA complex prior to the DNA induced conformational change were set to 1, and the relative intensities for all other clamp loaderDNA complexes were set to the v alue of 0.58 derived from the titration in Fig. 3 7 B. Concentrations of protein and DNA were fixed based on the concentrations determined experimentally from absorbances at 280 nm and 260 nm, mplex was preincubated with ATP, the incubate and dilute functions in DynaFit were used to that exist in the presence of ATP (84) These functions were used to: 1) mix a solution complex in half to the final concentration, and 3) incubate the solution for 1 s in silico to generate the equili brium populations of conformational states that is added to DNA to incubated with ATP, reactions were initiated in silico

PAGE 41

41 CHAPTER 3 RE SULTS Introduction ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze all three molecules of ATP (84 86) DNA complex. Thus the clamp loading reaction can be divided into two stages based on ATP requirements: 1) formation of a ternary complex requiring ATP binding, and 2) dissociation of the ternary first phase involving the formation of the ternary complex (81) High affinity binding clamp and to DNA requires ATP binding by the clamp loader (27,73,74) Presumably, ATP binding promotes conformational changes within the clamp loader that expose surfaces/residues that either the clamp or DNA; however, a productive clamp loading reaction most likely alone binds p/tDNA, the interaction with DNA triggers rapid ATP hydrolysis and dissociation of the clamp loader (75,76) Therefore, clamp loading would be more efficient if there were some mechanism to favor clamp binding bef ore DNA binding and productive clamp loading. One such mechanism would be a kinetic preference for the clamp loader to bind the clamp before DNA. This kinetic preference could be established simply by the clamp loader binding the clamp at a faster rate t han DNA. Alternatively, given that the clamp loader has three ATP binding sites, sequential filling

PAGE 42

42 of ATP sites and incremental conformational changes could allow the clamp loader to bind the clamp before DNA. In other words, a subset of sites bind AT P and induce ATP and make conformational changes to promote DNA binding. In this work, the rior to DNA and how the overall rates of ATP induced conformational changes contribute to the rates of clamp and DNA binding are addressed. The clamp loader when charged with ATP has a high affinity for the clamp. The equilibrium dissociation constant (KD (27) In previous studies, an anisotropy assa binding (61,72) but a limitation of this assay is that it is not sensitive enough to work at low nanomolar protein concentrations, where KD values are accurately determined. Therefore, a more sensitive intensity based fluorescence assay was developed to measure clamp loader clamp binding. In this assay, the Gln299 in was mutated to Cys, and Cys 299 was covalently labeled with pyrene (PY). Based on available structural data and similarities between the E. coli and yeast clamps and clamp loaders (33,34,45) residue 299 in is likely to be near a site where a subunit contacts the surface of the clamp in a clamp loaderclamp complex (Fi g. 3 1). Given the headto tail symmetry of the dimer and the anticipated contacts between complex and the fluorescence of PY is likely to be affected by complex binding only at the position near the middle subunit (Fig. 3 1). Although there ar clamp (one on PY PY f luorescence increases (Fig. 32 ). At saturating

PAGE 43

43 over two and a half times greater PY (81) This increase in PY fluorescence was used to measure equilibrium binding of complex to PY and calculate the dissociation constant for the interaction. A KD value PY (aver age values are shown in Fig. 33A ). This KD value is in agreement with the value of 3.2 nM previously reported (27) PY (80 nM) to determine the dissociation constant for the weaker ATP independent binding reaction (Fig. 3 3B filled circles) and to determine the stoichiometry of binding i n the presence of ATP (Fig. 3 3B filled squares). The KD value calculated for ATP independent binding was 135 32 nM, which is in agreement with the value 151 nM reported previously (72) 1:1 stoichiometry as indicated by achieving saturation at a concentration of around 80 n M D value of 2.9 n M for ATP dependent binding. Interesting ly, the maximum increase in PY fluorescence in assays without ATP is less than 2fold, whereas it is greater than 2.5fold in the presence of absence of ATP. ATP is shown to cause a conformational (27) The clamp is likely to be open in clamp loader clamp complexes in assays with ATP, whereas it is likely to be closed in clamp loader clamp complexes in assays without ATP (60)

PAGE 44

44 PY PY plex concentrations of 10, 20, 40, and 80 nM (Fig. 34 A). These time courses were empirically fit to exponential rises to calculate apparent rate constants, kobs. These kobs values were calculate an apparent onrate constant, kon, app, of 2.3 x 107 M1 s1 and an apparent dissociation rate constant, koff, app, of 0.14 s1 from the slope and y intercept, respectively (87) (Fig. 3 4 B). The apparent KD value calculated from these kinetic constants was 6 nM which is in agreement with the value determined from equilibrium measurements. A fluorescence intensity based assay was used in which the primer strand of p/t DNA is labeled with DCC three nu cleotides from the 3 end, p/t DNA DCC, was used to (61) When the clamp loader binds DNA at the primer/ template junction, the environment of DCC is altered and the DNA, but DNA binding triggers hydrolysis of ATP and dissociation of the clamp loader so that binding is transient (75,88) Ther DNA binding under equilibrium conditions was measured in assays with the nonhydrolyzable ATP analog, DNA DCC, the fluorescence is quenched and the emission maximum of DCC is blueshifted by 5 to 6nm (Fig. 3 5 A). To determine the magnitude of the fluorescence quench and relative quantum yield of a clamp loader DNA complex, p/t DNA the presence of 5 B). These titration data showed that the fluorescence of the pr otein DNA complex is 58% of the fluorescence of free p/t DNA DCC.

PAGE 45

45 time stoppedadded to a solution of p/t DNA DCC and ATP, and the intensity of DCC was measured as a function of time (Fig. 36 ). Binding rates were measured with increasing M In each case, a rapid ex binding p/t DNA DCC was followed by binding reaction became faster and the magnitude of t he fluorescence quench increased. However, the magnitude of the decrease in fluorescence at the lowest point was not as large as measure 5 B). Moreover, these reactions are not approaching a limiting value for the minimum fluor escence, but continue to decrease with increasing concentrations. Assuming that the magnitude of the quench in fluorescence is the same for clamp loader DNA complexes in assays with iation is fast relative to the DNA binding reaction so that only a fraction of p/t DNA DCC is bound when the fluorescence reaches a minimal value. Given a relative fluorescence of 58% for the clamp loader DNA complex, about 14% of the p/t DNA DCC is bound when the intensity reaches a minimum value of about 0.94 in the reaction containing 500 nM complex and p/t DNA DCC. ATP x that give it a high affinity for the clamp and DNA. It is possible that the rates of these conformational

PAGE 46

46 changes govern the rates of clamp and DNA binding to the clamp loader. To test this PY was measured in a complex was preincubated with ATP (Fig. 37 PY (Fig. 3 7 A, black trace). After mixing, both PY (200 nM (200 nM ), and ATP (0.5 m M This suggests that ATP binding and ATP are relatively rapid PY binding was measured in the absence of ATP (Fig. 37B, reaction with ATP (Fig. 37B, black trace). The s maller fraction bound is consi stent with the weaker ATP independent binding reaction (Fig. 33B ). This result provides additional support that ATP binding and ATP induced conformational changes that give relatively rapid. DNA Binding with and without ATP Preincubation To determine whether the rate of ATP binding and ATP induced conformational complex was added to ATP at the same time as p/t DNA complex and ATP was added to a solution of p/t DNA DCC and ATP (ATP preincubation), a rapid quench in fluorescence occurred that reached a minimum value in about 60 70 ms and was followed by an increase in fluorescence that reached a steady state lev el within about 500 ms (Fig. 38 black trace, gray line). In contrast, DNA DCC and

PAGE 47

47 ATP (no ATP preincubation), the decrease in DCC fluoresc ence is slower overall (Fig. 3 8 gray trace, black line). There is a short lag of at least 25 ms before fluorescence begins to decrease and the rate of decrease is slower, such that the maximal quench occurs between 150 and M ) and pt/ DNA DCC (200 n M ) were the same in both reactions. Because the overall binding rate DNA dissociation are likely to be the same, th instead of ATP to measure DNA binding in the absence of DNA triggered ATP hydrolysis and subsequent clamp loader diss ociation ( Fig. 3 9). Although the reaction in preincubated case, overall both reactions were much slower and took 6 8 s to reach completion. It is possible that DNA binding reacti ons are efficacy of ATP in promoting conformational changes that allow the clamp loader to bind DNA. We have also observed a decrease in compar ed with ATP (61) For this reason, we chose to focus on assays with ATP even though the kinetics are complicated by hydrolysis. In any case, a slow ATP complex is preincubated with ATP. DNA Binding Kinetics Either a slow ATP binding step or a slow ATP induced conformational change DNA binding were measured as a function of ATP concentration in

PAGE 48

48 and p/t DNA DCC was mixed with solutions of ATP, and DCC fluorescence was measured as a function of time. In Fig. 3 10, reactions containing 25 (A, red), 50 (B, M (D, blue) ATP are plotted in the same graph with a M ATP (black trace). At the lowest concentration of ATP used, the reactions approached a max imal rate, becoming slightly faster with M ATP. In all M ATP, therefore, ATP binding was unlikely to be ratelimiting. The slower rates of complex was not preincubated with ATP must be due to a slow ATP induced conformational change. Kinetic Modeling of DNA Binding Reactions DNA binding data were modeled to get an estimate of the rate of the ATP induced confor mational change that precedes DNA binding to determine whether there studies was adapted to fit these DNA binding data (84) The kinetic model was developed based on experiments measuring rates of ATP hydrolysis in reactions in iods of time before adding p/tDNA. The earlier studies revealed that the rate of ATP hydrolysi s was limited by an ATP dependent conformational change and suggested that a slow ATP induced conformational change activates the clamp loader for DNA binding. Experiments in Figs. 3 8 and 310 confirm this by establishing that a slow ATP induced conformat ional change precedes DNA binding. The complete kinetic mechanism is quite complex and contains too many forward and reverse rate constants to define in a single set of experiments. Therefore, the

PAGE 49

49 kinetic model was simplified by including only forward rat es for most of the steps and allowing three molecules of ATP to bind and three molecules of ADP to dissociate as a unit in a single step. All of the rate constants shown in Fig. 311 were derived by globall y fitting the data in Figs. 36, 3 8, and 3 10 to the model shown using DynaFit ( 89) The forward and reverse rate constants for the ATP induced conformational change obtained from DNA binding data were 3.3 and 1.7 s1 compared with values of 6.5 and 3.9 s1, respectively, obtained previously from fits of ATP hydrolysis data. After th e conformational change, p/t DNA binding occurred at a rate of 4.0 x 107 M1 s1. The rate of DNA binding is similar to the rate of clamp binding (2.3 x 107 M1 s1) and on the order of what would be expected for a diffusionlimited rate for two macromolec ules rate of DNA binding is limited by the slow, 3.3 s1 conformational change, whereas the DNA triggers a change in the clamp loader that makes the ATP sites competent for hydrolysis, and in the model used here, three molecules of ATP are hydrolyzed sequentially before DNA is released. ADP release allows the clamp loader to go through the cycle again. The DNA binding d ata do not provide a direct measure of ATP hydrolysis, and in terms of these data, the ATP hydrolysis steps in the kinetic model provide a time lag between observed DNA binding and release. The model used to fit DNA binding data here differs from that used to fit ATP hydrolysis data in that two molecules of ATP were hydrolyzed rapidly and the third slowly in the ATP hydrolysis model. The DNA binding data could be fit equally well by a model in which ATP hydrolysis occurred in two phases (data not shown), hy drolysis of two molecules of ATP rapidly before DNA

PAGE 50

50 release, and hydrolysis of one molecule of ATP slowly after DNA release as proposed previously based on ATP hydrolysis data (84) When DNA binding data were fit to this two phase model for ATP hydrolysis, similar rates for ATP binding (kon (ATP) M1 s1), the ATP induced conformational change (kconf = 3.3 s1, krev (conf) = 1.7 s1), DNA binding (3.9 x 107 M1 s1), and DNA release (88 s1) were obtained. Therefore, for the purposes of this study, which focuses on the overall rate of DNA binding, we used the simpler model in which all three molecules were hydrolyzed at the same rate. Discussion Clamp loaders catalyze the assembly of sliding clamps onto DNA for use by DNA polymerases. To load clamps, the affinity of the clamp loader for the clamp and DNA must be modulated. Initially, the clamp loader must have a high affinity for the clamp and DNA to bring these macromolecules together, but then the affinity must decrease so that the clamp loader can release the clamp onto DNA. This affinity modulation is achieved by ATP binding and hydrolysis. ATP binding activates the clamp loader for binding the clamp and DNA, whereas ATP hydrolysis deactivates the clamp loader rele asing the clamp and DNA. T he clamp loading reaction can be divided into two phases depending on the ATP requirements: 1) ATP binding dependent formation of a ternary clamp loader clamp DNA complex, and 2) ATP hydrolysis dependent decay of the ternary complex releasing the clamp on DNA. Much of our previous work focused on the second phase of the reaction by adding a preformed clamp loader clamp complex to DNA to rapidly form the t ernary complex via a single pathway. Here, we focused on the first phase of the reaction, formation of a ternary clamp loader clamp complex. We clamp loader with ATP to for m the ATP activated state affects the rates of clamp loader -

PAGE 51

51 clamp and clamp loader DNA binding. To measure the rate of clamp binding, a sensitive fluorescence intensity based clamp was covalently labeled with PY. The inte nsity of PY increases when the clamp loader binds the clamp. An advantage of this assay is that clamp loader clamp binding can be measured directly in solution and in comple millisecond time scale. Using this binding assay, a bimolecular rate constant of 2.3 x 107 M1 s1 indicates the binding interaction is limited by the rate of diffusion of the proteins. Two Sets of ATP induced Conformational Changes High affinity binding of the clamp loader to the clamp and to DNA requires the clamp loader to bind ATP first. Presumably ATP binding promotes conformational changes in the clamp loader that place amino acid residues and protein surfaces in the appropriate conformation to productively interact with the clamp and DNA (27,60,76) Inter estingly, our results strongly suggest that a different set of ATP induced conformational changes is required to promote binding of the clamp loader to the clamp versus binding to DNA (81) (Fig. 3 12). Given that A TP induced conformational changes must precede clamp or DNA binding, the overall rate of these conformational changes will contribute to observed before adding the clamp or DNA, these conf ormational changes can take place during the preincubation period and binding rates will not be influenced by the rate of the

PAGE 52

52 DNA binding was faster than for reactions in whi ATP before p/t DNA (see Fig. 3 8 ). DNA binding kinetics measured as a function of ATP concentration (see Fig. 310) demonstrated that the slower DNA binding kinetics were not due to a slow ATP binding reaction, but inst ead must be the result of slow ATP induced conformational changes. complex was preincubat ed with ATP or not (see Fig. 37 experiments without ATP pr eincubation is unlikely to be due to an ATP independent binding reaction because the ATP independent binding interaction is weaker (see Fig. 3 3B ) and could only account for a fraction of the binding events (see Fig. 37 B). ATP binding and subsequent ATP i nduced conformational changes are rapid relative to the induced conformational change, this would suggest that a different set of conformational binding (81) When equilibrated with ATP, the E. coli same is true for the eukaryotic clamp loader, RFC (90) Although these clamp loaders can bind either the clamp or DNA, productive clamp loading is likely to requi re these clamp loaders to bind the clamp first. In contrast, the bacteriophage T4 clamp loader can productively load clamps by binding either DNA or the clamp first (91,92) This may be due to differences in the sol ution structures of the clamps. The bacteriophage T4 gp45 clamp exists as an open ring in solution (93,94) whereas the clamp (95) and eukaryotic clamp are likely to exist as closed rings in solution (96)

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53 When bound to genomic D NA, the geometry of the clamp loader DNA complex likely prevents the clamp loader from productively binding a closed clamp. In addition, binding of the bacterial and eukaryotic clamp loaders to p/t DNA triggers rapid ATP hydrolysis and dissociation of the clamp loader from the DNA, such that the clamp loader DNA complex is transient and not likely to be long lived enough to efficiently bind clamps (75,97) Nonproductive interactions between the clamp loaders and DNA would reduce the overall efficiency of clamp loading by engaging the clamp loader in futile cycles of DNA binding and ATP hydrolysis. A mechanism that favored clamp binding prior to DNA binding would increase the overall efficiency of clamp loading. It is possible that the rate limiting ATP induced conformational changes that precede DNA binding but not clamp binding provide a kinetic preference for the clamp loader to bind the clamp before DNA and increase the overall efficiency of clamp loading. The fastest rate at which the clamp loader c an bind DNA is limited by rate of the ATP induced conformational change (3.3 s1), but binding could be slower if DNA concentrations are limiting. The rate of the conformational change that promotes high inding may be a function of the M (98) in a 1 x 1015 liter cell volume, and the bimolecular rate constant for binding, 2.3 x 107 M1 s1, to give an effective on rate of 11 s1. This effective on free to be loaded (59) Even at saturating concentrations of D of primer synthesis on the lagging strand could also provide a mechanism for regulating

PAGE 54

54 lished by synthesizing primers at a rate that would allow the clamp loader to bind a clamp before a new primed template site is formed.

PAGE 55

55 Figure 31. Fluorescence intensity clamp binding assay. Upper panel A clamp is sh own with one monomer in cyan and the other in magenta. GlutamineCys 260 and Cys 333 were converted to serine, so that Cys 299 could be selectively labeled with pyrene (PY). Lower panel clamp has a C2 axis of symmetry through the center of the ring such that two PY fluorophores (yellow starbursts) covalently attached to Cys 299 are located on opposite PY likely binds at or near a PY to alter its environment and increase PY fluorescence (larger yellow starburst). The second PY molecule is unlikely to porting on the binding interaction.

PAGE 56

56 Figure 32. Emission Spectra of pyrene fluorescence. PY were taken at excitation wavelength of 345 nm. The light gray trace is a scan of PY and the black trace complex and ATP. Final PY 240

PAGE 57

57 Figure 33. A complex to plex PY in assay buffer PY and 0.5 mM ATP in assay buffer. B the absence (circles) and presence PY in assay buffer. The PY and 0.5 mM ATP in assay buffer.

PAGE 58

58 Figure 34 PY A) The increase in PY intensity was PY and ATP (0.5 mM). Reactions PY and 10 nM (red), 20 nM (blue), 40 nM (green), or 80 through time courses are the result of an empirical fit of the data to an exponential rise. B) Observed rat e constants, kobs, calculated from exponential fits of the reaction time courses are plotted against the 1s1 and a y intercept of 0.14 s1.

PAGE 59

59 Figure 35. lex to DNA. A) Emission spectra of DCC were measured u sing an excitation wavelength of 455 nm. The black trace is a scan of free p/tDNA DCC, the dashed dark gray trace is a scan repeated after the light gray trace is a scan repeated after DNA DCC, 200 of DCC in a clamp loader DNA complex was determined by measuring DCC fluorescence as in DCC fluorescence was calculated by fitting these data to a quadratic equation ( Materials and Methods ). The solid line through the data is the result of the fit, which gave a value of 0.58 for the relative intensity of DCC in a clamp loaderDNA complex and a KD value of 375 7 nM for clamp loaderDNA dissociation. Final concentrations were 100 nM p/t DNA DCC

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60 Figure 36. binding to DNA T he binding to DNA was measured in to a solution of p/t DNA DCC and ATP. The relative fluorescence of DCC is complex and p/t DNA DCC and 0.5 mM ATP in assay buffer with 4% glycerol. darker indicates a higher concentration. Solid lines through the reaction time course were generated from the kinetic model illustrated in Fig. 311.

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61 Figure 3PY was measured in assays with (gray trace) and without (black trace) pre inc ATP. The relative intensity of PY is plotted as a function of time for a reaction PY add PY and ATP (black trace). The insert is the first 0.2 s PY were measured in the absence of ATP (gray trace) and in the presence of ATP but without the preomplex with ATP (black trace). Final buffer with 4% glycerol.

PAGE 62

62 Figure 38. ATP. The change in DCC fluorescence measured as a function of time. In one assay ( black trace complex and ATP was added to a solution of p/t DNA DCC and ATP. In the second ( gray trace added to a solution of p/t DNA DCC and ATP. The relative intensity of DCC is plotted as a function of time on a scale of 2 s. Final concentrations were 200 nM p/t DNA glycerol. Smooth solid lines thro ugh the data are from a fit to the model in Fig. 3 11.

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63 Figure 3DNA DCC. The measured as a function of time. In one assay ( b lack trace DNA the second ( gray trace was added to a solution of p/t DNA DCC is plotted as a function of time on a scale of 2 s. Final concentrations were 200 nM p/t DNA buffer with 4% glycerol.

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64 Figure 310 DNA binding kinetics. Rates of DNA binding were measured as a function of ATP concentration in a reaction and p/t DNA DCC was mixed with a solution of ATP and the DCC fluorescence was measured as a function of time. Final concentrations were 200 nM p/t DNA black trace ) in assay buffer with 4% glycerol. Smooth black lines through the data are from a fi t to the model in Fig. 311.

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65 Figure 311. Kinetic model fit to p/t DNA binding data. The model starts on the left hand side 33 lustrated in the step. ATP binding promotes a conformational change that allows the clamp loader to bind DNA. DNA binding induces a second conformational change that activates the ATP sites for hydrolysis and three molecules of ATP are hydrolyzed sequentially at the same rate. DNA and then ADP are released to and the relative intensities for all bound DN A species set at 0.58. The data in Figs. 3 6, 3 8, and 310 were fit to this model using DynaFit (89)

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66 Figure 31 2 Model for formation of a ternary clamp loader clamp DNAcomplex ATP binds the clamp loader and rapidly induces a conformational change ( yellow star state ) that enables the clamp loader to bind the clamp. A second conformational change ( green star state) occurs more slowly and enables the clamp loader to bind p/t DNA. This second conformational change could either occur within a clamp loader clamp complex when the clamp is present ( upper reaction scheme), or within the free clamp loader in the absence of the clamp ( lower reaction scheme) to promote p/t DNA binding. Note that the complex can bind three molecules of ATP, but that we have not determined how many molecules bind at each stage of the assembly reaction. Therefore, the stoichiometry of bound ATP molecules at each step is not given. It is possible that the slow conformational change opens an ATP site, or a subset of sites, to allow binding of additional ATP that promotes DNA binding.

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67 CHAPTER 4 DISCUSSION AND FUTURE STUDIES Given binding i t will be interesting to see which conformational changes promote clamp opening. The first fast conformational change could promote both clamp binding and clamp op ening as illustrated in Fig. 312. Alternatively, the second slower conformational change could promote clamp opening and subsequent DNA binding. We have recently developed a fluorescence intensity opening and closing. Using this assay, we could potentially define the rate of opening There are a number of int eresting observations in the literature that can be used to speculate about a mechanism by which two distinct sets of conformational changes ATP sites and sequential fillin g of these sites could promote sequential sets of conformational changes. A subset of ATP sites could fill first to induce conformational the remaining sites to fill with the conformational changes to occur so that DNA. Studies with arginine finger mutants of the E. coli and yeast clamp loaders, which bind but do not sense or respond to bound ATP, show that ATP binding at different sites has differential effects on clamp and DNA binding (72,99) Sim ilarly, studies of single Walker A mutants that reduce ATP binding to the yeast clamp loader showed that

PAGE 68

68 mutations of individual sites had differential effects on DNA binding (100) Both Arg finger (99) and Walker A (100) mutations that affect ATP sensing/binding to the replication factor C (RFC) 2 and RFC3 subunits of the yeast clamp loader gave the greatest decreases in DNA binding activities. Interestingly, Arg finger mutations in the E. coli he same position as RFC3 (darkest bl 1 2 adjacent to the subunit, greatly reduced DNA binding. In a crystal structure of the minimal E. coli 3 subunit does not co (44) Biochemical characterization showed that this minimal clamp l 3 3 important for DNA binding (61) That is not to say that a single subunit binds DNA, but instead that ATP binding at one (or a subset of sites) is important for inducing conformational changes within the clamp loader that as a whole increase DNA binding activity. If a slow conformational change were to open this site and cause it to fill last, then this could provide a mechanism for sequential binding of the clamp and DNA. The observation that the rate of p/t DNA binding is limited by the rate of ATP induced conformational changes is consistent with resu lts from previous work measuring rates of p/t DNA binding kinetics, we found that rates of DNA triggered ATP hydrolysis were faster when reincubation, and that the slower rate was not due to slow ATP binding (84) Based on rates of ATP

PAGE 69

69 hydrolysis as a function of preincubation time with ATP, a forward rate constant of 6.5 s1 and reverse rate cons tant of 3.9 s1 was calculated for the ATP induced conformational changes. These values are in good agreement with the values of 3.3 and 1.7 s1 for the forward and reverse rate constants calculated from kinetic modeling of the DNA binding reaction (see Fi g. 3 11 ) given both the complexity of this reaction and that the two data sets were fit independently. Both sets of experiments predict that after equilibration with ATP a little over 60% of the clamp loaders (63% from ATPase experiments and 66% from DNA b inding ex periments) exist in the conformational state that has high affinity for p/tconformatio nal state with a high affinity for DNA and shifting the equilibrium to favor this species (73) Studies with a minimal form of the 3 subunits support this idea. The minimal clamp loader is defective in ATP dependent dependent DNA binding activity of the minimal clamp loader most likely by stabilizing or promoting the conformational state with high affinity for DNA (61) of the clamp loader for DNA by directly interacting with the DNA duplex. The c entral (13) and these residues interact with the sugar phosphate backbone of the duplex (101)

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70 APPENDIX DEVELOPMENT OF A NEW FLUORESCENCE BASED ASSAY: MEASURI NG PCNA OPENING IN THE SACCHAROMYCES CEREVI SIAE DNA REPLICATION SYST EM Introduction In S. cerevisiae clamp loading is a dynamic, multistep process that leads to the formation of a ternary complex and ultimately the assembly of PCNA onto DNA to allow for processive replication (102) RFC must first be primed with ATP and undergo a conformati onal change that increases its affinity for binding PCNA. After binding ATP, RFC binds to PCNA and DNA to form a ternary RFC PCNA DNA complex. Alternatively, a similar set of conformational changes may trigger both PCNA binding and opening or PCNA opening and DNA binding at the same time or sequentially. The binding of RFC to DNA triggers hydrolysis of ATP in one or more of the ATP sites Hydrolysis of ATP most likely causes another conformational change in RFC, which prompts it to lose affinity for PCNA and DNA. PCNA closes around DNA and RFC dissociates from the complex. The eukaryotic clamp loader for replication, RFC, consists of five subunits (A E) that are found in a similar arrangement to that of the bacterial clamp loader. Three alternative RFC complexes also exist in which the RFC A subunit is replaced by another protein including Rad24 in S. cerevisiae (Rad17 in humans), Cft18, and Elg1. Rad24RFC functions at the DNA damage checkpoint during S phase (104,106) This clamp loader interacts with and loads onto DNA an alternative clamp called the 91 1 complex. The heterotrimeric clamp is composed of Rad9, Rad1, and Hus1 in humans and S. pombe (115,116) and Ddc1, Rad17, and Mec3, respectively in S. cerevisiae (117) The loading of the 91 1 complex by Rad24RFC starts the DNA damage checkpoint activation and signals the recruitment of ATR to the site of damage (118) Rad24 RFC

PAGE 71

71 can bind to PC NA and unload it from DNA but cannot productively load PCNA onto DNA (115,119) The other two alternative subunits are not well understood (103 106) The Ctf18RFC complex is required for sister chromatid cohesion. Two additional proteins, Ddc1 and Ctf8, bind to Ctf18 and are recruited to RFC to form a sevensubunit complex (105,107,108) Ctf18 RFC, both the five and sevensubunit complexes, can load and unload PCNA onto DNA (109111) The Elg1RFC alternative complex suppresses chromosomal rearrangements and play s a role in chromosome stability (112114) A productive Elg1RFC PCNA interaction has not been demonstrated in vitro although protein interaction between Elg1 and PCNA was confirmed by co immunoprecipitation (112) It is still unclear whether there is an alternative clamp that mainly interacts with this clamp loader or if PCNA is the main target for interaction. Different RFC A subunits can be swapped out in an RFC complex to have different functions in the cell. Each new RFC complex has specificity for and interacts with PCNA or an alternative clamp in some way. In this work, the question of the contribution of the RFC A subunit in the RFC complex to the interaction with PCNA will be addressed by measuring clamp loader clamp opening and closing A fluorescenceintensity based assay to measure opening and closing of an RFC PCNA complex A sensitive intensity based fluorescence assay was developed to measure clamp loader clamp opening and closing in the S. cerevisiae replication system In this assay, residues Ile111 and Ile 181, located on opposite sides of the interfaces in PCNA (Fig. A 1), were mutated to Cys, and Cys 111 and Cys 181 were covalently labeled with Alexa Fluor 488, C5 maleimide ( AF488 ). These mutations were placed in a PCNA mutant with

PAGE 72

72 three of the four naturally occurring Cys residues (Cys 22, Cys 62, and C ys 81) converted to Ser to allow for selective labeling. The PCNA mutant was labeled in the presence of a reducing agent tris (2 carboxyethyl) phosphine (TCEP), which does not react with maleimides, to prevent disulfide bond formation between the closely s paced cysteines. Two AF488 fluorophores are able to stack and self quench when in physical contact. Based on available structural data for the yeast clamp, carbon atoms of residues 111 and 181 are located within 5.5 from each other. Each PCNA monomer is labeled with two fluorophores per monomer. The homotrimer contains six fluorophores with a pair located at each monomer interface. Physical interaction of the fluorophores on either side of a monomer interface will quench AF488 fluorescence, and physic al separation of these fluorophores should increase fluorescence One mechanism to physically separate the fluorophores is by physical denaturation with a detergent. When a 5% SDS solution was added to PCNA AF488, the fluorescence increased. The fluoresc ence of the denatured PCNA AF488 in SDS was 8.6 fold greater than the fluorescence of AF488 in the native protein (Fig. A 2). The clamp loader can physically separate fluorophores on either side of a monomer interface by opening the clamp. When RFC binds to PCNA AF488 in the presence of ATP AF488 fluorescence increases due to the formation of an open clamp loader clamp complex (Fig. A 3, red). At saturating concentrations of RFC, the intensity of AF488 is about twofold greater A control experiment in which buffer was added in place of RFC gave a decrease in fluorescence due to dilution (Fig. A 3, blue). The fluorescence intensity of an open RFC PCNA complex is less than the denatured

PAGE 73

73 clamp, which is presumably due to only one of the three interfaces opening and showing a relief of quench. The increase in AF488 fluorescence was used to measure equilibrium binding of RFC to PCNA AF488 and calculate the dissociation constant for the interaction. A KD value of 13.0 2.4 nM was calculated from three independent experiments at 10 nM PCNA AF488 (av erage values are shown in Fig. A 4 ). Measuring Rad24RFC PCNA Interactions using the PCNA AF488 Opening Assay Previous studies have shown that Rad24RFC can unload PCNA from a circular DNA plasmid but cannot load PCNA onto DNA (119) Even though there was no loading detected, the Rad24RFC ATPase was stimulated by PCNA (117) ; therefore, there is some interac tion between Rad24RFC and PCNA. The unloading of PCNA from a plasmid DNA was used as an indirect measurement of PCNA opening to determine whether Rad24RFC binds and then opens the clamp. One disadvantage of using an indirect method to measure opening, is that stable open complexes of Rad24RFC PCNA maybe not be forming. Simply, PCNA may be transiently opening and closing and can easily slip off of the DNA or that Rad24RFC transiently opens PCNA The newly developed PCNA AF488 opening assay would be useful to measure directly if Rad24 RFC opens PCNA to form a stable open complex. A preliminary opening assay experiment was performed and the AF488 fluorescence was used to measure equilibrium binding of wild type RFC or Rad24RFC to PCNA AF488. The calculated dissociation constant s for the interactions were then compared between the two RFC complexes A KD value of 76 nM was calculated for Rad24RFC PCNA while a KD value of 13 nM was calculated for wild type RFC at 10 nM PCNA AF488. The Rad24 RFC complex has about a six fold weaker interaction with PCNA than wild type RFC. Based on the relative fluorescence intensity differences,

PAGE 74

74 Rad24RFC has a reduced ability to open PCNA compared to RFC. We currently cannot determine the absolute percentage of stable open clamp complexes in either the wild type RFC or Rad24RFC cases but the data suggests that there is about five times more open PCNA in a complex with RFC than in a complex with Rad24RFC. This will be investigated further and will be interesting to see if we can further quantify the reduced amount of opening by Rad24RFC compared to wild type RFC. Discussion and Future Studies The PCNA AF488 opening assay will be useful for studying the initial and final steps of the clamp loading reaction: 1) binding and opening and 2) closing and release in steady state and pre steady state experiments. Although basic characterization of this new mutant PCNA has been done, additional experiments are required to determine if PCNA AF488 has wild type PCNA activity. One such experiment would be a competition binding assay in which the labeled clamp competes with the unlabeled clamp to bind RFC. Many proteins that bind clamps interact through a conserved peptide sequence motif. Based on sequence alignments and binding studies, this motif is proposed to be Qxx (I/L/M) xxF (F/Y) for PCNA, termed the PCNA Interacting Peptide box (PIP box) (29 31) An important feature of this binding motif is the presence of hydrophobic amino acid residues including phenylalanine and tyrosine, which come in contact with the hydrophobic pocket on the clamp under an IDCL (32 34) Through sequence alignment studies, the RFC1 (A) subunit in yeast has a proposed PIP motif of NMSVVGYF Using an additional method to test the importance of the RFC A subunit in the opening of PCNA, the two aromatic residues (tyrosine and phenylalanine) of the RFC1 (A) PIP motif will be mutated to alanine residues to disrupt the binding interaction with PCNA. If

PAGE 75

75 the RFC A subunit is essential for opening PCNA, this RFC1 (A) PIP mutant will have a reduced opening ability, which will be t ested using the PCNA AF488 opening assay. We anticipate that the mutant will have a reduced ability to open PCNA as we have observed for Rad24RFC, which has the RFC A subunit replaced with Rad24. In addition to the PCNA AF488 opening and closing assay, i t will be useful to develop a PCNA binding assay as a method to compare the different steps in the clamp loading mechanism. Based on the available yeast RFC PCNA crystal structure, a proposed site of mutation would be serine 43 on PCNA, which is at a site of interaction with RFC1. The Ser 43 residue would be mutated using sitedirect mutagenesis to a cysteine into a PCNA with three of the four naturally occur ring Cys residues (Cys 22, Cys 62, and Cys 81) converted to Ser to allow for selective labeling. The Ser 43 Cys residue would be labeled with an environmentally sensitive fluorophore that would potentially change in fluorescence upon binding to RFC. It wil l be interesting to use both the binding and opening assays in steady state and presteady state conditions to define the role of the RFC A subunit in the clamp loading reaction.

PAGE 76

76 Figure A 1. Crystal Structural Model of PCNA highlighting the residues mutated for the PCNA opening assay Residues Ile111 and Ile181 are located on opposite sides of the PCNA monomer interfaces. carbon atoms is about 5.5 These residues were mutated to cysteines. Three of the naturally occurring Cys residues in PCNA were mutated to Ser to allow for selective labeling of these two residues. This creates two labeled residues per monomer, and a pair of fluorophores on each tr imer interface. Structures were generated using PDB file 1SXJ (33)

PAGE 77

77 Figure A 2. Relief of the AF488 Fluorescence quench in the presence of SDS. Denaturation of PCNA AF488 in 5% SDS solution increases the fluorescence of AF488 in the doubly labeled clamp by about 8.6fold. Solutions contained 10 nM PCNA AF488 5% SDS.

PAGE 78

78 Figure A 3. Emission spectra of PCNA AF488 were taken at excitation wavelength of 495 nm. The green trace is a scan of free PCNA AF488, the red trace is after the addition of RFC and ATP, and the blue trace is after the addition of storage buffer and ATP instead of RFC. Final concentrations were 10 nM PCNA AF488, 285 nM RFC and 0.5 mM ATP.

PAGE 79

79 0.8 1 1.2 1.4 1.6 1.8 2 2.2 0 100 200 300 400 500 600Relative IntensityConcentration of RFC (nM) Figure A 4. Equilibrium binding and opening of RFC PCNA. The equilibrium dissociation constant was determined by measuring the intensity of AF488 as a function of RFC concentratio n, where RFC was titrated into PCNA AF488 in assay buffer containing ATP. Final concentrations were 10 nM PCNA AF488 and 0.5 mM ATP in assay buffer.

PAGE 80

80 0.8 1 1.2 1.4 1.6 1.8 0 100 200 300 400 500Relative Intensity Concentration of RFC or Rad24-RFC (nM) Figure A 5. Equilibrium binding and opening of Rad24 RFC PCNA. The equilibrium dissociation constant f or Rad24RFC PCNA ( red squares ) was compared with wild type RFC PCNA ( black circles) and was determined by measuring the intensity of AF488 as a function of Rad24 RFC or RFC concentration, where Rad24RFC or RFC was titrated into PCNA AF488 in assay buffer containing ATP. Final concentrations were 10 nM PCNA AF488 and 0.5 mM ATP in assay buffer.

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88 BIOGRAPHICAL SKETCH Jennifer Thompson was born in 1985 in Long Island, New York. The middle child of three children, she has an older brother and a younger sister. For the first 12 years of her childhood, she lived in the Poconos in Pennsylvania. In 1998, her family moved to Bradenton, Florida where she graduated from Lakewood Ranch High School i n 2003. She earned her B.S. in microbiology and cell s cience from the University of Florida in 2007. In her last semester of her undergraduate career, Jennif er began research in Dr. Linda Blooms biochemistry laboratory. She continued working in the lab as a technician until she started the masters program in the Biochemistry and Molecular Biology Department at U.F. in the fall of 2008. After completing the masters program, Jennifer will continue working on her research in Dr. Blooms lab.