Two proteins from Ustilago maydis

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Title:
Two proteins from Ustilago maydis purification and interaction with DNA
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xiii, 143 leaves : ill. ; 29 cm.
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Rusche, James Robert, 1954-
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DNA   ( mesh )
Ustilago   ( mesh )
Immunology and Medical Microbiology thesis Ph.D   ( mesh )
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Notes

Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 129-142.
Statement of Responsibility:
by James Robert Rusche.
General Note:
Photocopy of typescript.
General Note:
Vita.

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University of Florida
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\TWO PROTEINS FROM USTILAGO MAYDISt
PURIFICATION AND INTERACTION
WITH DNA








BY

o /
JAMES ROBERT RUSCHE


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


UNIVERSITY OF FLORIDA


1981



























To my family.














ACKNOWLEDGMENTS


I would like to express my thanks to my advisory

committee, Doctors Ken Berns, Bert Flanegan, Bill Hauswirth,

Ed Siden, Dick Boyce and Nick Muzyczka. I am very grateful

to my mentor Dr. Bill (ACE) Holloman for his friendship,

excellent guidance and endurance throughout my graduate

training. I would like to thank Dr. George Gifford for

his academic and personal interest In my education.

An optimal environment for research was developed and

maintained by Greg Chomic, Tom Rowe, Don Goodroe, Laura Prall,

Michael Brougham, Sherin Smallwood, and Eric Kmiec. I am

deeply indebted to these people for their patience, under-

standing and humor. I would especially like to thank Tom

(TR, Tommy Wong, Johnny Ciringo) Rowe for his personal and

academic companionship as well as his laugh. Thanks go to

Sherin for her friendship, technical assistance and excellent

graphic skills. Also I would like to thank Diane Blucher

for her work on this manuscript.

The love and support of my family was an inspiration

throughout this work. The constant encouragement of my parents

John and Mary was indispensable. I also profited from the

unique opportunity to have my grandparents Al,Marie, John

and Mil for friendship and support.








Finally I would like to thank Dr. Marty Venneman for

his advice to me in 1976, "Hell, why not go for it?"















TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS..................................... iii

LIST OF TABLES...... ......................... ....... vii

LIST OF FIGURES...................................... viii

ABBREVIATIONS USED................................... X

ABSTRACT........................................... xii

CHAPTER I INTRODUCTION..................... ....... 1

Proteins That Bind to DNA........... 2
Nucleolytic Alterations
in DNA Structure.................... 8
Ustilago maydis as a Model
System of Eucaryotic
DNA Metabolism...................... 10

CHAPTER II METHODOLOGY............................ 12

Cell Culture......................... 12
Nucleic Acid Preparation............ 13
Assays...... ......................... 17
Protein Purification................ 22
Materials........................... 25

CHAPTER III PURIFICATION AND CHARACTERIZATION
OF NUCLEASE B.......................... 26

Purification........................ 27
Reaction Conditions.................. 36
Mode of Hydrolysis.................. 47
Products of Hydrolysis.............. 57
Other Activities .................... 65


.......... 65









CHAPTER IV PURIFICATION AND CHARACTERIZATION
OF DBP III.............................. 72

Purification........................ 72
Reaction Conditions For DBP III..... 82
Characterization of Protein
Binding to DNA...................... 89

CHAPTER V DISCUSSION.... ........................ 111

Characterization of Nuclease 8..... 111
Characterization of DBP III........ 118

LITERATURE CITED.................................... 129

BIOGRAPHICAL SKETCH................................. 143













LIST OF TABLES


Table Page

I. Purification of nuclease ..................... 28

II. Reaction rates of nuclease .................... 58

III. Mononucleotide isomers of digestion products.... 62

IV. Activity of snake venom phosphodiesterase
on DNA partially digested by nuclease ......... 66

V. 5'-nucleotidase activity of nuclease .......... 67

VI. Effect of small molecules dh nuclease
activity.................... ..................... 77

VII. DNA binding activity in purified
nuclease preparations......................... 81

VIII. Effect of small molecules on DNA binding........ 86


vii














LIST OF FIGURES


Figure Page

1. Gel electrophoresis of nuclease a................ 31

2. SDS-gel electrophoresis of nuclease B............ 33

3. Sedimentation of nuclease in a
glycerol gradient................................. 35

4. Determination of Stokes radius for
nuclease B........................................ 38

5. Isoelectric focusing of nuclease B............... 40

6. Time course of hydrolysis of DNA
by nuclease ................ .................... 42

7. Effect of pH on nuclease S activity.............. 44

8. Effect of temperature and ionic
strength on nuclease B activity.................... 46

9. Effect of small molecules on
nuclease a activity.............................. 49

10. The action of nuclease S on
terminally labeled DNA........................... 51

11. Distributive mode of DNA hydrolysis
by nuclease .................................... 54

12. Sucrose gradient sedimentation of
*X174 phage DNA after treatment
with nuclease a................................... 56

13. Size determination of the products
of reaction ...................................... 60

14. Chromotography of 5'-and 3'-
mononucleotides on Dowex-1....................... 64


viii








15. Identification of the initial product
of reaction with 5'-terminally labeled DNA....... 70

16. Chromatographic separation of
DNA binding activity............................ 76

17. PH optimum of nuclease activity.................. 79

18. Optimal pH of DBP III activity................... 85

19. Effect of temperature on DBP III activity........ 88

20. Activity with form I, form II or
form III DNA as substrate........................ 91

21. Agarose gel electrophoresis of
DBP III-DNA mixtures ............................. 93

22. DBP III activity with form I
or form IV DNA as substrate...................... 96

23. Effect of ethidium bromide
intercalation on DBP III activity................ 98

24. Effect of strand breakage on
preformed DBP III-form I DNA complexes............ 101

25. Binding competition with
various forms of 4X174 DNA....................... 104

26. Binding competition with deoxyhomopolymers....... 106

27. Binding competition with other form I DNA........ 109















ABBREVIATIONS USED


ATP............adenosine-5'-triphosphate

BSA...........bovine serum albumin

OC............degree centigrade

Ci............Curie (unit of radioactivity)

cpm...........counts per minute

d.............dalton

DBP...........DNA binding protein

DNA........... deoxyribonucleic acid

DNAse.........deoxyribonuclease

EDTA..........ethylenediamine tetra-acetic acid

form I........negatively supercoiled, covalently
closed DNA

form II.......circular duplex DNA containing
a strand break

form III......linear duplex DNA

form IV.......relaxed covalently closed DNA

g.............gram

h............. hour

HDP...........helix destabilizing protein

L..............liter

mamp...........milliampere









min...........minute

MOI...........multiplicity of infection

NAD..........nicotinamide adenine dinucleotide

PEG...........polyethylene glycol

poly [dA].....polydeoxyadenylic acid

poly [dG].....polydeoxyguanylic acid

poly [dT].....polydeoxythymidylic acid

psi...........pounds per square inch

RF I.......... replicating form I

RNA............ribonucleic acid

RNAse..........ribonuclease

rpm...........revolutions per minute

S...............Svedberg (unit of sedimentation)

SDS........... sodium dodecyl sulphate

SSC............standard saline citrate
(0.15 M NaCl, 15 mM sodium citrate)

V.............volts














Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


TWO PROTEINS FROM USTILAGO MAYDIS:
PURIFICATION AND INTERACTION
WITH DNA

By

James Robert Rusche

December, 1981

Chairman: William K. Holloman
Major Department: Immunology and Medical Microbiology

Cell extracts of Ustilago maydis were examined for pro-

teins that alter the structure of DNA. Two proteins were

isolated and characterized that interact with DNA. An activ-

ity that hydrolyzes DNA, termed nuclease 8, was purified

and the mode of DNA digestion by nuclease 6 was investigated.

Also a DNA binding activity, termed DBP III, was isolated

by utilizing the property of protein dependent retention of

DNA on nitrocellulose filters. The in vitro characterization

of these activities was performed to enrich the present under-

standing of eucaryotic DNAmetabolism.

The single strand specific nuclease B has been purified

11,200-fold from crude extracts to about 70% homogeneity.

The enzyme has a sedimentation coefficient of 4.3 S and a
Stokes radius of 36A consistent with a native form of a
Stokes radius of 36A consistent with a native form of a


xii








single polypeptide of 68,000 daltons. The enzyme is fully

active in the presence of chelating agents but is strongly

inhibited by nucleotides. Maximal activity is seen at pH

6.0. The enzyme hydrolyzes linear DNA in an exonucleolytic

fashion from the 5'-end liberating 3'-mononucleotides and

small oligonucleotides. The enzyme is also active on both

single stranded and duplex circular DNA, dephosphorylates

5'-mononucleotides, and hydrolyzes RNA.

A DNA binding activity, termed DBP III, was found to

remain bound to DNA in the presence of 1.5 MNaCl. Purifi-

cation of DBP III was accomplished by assaying for activity

that retained DNA on nitrocellulose filters in the presence

of 1.5 M NaCl. Purified preparations of DBP III contained

one major polypeptide of 47,000 daltons. Protein binding

to DNA required a minimum temperature of about 250C.

DBP III retained covalently closed DNA (either relaxed or

negatively supercoiled) but did not retain nicked circular

or linear DNA to nitrocellulose filters. Titration of form

I DNA with ethidium bromide did not alter the amount of DNA

bound by DBP III. DBP III-DNA complexes appeared to dissoci-

ate if the DNA was nicked after complex formation. Competi-

tion experiments suggest that DBP III has affinity for single

stranded DNA, especially the homopolymer poly (dT). A

plasmid containing an A*T rich DNA insert was also an effec-

tive competitor.


xiii














CHAPTER I
INTRODUCTION

The intimate association of DNA with protein confused

early attempts at identifying which compound was the genetic

material. The accumulation of experimental evidence from

1944 (Avery et al., 1944) to 1953 (Watson and Crick, 1953)

indicated that deoxyribonucleic acid was the genetic material.

Proteins possess the capacity to facilitate gene expression

and replicate the genetic material. The molecular mechanisms

by which proteins accomplish this manipulation of genetic

material is the subject of extensive research. Although the

secondary structure of DNA invivo isprobably a duplex right-

handed helix, extensive tertiary structure is brought about

by the interaction of proteins with DNA (Bak et al., 1977).

It appears that the secondary and tertiary structure of DNA be-

comes altered during replication, recombination and repair. For

example, replication of duplex DNA requires that the parent strands

be unwound to allow them to act as templates (for example see

Alberts and Sternglanz, 1977). Repair of DNA damage in the

absence of light requires strand incision and removal of

damaged bases (for review see Grossman et al., 1975;

Hanawalt et al., 1979). Recombination between duplex mole-

cules appears to occur through the breakage and reunion of

DNA strands (Tomizawa, 1967; Meselson, 1967). A final

example is that DNA replication in some eucaryotic viruses








seems to require nucleolytic incision of covalently linked

DNA termini (Berns et al., 1978). Recent research on DNA

metabolism has focused on the molecular mechanisms by which

proteins make these alterations in DNA structure. One method

used to identify the proteins involved in DNA metabolism is

to isolate cell constituents and characterize their interac-

tion with DNA in vitro.

A goal of this project was to isolate and characterize

proteins that alter the structure of DNA. Previously isolat-

ed DNA binding proteins alter the structure of DNA by funda-

mentally different processes. The following discussion of

proteins that bind to DNA provides a reference by which to

compare a binding protein I have isolated from Ustilago

maydis.

Proteins that Bind to DNA

Proteins that bind to DNA can most easily be separated

into three types: i) proteins that bind to specific sequences

in DNA ii) proteins that maintain the structure of chromatin

and iii) proteins that alter the tertiary structure of DNA.

Many of these proteins appear to be multifunctional and could

be listed in more than one of the above categories.

Sequence Specific Binding Proteins

Proteins that bind to specific sequences in DNA have

been isolated from both procaryotes and eucaryotes. These

proteins seem to function in controlling gene expression and

performing some functions in DNA replication. For example,

the lac repressor of E. coli (Riggs et al., 1970)controls the








transcriptional activity of the lactose operon by binding to

transcriptional operator sequences (for review see Beckwith

and Zipser, 1970). Repressor protein-operator DNA com-

plexes prevent the initiation of transcription of the lac-

tose operon by RNA polymerase, another sequence specific

binding protein (Hinkle and Chamberlin, 1972). The CI gene

product of bacteriophage Lambda (Ptashne, 1971) and the lex

A gene product of E. coli (Little et al., 1981; Brent and

Ptashne, 1981) are also transcriptional repressor proteins

that bind specific sequences. DNA restriction-modifiction

enzymes (Modrich, 1979; Yuan, 1981) from procaryotic cells

are another example of site specific binding proteins.

Eucaryotic cells also possess sequence specific bind-

ing proteins. For example, a binding protein isolated

from Drosophila melanogaster (Hsieh and Brutlag, 1979a) binds

specifically to sequences found in satellite DNA from this

organism (Hsieh and Brutlag, 1979b). The eucaryotic virus

SV-40 codes for a protein (gene A protein or T antigen) that

binds to specific sequences on the viral genome (Reed et al.,

1975). T antigen is a multifunctional protein thought to be

involved in controlling both gene expression (Cowan et al.,

1973) and replication (Tegtmeyer, 1972) of SV-40 DNA. Spe-

cies specific DNA binding proteins have also been observed

in eucaryotic cells that have been chemically transformed

(Sen and Todaro, 1978).








Proteins that Maintain the Structure of DNA in Vivo

Proteins are thought to induce the compact structure

of DNA in vivo (von Hipple and McGhee, 1972). The primary

level of chromatin structure is maintained by a class of

proteins called histones (Elgin and Weintraub, 1975).

Histones are arranged on the DNA in approximately 200 base

pair repeating units called nucleosomes (for review see

Kornberg,1977; McGhee and Felsenfeld, 1980). Examination of

isolated chromatin at physiological ionic strength suggests

that the nucleosomes further compact DNA by wrapping in a

superhelix or solenoid (Bak et al., 1977). The nonhistone

chromosomal proteins also play a role in DNA compaction.

Glycoproteins have been isolated from a variety of eucaryotic

cells that may function in the condensation process of DNA in

vivo (Champ et al., 1978).

Chromosomal DNA that has been depleted of histones re-

mains a compact structure (Ide et al., 1975; Berezney and

Coffey, 1976; Paulson and Laemmli, 1977). The DNA appears

to be held in negatively supercoiled loops attached to a

structure called the nuclear matrix (Benyajata and Worcel,

1976; Cook et al., 1976; for review see Georgiev et al.,

1978). Proteins extracted from the nuclear matrix have

been shown to bind DNA in vitro (Comings and Wallack, 1978).

The experiments of Pardoll et al. (1980) suggest that newly

replicated DNA is preferentially associated with the nuclear

matrix. Thus the nuclear matrix may function in both main-

taining the DNA superstructure of chromosomes and providing

a site for DNA replication.








Proteins that Alter DNA Structure

The processes of DNA replication and recombination

require that the secondary structure of DNA be altered. For

example, the unwinding of duplex DNA is necessary for the

semi-conservative replication of DNA (for review see Gefter,

1975; Alberts and Sternglanz, 1977; Champoux, 1978). Pro-

teins termed helix destabilizing proteins (HDP) have been

isolated from a variety of procaryotic (Sigal et al., 1972;

Alberts and Frey, 1970) and eucaryotic (Banks and Spanos,

1975; Herrick and Alberts, 1976; Schechter et al., 1980;

Riva et al., 1980) cells. Thesebinding proteins alter the

temperature required for renaturation and denaturation of

DNA in vitro (for example see Alberts and Frey, 1970).

Studies in vitro show that helix destabilizing proteins

interact with other DNA enzymes. The HDP from T4 infected

E. coli cells (T4 gene 32 protein) specifically stimulates

the synthetic capacity of the T4 coded DNA polymerase

(Huberman et al., 1971). Specific stimulation by HDP of the

homologus polymerase has also been observed with protein from

E. coli (Molineux et al., 1974), T7 infected cells (Reuben

and Gefter, 1973), Ustilago maydis (Yarranton et al., 1976)

and Hela cells (Riva et al., 1980). The formation of a

physical complex between DNA polymerase and HDP has been

demonstrated for proteins isolated from E. coli (Molineux

and Gefter, 1975) and T4 (Huberman et al., 1971) or T7

(Scherzinger et al., 1973) infected cells. The HDP of E.

coli also forms a complex with exonuclease I of E. coli.








Genetic studies with E. coli (Meyer et al., 1979;

Johnson, 1977) and bacteriophage T4 (Breschkin and Mosig,

1977) suggest that helix destabilizing proteins are neces-

sary for both replication and recombination in vivo. The

Adenovirus HDP seems to be involved in both the initiation

and chain elongation processes of DNA replication (van der

Vliet et al., 1977).

Proteins that enzymatically alter the structure of DNA

have also been isolated. The helical winding number of

circular DNA can be changed by nicking and resealing the

phosphate backbone of DNA with enzymes called topoisomerases

(Wang and Liu, 1979). Nicking-sealing enzymes seem to be

involved in transcription and some types of recombination

(for review see Gellert, 1981).

Proteins that unwind DNA by utilizing the energy of

ATP hydrolysis have also been isolated (Scott and Kornberg,

1978; Abdel-Monem et al., 1976; Mackay and Linn, 1976;

Rosamond et al., 1979). The rep gene product of E. coli is

an example of enzyme catalyzed unwinding of DNA chains

(Duguet et al., 1978). Rep protein has been implicated in

the process of chain elongation during replication of

bacteriophage (Denhardt et al., 1972) and cellular (Lane

and Denhardt, 1975) DNA.

Alterations in DNA Structure During Recombination

Homologous recombination is thought to occur by the

exchange of strands between two duplex DNA molecules

(Whitehouse, 1963; Holliday, 1964). The process of strand








exchange requires that the DNA duplexes be unwound and re-

wound to form a heteroduplex. The rec A protein of E. coli

is a DNA-dependent ATPase that can reanneal homologous

single strands of DNA (Weinstock et al., 1979; for review see

Radding, 1981). Recent studies in vitro demonstrate that

rec A protein can catalyze strand exchange between partially

duplex molecules (Cunningham et al., 1980; Cassuto et al.,

1980). The importance of enzyme catalyzed strand exchange

is suggested by the observation that rec A deficient mutants

have a severely reduced frequency of recombination (Clark,

1973). Rec A protein catalyzed Strand exchange requires

that one DNA molecule be at least partially single stranded

and that either DNA molecule contain a free end (Cunningham

et al., 1980; West et al.,1981). DNA damage induced recom-

bination in the absence of replication appears to be initiat-

ed by the nucleolytic formation of nicks or gaps in the DNA

(for review see Witkin, 1976; Howard-Flanders, 1973).

The preceding discussion illustrates that many types

of structural alterations of DNA can be induced by protein-

DNA interactions. Another source of protein induced alter-

ations in DNA structure is the hydrolysis of the phosphate

backbone of DNA by enzymes termed nucleases. Another goal

of this project was to purify and characterize a nuclease

activity I had observed in crude extracts of Ustilago maydis.

The following is a discussion of the possible involvement of

nuclease activities in DNA metabolism.








Nucleolytic Alterations in DNA Structure

The structural alteration of DNA by nucleolytic cleavage

plays an important role in replication,repair and recom-

bination. For example, the accumulation of progeny virus in

bacteriophage ZX174 infections requires the site specific

cleavage of replicating form molecules by the OX174 gene A

protein (Eisenberg et al., 1977). Another example is that

nuclease activities associated with DNA polymerase I of E.

coli appear to be important in replication and repair of

DNA in vivo (for review see Kornberg, 1980). Nucleases that

can hydolyze damaged DNA have been isolated from a variety of

sources (Kushner and Grossman, 1971; Simon et al., 1975;

RiazuddinandGrossman, 1977; for review see Grossman et al.,

1975; Hanawalt et al., 1979). These damage specific

nucleases probably stimulate recombination via the formation

of nicked or gapped DNA molecules (see above).

Involvement of Nucleases in Recombination

Current models of recombination by strand exchange

(Holliday, 1964; Meselson and Radding, 1975; Wagner and

Radman, 1975) suggest a role for nucleases at multiple stages

of the recombination process. The Meselson-Radding model

(1975) proposes the incision of a duplex molecule that has

assimilated a third DNA strand (D-loop). D-loop molecules

have been formed nonenzymatically in vitro (Holloman et al.,

1975). Specific cleavage of the displaced strand in D-loop

molecules was demonstrated (Wiegand et al., 1977) using

enzymes with single strand specific endonuclease activity








(ie.,SI nuclease or rec BC nuclease). The nicking of duplex

DNA that has assimilated a third strand would allow for

reciprocal strand exchange between the two molecules

(Meselson and Radding, 1975). Experiments in vivo show that

duplex circular DNA becomes nicked when cells also contain

homologous DNA containing damage (Howard-Flanders et al.,

1978). The enzyme responsible for this "cutting in trans"

activity has not been identified.

Strand exchange between DNA molecules that contain a

single base difference could result in the formation of

heteroduplex DNA containing a mismatched base-pair. The

excision repair of mismatched bases is thought to result in

gene conversion (Whitehouse, 1963; Holliday, 1964). Gene

conversion describes the genetic observation of nonreciprocal

allelic recombination. Single strand specific nucleases are

candidates for this excision repair because of their ability

to recognize structural distortions in duplex DNA. Cleavage

of DNA containing structural distortions has been demon-

strated with a variety of endonucleases including enzymes

from Aspergillus (Wiegand et al., 1975), mung bean

(Kowalski et al., 1976), T7 bacteriophage (Center and

Richardson, 1970) and Ustilago (Holloman et al., 1981).

Ustilago mutants deficient in gene conversion were shown to

have reduced levels of the single strand specific nuclease a

(Holloman and Holliday, 1973).








Ustilago maydis as a Model System of Eucaryotic DNA Metabolism

Lower eucaryotes have proven to be valuable systems for

the biochemical analysis of eucaryotic DNA metabolism. The

ability to grow yeast and fungi on chemically defined media

(solid or liquid) and the ability to isolate mutants in DNA

metabolism havemade these organisms popular systems for bio-

chemical studies (for example see Holliday, 1974). The

studies to be presented below were done with proteins

isolated from the basidiomycete fungus Ustilago maydis.

A number of enzymes involved in DNA metabolism have

been isolated from Ustilago maydis. For example, a DNA

polymerase was isolated and characterized (Banks et al., 1976)

from wild type cells and a mutant cell type that was temper-

ature sensitive for DNA synthesis (Jeggo and Banks, 1975).

The polymerase from the mutant strain was temperature sensi-

tive for polymerizing activity suggesting this polymerase

may be the major polymerase involved in chromosomal replica-

tion in U. maydis. A nuclease termed nuclease a has also

been purified that appears to be involved in gene conversion

(see above). Two proteins which bind to DNA have been

isolated and characterized. DNA binding protein I (DBP I)

was shown to be a helix destabilizing protein (Banks and

Spanos, 1975). DBP I interacts with the DNA polymerase of

Ustilago and reduces the Km of the polymerase for both

substrate and template (Yarranton et al., 1976). DBP II

from Ustilago can bind to DNA forming a protein-DNA

complex that is soluble in dilute acid (Holloman, 1975;








Unrau et al., 1980). DBP II is a glycoprotein thought to be

involved in the condensation process of nucleic acids in vivo

(Champ et al., 1978).

Originally I was examining extracts of U. maydis for an

activity that could catalyze strand assimilation of a DNA

fragment into a homologous duplex DNA molecule. During these

investigations I observed a nuclease activity and DNA binding

activity. The work described in the subsequent chapters

deals with a nuclease, termed nuclease 8, and a DNA binding

protein termed DBP III. The goals of this project were to

purify the nuclease and DNA binding protein activities and

characterize their interaction with DNA. Some of this work

has previously been published (Rusche et al., 1980).














CHAPTER II
METHODOLOGY

Cell Culture

Strains

Ustilago maydis al, b2 was obtained from Dr. Robin

Holliday, National Institute for Medical Research, London.

Cells were grown in a medium composed of 2% peptone, 2% suc-

rose, and 1% yeast extract in a Vir-Tis fermentor under

vigorous aeration. When the density reached 5 X 10 cells/

ml the cells were harvested by centrifugation and stored

frozen at -700C. Bacteriophage 0X174 am3 and its host

Escherichia coli HF4704 were obtained from Dr. Charles

Radding, Yale University. Phage P22 and its host Salmonella

typhimurium DB25 were obtained from Dr. David Botstein,

Massachusetts Institute of Technology. Escherichia coli HB101

containing the plasmid aDM23-24 (Hsieh and Brutlag, 1979a)

was obtained from Dr. Douglas Brutlag, Stanford. HB101 con-

taining pBR322 (Bolivar et al., 1977) was obtained from Dr.

Susan Lederberg, Stanford University.

Media

Minimal media was used for the in vivo labeling of phage

DNA. The P22 phage host cell strain DB25 was grown in LCG

medium (Botstein, 1968). This medium contained 0.5 g NaC1,

8 g KC1, 1.1 g NH4C1, 5g casein hydrolysate, 0.12 g MgSO41

0.055 g CaC12, 12.1 g Tris base, and 10 pg of FeS04 per liter of








solution and was adjusted to pH 7.5. The zX174 phage host

cell strain HF4704 was grown in TPA medium (Benbow et al.,

1974). This medium contained 12.1 g Tris base, 2 g casein

hydrolysate, 1 g NH4C1, 0.3 g MgSG4, 0.24 g NaC1, 0.3 g

KC1, 0.22 g CaC12, and 10 pg of FeS04 per liter of solution

and was adjusted to pH 7.5. These media were supplimented

with glucose, thymine and phosphate as indicated below.

Strains of E. coli HB101 containing plasmid DNA were grown

in Luria broth medium that contained 5 g NaC1, 10 g tryptone,

and 5 g yeast extract per liter. The pH of the media was

adjusted to 7.5 with 4.5 mmol of NaOH.

Nucleic Acid Preparation

Isolation of DNA Made in Vivo

Bacteriophage P22 DNA was made as described by Carter and

Radding (1971). Strain DB25 was grown in 1 liter of LCG medi-

um supplimented with 1 mM KPO4, 0.2% glucose, and 3 mg/ml

thymine. When the cell density reached 4 X 10 cells/ml the

culture was infected with P22 phage at a multiplicity of in-

fection (MOI) of 5. Five milliCurie of [ H] thymidine was

added and incubation continued for 2.5 h. DNAse and RNAse

(100 ug of each) were added and the solution incubated an addi-

tional 10 min. To this mixture was added 29 g of NaCl and

110 g of PEG 6000 and the mixture was stored at 4C overnight.

The resulting precipitate was collected. This was washed and

pelleted three times with 10 mM Tris-HC1, pH 7.5, collecting the

supernatant each time. The phage was then banded in a CsCl

step gradient of 1.35 g/ml and 1.5 g/ml. The DNA was obtained

by phenol extraction followed by ethanol precipitation.








Bacteriophage sX174 DNA was made using a method described

by Beattie et al. (1977). Strain HF4704 was grown in TPA me-

dium supplimented with 3 yg/ml thymine, 0.2% glucose, and 1 mM

KPO4. At a cell density of 5 X 10 cells/ml 0X174 am3 phage

was added at a MOI of 10. At 3 min. post infection 60 mg

per liter of chloramphenicol was added and after an additional

7 min., 5 mCi of [ H] thymidine. This was incubated for 2 h.

and the cells were pelleted. The cells were resuspended in

15 ml of a solution containing 50 mM Tris-HCl 7.5, 10 mM EDTA,

and 10% sucrose. To this solution was added 30 mg of lysozyme

and the cells were incubated on ice for 15 min. Then 0.8 ml

of a 10% sarkosyl solution was added and the mixture was

centrifuged for 1 h. at 25,000 rpm in a SW27 rotor. The super-

natant was phenol extracted and ethanol precipitated. The

precipitate was resuspended in 2 ml of 10 mM Tris, 1 mM EDTA

and treated with 0.1 mg of RNAse for 15 min. The RFI DNA was

isolated by centrifugation through a 35 ml 5-20% sucrose

gradient for 18 h. at 25,000 rpm in a SW27 rotor. One milli-

liter fractions containing radioactivity were pooled and

precipitated with 2 volumes of ethanol.

HB101 cells containing a plasmid were grown in 1 liter

of Luria broth medium to a density of 4 X 108 cells/ml. Chlor-

amphenicol was added to 150 y g/ml and the culture incubated

18 h. (Clewell, 1972). The cells were pelleted and the

plasmid DNA purified as described for zX174 RFI DNA. As an

alternative to the final step of sucrose gradient sedimenta-

tion, the form I DNA was purified by chromatography through








an affinity column containing acridine yellow (Boehringer

Mannheim). After RNAse treatment the solution was diluted

to 10 ml with a buffer containing 0.5 M NaC1 and 20 mM Na

citrate, pH 6.0 as suggested by D. Rawlins of this department.

This was applied to an acridine yellow column (20 ml bed

volume) equilibrated with 0.5 M NaC1, 20 mM Na citrate, pH

6.0 and washed with the same buffer (100 ml). The RFI was

eluted by washing the column with 20 mM Na citrate, pH 6.0,con-

taining 1.5 NaC1. This wash was collected in 5 ml fractions

and DNA eluted in fractions 5 and 6. This DNA was dialyzed

versus 10 mM Tris-Hcl 7.5, 1 mM EDTA and precipitated with

ethanol.

Modification of DNA Substrates

The units indicated for enzymes obtained commerically

are those described by the manufacturer. Linear DNA

(RFIII) was prepared by cleavage of ZX174 RFI DNA with restric-

tion endonuclease Pst I. One mole of 5X RFI DNA was incubated

with 25 units of Pst I at 300C for 8 h. in 10 mMTris-HCl, pH 7.5,
32
10 mM MgCl2, and 10 mM B-mercaptoethanol. The 5'- P-labeledDNA

was prepared by dephosphorylating DNA with bacterial alkaline

phosphatase and rephosphorylating with polynucleotide kinase
32
in the presence of [y- 32 ATP as described by Weiss et al.

(1968). A reaction mixture (0.5ml) containing 0.5 pmol of

dephosphorylated DNA, 1 mM [y 32P ATP (200 mCi/nmol), 50 mM

Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, and 10

units of polynucleotide kinase was incubated at 370C. After

1 h. EDTAwas added to 20mM and the mixture was held at 650C

for 15 min. Thiswasappliedto a Sephacryl S-200 column (10 ml










bed volume) equilibrated with 10 mM Tris-HCl, pH 7.5, 1 mM
32
EDTA, 0.1 M NaCl and the unincorporated [y- P] removed

from the labeled polynucleotide. DNA was precipitated with

ethanol and resuspended in 10 mM Tris, pH 7.5, and 1 mM

EDTA. The 3'- 32P-labeled DNA was preparedas described by

Nossal and Hershfield (1971). A reaction mixture (0.3 ml)

containing 0.2 pmol of XX174 RFIII DNA, 100 DM [a-32P]dGTP

(10 yCi/nmol), and 5 units of T4 DNA polymerase in 67 mM

Tris-HCl, pH 8.8, 6.7 mM MgC12, 10 mM 8-mercaptoethanol,

and 15 mM (NH )2S04 was incubated at 150C for 30 min. The

reaction was terminated by addition of EDTA to 10 mM and heat-

ing at 65C for 15 min. The DNA was purified by chromato-

graphy through Sephacryl S-200 as described above. When

necessary, DNA was denatured by heating to 1000C for 2 min.
32P-labeled deoxynucleotides were prepared by exhaustive

digestion of [32 P] DNA with panceatic DNAse and snake venom

phosphodiesterase. A reaction mixture containing 0.46 imol

of OX[32 P DNA (2 X 104 cpm/nmol) and 25 pg of pancreatic

DNAse in 10 mM Tris-HCl, pH 7.5, and 10 mM MgC12 was incubated

at 37C. After 30 min. the mixture was brought to pH 9.0 by

addition of sodium glycinate buffer and 10 units of snake

venom phosphodiesterase were added. The reaction was termin-

ated after 60 min. by the addition of a stoichiometric amount

of EDTA followed by heating at 1000C for 10 min. All nucleic

acid concentrations are expressed as nucleotide residues.

Relaxed convalently closed 0X174 DNA (RF IV) was prepared

using partially purified Ustilago topoisomerase (Rowe et al.,


al.,







1981). A reaction mixture (0.25 ml) containing 200 nmol of

[H] 3 X174 RFI DNA, 50 mM Tris-HC1, pH 7.5, 10 mM MgCl2

and 300 units topoisomerase was incubated at 220C for 1 h.

One unit of topoisomerase is that amount necessary to relax

1 nmole of RFI DNA in 1 h. EDTA was then added to 20 mM and

the solution extracted with phenol and the DNA precipitated

with ethanol.

Assays

Nuclease Assays

The deoxyribonuclease assay measures the conversion of

DNA to an acid soluble form. Standard assay mixtures (0.1 ml)

containing 0.1 M potassium acetate, pH 6.0, and about 5 nmol

of denatured P22 [ H] DNA (1-3.5 X 104 cpm/nmol) were incubat-

ed with enzyme at 370C for 30 min. Reactions were terminated

by addition of 0.4 ml ice cold carrier salmon sperm DNA (1 mg/

ml) and 0.5 ml of 5% trichloroacetic acid. Tubes were held on

ice for 10 min., then centrifuged at 8000 rpm for 10 min. An

aliquot (0.5 ml) of supernatant was removed and counted in

5 ml Triton scintillation fluor (New England Nuclear,

Formula 950A). One unit of activity is that amount of

enzyme necessary to render 1 nmol of DNA acid soluble under

these conditions. The activity was proportional to enzyme

concentration at levels of 0.01 to 0.25 units. The extract

was diluted when necessary with buffer containing 50 mM

Tris-HCl pH 7.5, 0.1 mg/ml bovine serum albumin, 50%

glycerol.









The endonuclease assay measures the conversion of super-

helical OXRFI[3H] DNA (1-4 X 104 cpm/nmol) to an open "nicked"

form and was performed as described by Kuhnlein et al. (1976).

One unit is defined as the amount of enzyme required to cause

retention of 1 nmol of DNA (as nucleotide) on a nitrocell-

ulose filter.

A nuclease 8 digest of denatured [ H] DNA was redigested

with snake venom phosphodiesterase with or without prior

treatment with alkaline phosphatase. If the oligonucleotides

contained 3'phosphates the phosphatase treatment would make

them a more suitable substrate for the venom phosphodiesterase

(Turner and Khorana, 1959). In an experiment performed by

William Holloman, a reaction mixture (0.12 ml) containing 63

nmol sX174 [3H] DNA (2.7 X 104 cpm/nmol) in 0.1 M sodium

acetate buffer, pH 6.0, was incubated with 20 units of nuclease

8. After 30 min. at 370C, 0.1 ml of 10% trichloroacetic acid

was added and the mixture was held on ice for 10 min. After

centrifugation, the supernatant was discarded and the pellet

was washed with 1 ml of 0.02 M HC1. The precipitated DNA

was dissolved in 50 1 of 0.1 M NaOH and the solution neutra-

lized by addition of 5 1 of 0.1 M Tris-HC1, pH 8.0 and 5ul

of 1.0 M HC1. The mixture was split into two equal portions

each containing 18 nmol of digested DNA. One portion was

dephosphorylated by incubation at 650C for 10 min. with 2 ug

of bacterial alkaline phosphatase. The activities of the

treated and untreated DNA samples as substrates for snake

venom phosphodiesterase were then determined. Reaction








mixtures (1.0 ml each) containing 50 mM Tris-HCl, pH 8.8,

10 mM MgCl2, 10 mM NaC1, 1.7 pg snake venom phosphodi-

esterase, and either the dephosophorylated or untreated DNA

samples were incubated at 370C. Initial rates of digestion

were determined by removing aliquots of 0.2 ml at intervals

and measuring the amount of DNA made acid soluble. Reactions

were linear for 10 min.

5'-Nucleotidase Assay

5'-Nucleotidase activity was assayed by measuring the

conversion of 32P-labeled deoxynucleotides to a form no long-

er able to adsorb to norite. ReaCtion mixtures were the same

as described for the deoxyribonuclease assay except that 5

nmol of 32P-labeled 5'deoxynucleotides (0.5-2 X 104 cpm/nmol)

were used in lieu of DNA. Reactions were terminated by addi-

tion of 0.3 ml bovine serum albumin (2 mg/ml), 0.3 ml salmon

sperm DNA (1 mg/ml), and 0.3 ml of norite (20% w/w) in 1 M

HC1, 50 mM KH2P04. Norite was removed by centrifugation and

an aliquot (0.5 ml) of the supernatant was removed and counted

in Triton fluor.

Protein Binding to DNA

The binding of protein to DNA was observed by measuring

the retention of labeled DNA on a nitrocellulose filter

(Riggs et al.,1970). A reaction mixture (0.1 ml) containing

0.1 M potassium acetate, 1 mM EDTA, 1 nmol [ H] DNA (2 X 104

cpm/nmol), and extract was incubated at 37*C for 10 min.

The reaction was terminated with 1 ml of 5 X SSC (Standard

Saline Citrate: 0.15 M NaC1, 15 mM Na citrate)








and the solution was filtered through BA85 nitrocellulose

filters at a flow rate of 3 ml/min. The filters were rinsed

with two 1 ml aliquots of 5X SSC and dried. Radioactivity

remaining on the filters was counted in 2 ml of a toluene

fluor (New England Nuclear Econoflouor). For labeled single

stranded DNA the filters were treated with 0.5 M KOH for 15

min. prior to use. The filtration buffer used was 0.5 X SSC.

The DNA from the above reaction could be directly visual-

lized by gel electrophoresis. Identical 0.1 ml reaction mix-

tures were loaded directly to a 1.4% agarose gel containing

5 mM sodium acetate, 40 mM Tris-HC1, pH 7.9 and 1 mM EDTA.

Gels were run at 40V and 25 mamp overnight and DNA visual-

lized by staining with ethidium bromide.

Protein Determinations

The amount of protein was determined using the method of

Bradford (1976) using bovine serum albumin as a standard.

Low amounts of protein (< 0.025 mg/ml) were determined by

measuring the absorbance of 280 nm light in a Zeiss PMQ III
0.1%
spectrophotometer. Protein was calculated using e 280 = 1.0.

Gel Electrophoresis of Proteins

Electrophoresis of native enzyme was carried out with

the discontinuous buffer system described by Reisfeld et al.

(1962) in tube gels. Running gels (3 X 70 mm) containing

5.0% acrylamide, 0.2 N,N'-methylenebisacrylamide were over-

laid with a 10 mm stacking gel. A sample of 100 ul contain-

ing 10 ug cytochrome c as marker was added and a current of









3 mamp was applied until the cytrochrome c had migrated to the

bottom of the gel. Cooling was provided by circulating ice

water around the gel during the course of the run.

Isoelectric focusing of protein samples was performed

in polyacrylamide tube gels (O'Farrell, 1975). The gels (3 X

70 mm) contained 5% acrylamide, 0.15% N,N'-methylenebisacryla-

mide, 2% ampholines (Pharmalyte, pH 3-10), and 0.03%ammonium

persulfate. Protein samples (100 pl) made 1% in ampholines

were layered on top of the gel. The cathode buffer (60 mM

sulfuric acid) and the anode buffer (40 mM NaOH) were added

to the reservoirs and the samplescelectrophoresed for 15 min.

at 75 volts. Electrophoresis was continued for 30 min. at

350 volts. Two parallel gels were electrophoresed and both

were cut into 2.5 mm slices. The slices from one gel were

soaked in 0.5 ml of H20 and the pH of the eluates determined.

The slices from the other gel were eluted in 100 pl of 50 mM

Tris-HC1, pH 7.0, 0.1 mg/ml BSA, 1 mM EDTA and 10% glycerol

overnight at 40C. These eluates were assayed for single

stranded DNA hydrolysis activity.

Electrophoresis in the presence of sodium dodecyl sulfate

was done according to Laemmli (1970) in slab gels of 10%

acrylamide, 0.27% N,N'-methylenebisacrylamide. Gels were

fixed in a solution of 40% ethanol, 7% acetic acid, stained

with Coomassie brilliant blue and destined by soaking over-

night in several changes of 5% ethanol, 7% acetic acid.








Protein Purification

Purification of Nuclease B

Wild type haploid cells of Ustilago maydis al b2 were

grown as described earlier. All operations were carried out
0 o
at 0 -4 C. Frozen cells (100 g) were suspended and thawed in

200 ml of 50 mM Tris-HCl, pH 7.5, 10% glycerol and sonicated

for 1 minute in order to disperse clumped cells. The cells

were crushed at 20,000 psi by passage through a French pres-

sure cell and the broken cell suspension was centrifuged at

14,000 rpm for 30 min. in a JA 20 rotor of a Beckman J21-C

centrifuge (Fraction I). Floating lipid was removed from the

supernatant and 68 g of solid ammonium sulfate were added.

After 30 min. the solution was centrifuged at 10,000 rpm for

10 min. Additional ammonium sulfate (30 g) was added and

after 90 min. the precipitate that formed was removed by

centrifugation. The supernatant (Fraction II) was applied

directly to an Octyl-Sepharose column (50 ml bed volume) pre-

viously equilibrated with a 70% saturated ammonium sulfate

solution containing 50 mM Tris-HCl, pH 7.5, 10% gylcerol.

After the column was washed with 100 ml of equilibration

buffer,the enzyme was eluted with the same buffer but 30%

saturated with ammonium sulfate. Fractions of 7 ml were

collected and those containing activity greater than 50 units/

ml were pooled and dialyzed against three 2 L changes of 20 mM

potassium phosphate pH 7.5, 10% glycerol (Fraction III). A

column of Affi-Gel Blue agarose (20 ml bed volume) equili-

brated with the phosphate buffer was loaded with Fraction III,









washed with 100 ml of buffer, followed by a linear gradient

from 0 to 0.7 M NaCl (200 ml total). A single peak of ac-

tivity eluted halfway through the gradient. Fractions con-

taining activity greater than 50 units/ml were pooled and

dialyzedagainst 2 L of 20 mM potassium phosphate pH 7.5,

10% glycerol (Fraction IV) and loaded onto a column of

phosphocellulose (7 ml bed volume) equilibrated with the same

buffer. The column was washed with 50 ml of phosphate buffer

followed by 100 ml of a linear gradient from 0 to 0.6 M NaCl.

The enzyme eluted as a single peak of activity at 0.3 M NaCl

(Fraction V). Fraction V was applied directly to a column

of hydroxylapatite (1.5 ml bed volume) equilibrated with 20

mM potassium phosphate pH 7.5, 10% glycerol. The column

was washed with 20 ml of buffer and then 40 ml of a linear

gradient of potassium phosphate buffer pH 7.5 (0.02 to 0.75 M)

containing 10% glycerol. Fractions (1.5 ml) were collected

into polyallomer tubes to minimize loss due to adsorption to

glass. Activity emerged as a single peak at 0.3 M potassium

phosphate. The peak fractions were pooled and dialyzed

against 20 mM Tris-HCl pH 7.5, 50% glycerol (Fraction VI).

Purification of DNA Binding Protein III

Ustilago maydis cell (100g)strain al b2 were suspended in

buffer A (20 mM Tris-HCl, pH 7.5, 1 mM EDTA) that contained

1 M NaCl. Cells were broken by passage through a French

pressure cell at 20,000 psi. Cell debris was removed by

centrifugation and the resulting supernatant of 210 ml

(Fraction I) was made 9% in polyethylene glycol (PEG 6000).

After 15 min. on ice the precipitate that formed was removed










and the supernatant dialyzed versus three 2 L changes of

buffer A. The dialyzed solution of 400 ml (Fraction II) was

applied to a 200 ml column of DEAE-cellulose equilibrated

with buffer A. Two column volumes of buffer A were washed

through the column followed by a linear 2 L gradient of 0-0.5

M NaCI in buffer A. DNA binding activity eluted from 50 to

150 mM NaCI and these fractions were pooled. The pooled

activity was dialyzed versus 2 L of buffer A yielding a volume

of 335 ml (Fraction III). A 32 ml phosphocellulose column

equilibrated in buffer A was used to further purify the DNA

binding activity. After passing the extract through the phos-

phocellulose the column was washed with 100 ml of buffer A

and eluted with a 300 ml 0-0.4 M NaCI gradient containing buf-

fer A. A single peak of activity emerged between 100 and 150

mM NaCl. Fractions containing activity were pooled yielding

a volume of 70 ml (Fraction IV). Half of the phosphocellulose

pool was concentrated ten-fold by bringing the solution to

60% saturation in ammonium sulfate and collecting the result-

ing precipitate. This pellet was resuspended in 3.4 ml of

buffer A containing 20% glycerol and applied to a Sephacryl

S-300 column. The column of 290 ml had an exclusion volume

of 117 ml as determined using blue dextran. Cytochrome c

eluted after 270 ml were collected. Three milliliter fractions

were collected and the activity found to elute in the excluded

volume. The three peak fractions were pooled and stored

(Fraction V). Binding activity in this fraction was stable
for at least one month when stored at 4C.
for at least one month when stored at 4 C.








Materials

Chromatographic Media and Chemicals

Affi-Gel Blue, hydroxylapatite agarose and Dowex-1

(X8) were purchased from Bio-Rad Laboratories. Octyl-

Sepharose, Pharmalytes (pH 3-10), Sephacryl S-200 and S-300

were from Pharmacia Fine Chemicals; DEAE-cellulose and

phosphocellulose were from Whatman. Ethidium Bromide,

activated charcoal and sodium dodecyl sulfate (SDS) were

from Sigma. Sarkosyl NL 30 was from ICN Parmaceuticals.

Scintillation fluors Econofluor and Formula 950-A were from

New England Nuclear. Nictrocelluiose filters type BA85 were

purchased from Schleicher & Schuell.

Enzymes and Radioisotopes

Pancreatic DNAse, bacterial alkaline phosphatase and

snake venom phospodiesterase were purchased from Worthington

Biochemicals Inc. Nuclease Pl from Penicillium citrinum was

purchased from Calbiochem-Behring Corp; Snake venom 5'-

nucleotidase, bovine serum albumin, lysozyme, cytochrome c and

the synthetic polynucleotides of dT and dG were from Sigma

Chemical Co. Restriction endonuclease Pst 1 and polynucleo-

tide kinase were from Bethesda Research Laboratories. Poly

[dA] was obtained from Miles. T4 DNA polymerase was prepared

as described by (Nossal and Herschfield, 1971). Ustilago

topoisomerase (hydroxylapatite fraction; Rowe et al., 1981)

was gift a from Tom Rowe and Michael Brougham; [3H]
32
thymidine was from Schwarz Mann; [ 32 orthophosphate,
32 32
[y- P] ATP and [c- P] dGTP were from New England Nuclear.















CHAPTER III
PURIFICATION AND CHARACTERIZATION OF NUCLEASE 0


Nucleases have been implicated in recombinational pro-

cesses in both procaryotic and eucaryotic cells (see Chapter

I). In vitro characterization of nucleases can often explain

observations of normal or mutant strains. For example, a

recBC mutant of E. coli (recB270, recC271) demonstrates a

reduced recombination frequency at $30C. Purification of the

recBC coded nuclease from this mutant revealed that only the

ATP-dependent exonuclease activity was thermolabile (Kushner,

1974). Protein purification and characterization made possible

the correlation of a specific phenotype with a defined en-

zymatic activity.

This project was aimed at isolating and characterizing a

nuclease from Ustilago maydis. A nuclease active on single

stranded DNA has previously been isolated and characterized

from U. maydis (Holloman and Holliday, 1973). The nuclease,

termed nuclease a, shows increased activity upon duplex DNA

substrates containing structural distortions (Holloman et al.,

1981). Ustilago mutants deficient in spontaneous or damage

induced allelic recombination (gene conversion) were found to

have reduced amounts of nuclease activity (Badman, 1972).

In particular, these mutant cells had reduced levels of

nulease a (Holloman and Holliday, 1973).








Nuclease activity was examined in extracts of U. maydis

that had been fractionated by chromatography on DNA-cellulose.

A single strand specific nuclease activity was observed that

had different characteristics than the previously isolated

nuclease a. This chapter describes the purification and

characterization of this enzyme, termed nuclease 8.


Purification

Fractionation of Nuclease B

The assay used to purify nuclease activity measures the

conversion of [3H] DNA to a form soluble in dilute acid (see

Chapter II). Nuclease B was purified by the fractionation

procedures outlined in table I. A detailed description of

enzyme isolation is given in Chapter II. Most proteins (90%)

present in crushed cell extract could be precipitated by

bringing the solution to 70% saturation in ammonium sulphate.

Nuclease activity remaining in the supernatant chromatographed

as a single peak of activity on all the subsequent steps in

purification. Ammonium sulphate fractionation of crude ex-

tracts would allow rapid screening of mutant strains for

nuclease B. The highly concentrated salt solution containing

nuclease activity was applied directly to an Octyl-Sepharose

column. Nuclease was eluted by washingthe columnwith buffer

containing 30% ammonium sulphate. The eluate was dialyzed to

remove the salt and applied to an Affigel-Blue agarose column.

Enzyme activity was eluted with a NaC1 gradient and the frac-

tions containing nuclease were pooled and dialyzed. Nuclease

8 was furhter purified by chromatography on phosphocellulose















dP 0 0 M C0 r-- 0

r-H Hl



H T H H C 0 0
H c00 0 0
0 CN

CN H
r-A


-40
It





P40






*H -




P40



a)

-I


0
O
4-1



-H
04














O
0 >


























H
-H
a )
















u
r1
U







-4

5-'


Cp o







1 0

-rl

Ln


r-- Li 0
o w to
N CN



0a
4-,
4'





-4
5
-H
)






HH
Hr


co o
, (,


o 0
0 0


CO CN
r (N


co m r-i
Ln o u 4


a)

m -
0 a)




0 a4
0H 3
>9 -4

O


H >
H H
H


> H


00

r-4 r-- M r- Co
4J r r-4Cm 0
4-0
-rl LO
a:

4-4



o- E
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040
.4 -4
4-1H


uoa







( &
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-H 4-1






(
a)

rc a











*H M

0 +
V*i





















H
S-4



0












Ha)O
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rdtn
12,




















H
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Sa,
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and hydroxylapatite. This fractionation procedure purified

nuclease 8 to a specific activity of 142,000 units/mg pro-

tein. The enzyme has been stable for 2 years at -200C.

Homogeneity

Fraction VI represents an 11,200-fold increase in spe-

cific activity compared to crude extracts. Electrophoresis

of purified nuclease in acrylamide gels under nondenaturing

conditions revealed two major protein bands after the gel

was stained with Coomassie blue (see Chapter II). The

slower migrating band, which accounted for approximately two-

thirds of the stain, comigrated with nuclease activity (fig-

ure 1). Further chromatography of fraction VI upon DNA-cell-

ulose, carboxymethyl cellulose or QAE-Sephadex did not separate

the two proteins present in this fraction. Storage of fraction

VI for 6 months caused a change in the relative amounts of

the two bands visible after native gel electrophoresis. The

faster migrating band now contained about 60% of the Coomassie

stain. Perhaps the contaminant observed by native gel elec-

trophoresis arises from proteolytic degradation of the band

containing nuclease activity.

Molecular Size and Isoelectric Point

The band from native gel electrophoresis that co-migrated

with nuclease activity was electrophoresed under reducing

conditions in a SDS-gel (see Chapter II). The protein migrat-

ed with a molecular weight of 68,000 when compared with stand-

ard proteins electrophoresed in parallel (figure 2). Purified

nuclease 8 had a sedimentation coefficient of 4.3 S (figure 3)























4 o
-o OM 0



a) Q) i-l o i 0
0 0 0 H ,.
rd O 0 r c
( ,0 0 U U ( 4-4
0 4d Ln O o r 4O t
0 rd r 0 0 U


0 Z 4J Q P .
I -) 3 ) U F *H-Z
H H .4 1 -m4 -) *H


a, uc a a c a a) 0
S00 41 ( 0 Q4
to H C 0 r ) r*


0 )I Hfl O *H .Q
ri ( p r-Sl p Ua 43 r-i (
r4 *H 4 (1 0 m
mm < 0 -0 0 4 04

0 H 0 0Ei < -rl 0
H0 C) )O ( 0

.4 r0 4- dI
a., 0;o 3
0) 0 1 > i *P i


4J mk. pp .-n rd
. 4-0 O EA +4- 04r-O
30 ,- 0H ) 4 C
r) l 0 H ) -H .4
d H 0d *ro > 0)






H Q 0 r-l 0 4-C d
Uf -4 )H 0 0
0 H3(0)0





p W-i 4-a) 0-i X u I
0 w QM 0 U Q) (U
OuWN EOMCuo
04002 020Q0 0








C f -rC d 0 4
C : 4 U O w U
0 0 >m u I Q 0
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4J 0) 0 0-HU
a, 0 03 q 01 Q (3 > i
000) p Q) 0q
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ijs/s!un) A,!AipOV

x SDo|lonuopu3
o o
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pa@ojaqn bI


to sap!ioalonN


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Figure 2

SDS-gel electrophoresis of nuclease 8. Fraction VI
was subjected to discontinuous gel electrophoresis
and the gel was sliced as described in the legend to
fig. 1. The slices corresponding to those containing
nuclease 8 activity as determined in a parallel gel
were eluted in 50 mM Tris-HC1 pH 6.8, 5% 2-mercapto-
ethanol, 2% SDS, 10% glycerol, the eluates pooled and
boiled for 2 min.and subjected to electrophoresis as
described in Chapter II. Parallel slots contained
the following protein standards: phosphorylase b,
bovine serum albumin, ovalbumin, chymotrypsinogen,
soybean trypsin inhibitor. The arrow indicates where
the nuclease 8 protein band migrated. Mobilities are
relative to a bromphenol blue tracking dye.















100

80

60


40




20





.2 .4
R
f


I I !


6 .8 1.0
























4-4 Ord
0 "
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0 0 4- 0 .
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o 7 ao r o T*
0 rO (U 4 1 C

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O o 0 O 0 CJ
*, 9 L* O4 -Q 0



0 d rd- Q) -l *H 0
4H 02 4-C 4 0 r0
0 4 04-4Q r U-
< >1 r- u0 4
r13i IH' m *d c
:1 : 1 -i f a a o
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So -r-4 r-lO 0
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4->1 >r-l r4 U (1c
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-1: 1 *-1 ; r-l ( C *
4-4 4 CO *4J 0 1 4-J
-3 CN o U H4-4 4-4
P C4 g n a)
4-) 73 -) -H c -i
*4 .0 a) O0
U4J d rnr- Q) d 4J
CD *H OHO 0 ,m


-i
a) L { *n o k c O
4 0 a o) 0
4J rd Ln r-( H M 41













(Uo0!1DJ/Sl!un) AI!A!J3V

zpi x aSDalpnuopu3


o0 0
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paIDJaq!-l


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(uo! jDJt/sl!un) A!^!AIOV








as determined by centrifugation through glycerol gradients

(Martin and Ames, 1961). The Stokes radius of the native

enzyme was determined by gel filtration (Siegal and Monty,

1966) to be 36A (figure 4). An examination of the size,

shape and electrophoretic properties of nuclease 8 suggests

the enzyme exists in solution as a globular polypeptide of

68,000 daltons. An isoelectric point of pH 5.7 (figure 5)

was determined by electrophoresis of nuclease 8 in acryl-

amide gels containing ampholines (O'Farrell, 1975).

Reaction Conditions

Optimum Conditions for Hydrolysis of DNA

Nuclease 8 was highly active on single stranded DNA.

Denatured DNA was hydrolyzed about 1,000 times faster than

native duplex DNA (figure 6). The optimum pH for nuclease

activity in acetate, imidazole, or 2(N-morpholino) ethane sul-

fonate buffers was 6.0 (figure 7). Nuclease 8 was most ac-

tive in sodium or potassium acetate. Enzyme activity in ace-

tate buffer at pH 5.0 or sodium piperazine-N,N'-bis[2 ethane

sulfonic acid] buffer at pH 7.0 was only half the amount

observed at pH 6.0. The enzyme was not active in Tris-HCl

buffer at pH 7.0.

Nuclease 8 was active over a wide range of temperature

(figure 8A) with maximal activity at 420C. DNA hydrolysis

by the enzyme was optimal at an ionic strength of 0.2 M po-

tassium acetate (figure 8B). The concentration of the mono-

valent cation appeared to be the critical component. The

amount of nuclease activity in 0.2 M potassium acetate was

identical to that observed in 0.05 M potassium acetate that






























Figure 4

Determination of Stokes radius for nuclease 8. A
column of Sephacryl S-200 (2.5 X 60 cm) was equilibrated
with 50 mM potassium phosphate pH 7.5, 0.2 M KC1, 10%
glycerol. The excluded volume of 124 ml and the included
volume of 287 ml were determined with the use of 3H-
labeled ZX174 phage and 32P04-3. Proteins used to
calibrate the column included catalase, bovine serum al-
bumin, ovalbumin, pancreatic deoxyribonclease, and cyto-
chrome c. Approximately 200 units of nuclease 8 were
loaded on the column. Recovery was 5%.












0
0.8- \
0




0.6










2 I!
0.4 o





0.2
20 30 40 50

Stokes radius (A)

























H )
> 6 ft
o *r *
0 n


(0 ) a)-4 r t* -I H
0 4 U2 a)
-i rO CN 4 r
0 w Z ra W Z
4C4 0 4 a Q 4
4.0 0O 0() U
OO > 44 4O
p -, to





0 01 0
in rH C -.
SU 0) a -,4
a 0 r-4 r U
S-0 )r-1 0
p r- 4 0 (o
H U P) (1 Z *r

O+1 *rO 0
(U 6> >1
4-01 )H 0 4
00 00 a*.-I
0' g -i (U) 0) -H
Z ra Q) o'0 4- 4
*H r- 4J 4-4m
U2 >1 044-4 U) 3 (0


4 0 )-HM a
0.-H4-) a)
4-1 C -ra z

U 0 0

0) 0 oJ 4 ) C;
U) () 0 a
HO -d U 0- 0)
H (am n C













Hd


0 10 0
OC u
2 l1 x wd IAIV


c-
0


LL
a_



























r-


U 3 r-*H -
l4 CO-
0 U0 0 0


a)*-I4 0
r -l 0 (Ts
U) O) 0 0I H
() O A -HO)
.-4 ()C -C a)
0Z 0 4 c0 rW Z



-44
0 04 u -H
o U z -P
> O a o -H



0 Q N r-l P 44-
tp (1) c3 0
-1 4-1 &PX 0
$- 0 Z *iW -)
-H H Lo&

-H *H (N *- HO
En i i


M 4 U rcl
S-4 4-) 4
o0 Z a 4 0


C:) (0 a
u) > E
Em M 0 4C


tR J W6 (D 0*
0u m w (
o4 C




W 4J> CO Z W
E X 4J > 0
-r*H *r H -H 4- (0
E-1 g 0 ) 0 2 3






































0
6
u)
(S|ol0Uu) 9lqnloS


0
ro)








CM
0



c'



a,
o E
-I


VNa


0
!o
in

PKPV



























Figure 7 -

Effect of pH on nuclease 0 activity. Reaction mixtures
(0.1 ml) contained 3 nmol of denatured P22[3H] DNA (2 X
104 cpm/nmol) and 50 mM of the following buffer at the
pH indicated. (A) K acetate; (X) Na citrate; (0) 2
(N-morpholino) ethane sulfonate; (o) sodium piperazine-
N,N'-bis [2 ethane sulfonic acid] (A) imidazole-HCl.
Reactions were incubated for 10 min.at 370C with 2 units
of fraction VI and nuclease activity was measured as
described in Chapter II. Activity at pH 6.0 in acetate
buffer was taken as 100%.














I00- a

S80
0 60 Q
E 60- o

0 /0
r 40 -

o 20-

I I I AA I
4.0 5.0 6.0 7.0 8.0

pH






























Figure 8

Effect of temperature and ionic strength on nuclease 8
activity. A. Reaction mixtures (0.1 ml) containing
2 nmol denatured P22[3H] DNA (2 X 104 cpm/nmol) and
0.1 M potassium acetate were equilibrated at the
temperature indicated. Three units of fraction VI
nuclease were added and after 10 min. the amount of
acid soluble DNA was determined, B. Reaction
mixtures (0.1 ml) containing 2 nmol of denatured P22
[3H] DNA and the indicated concentration of potassium
acetate buffer were incubated for 10 min. at 370Cwith
3 units of fraction VI nuclease. Acid soluble DNA
was determined as described previously.








-5
0

(I)
cn
a)
0
E
C


40


Temperature


oc


60


0.I


KAc (pH 6.0) Molarity


A





I I


0.4


0.2


20


I I I


0.6


0.4


0.2


a)

0
U)
a
0

C)
0
E
C:


a.


0.5


I _


I


I








contained 0.15M sodium orpotassium chloride. Hydrolysis of

DNA by nuclease 8 was not affected by divalent cations
++ ++ ++ ++
(eg. Mg Ca Co Mn ) and was not inhibited by the

chelating reagent ethylenediamine tetra-acetic acid (EDTA).

Sulfhydryl reducing agents such as 8-mercaptoethanol and

dithiothreitol did not alter enzyme activity. N-ethylmal-

eimide, a sulfhydryl blocking reagent, did not affect nuclease

8 activity.

Inhibitors of Nuclease 8

Nucleotides are potent inhibitors of nuclease 8. ATP or

dATP at 1 uM reduced nuclease activity 50% (figure 9). Phos-

phate and pyrophosphate were less effective inhibitors than

the nucleoside deoxyadenosine. S 1 nuclease from Aspergillus

oryzae is also strongly inhibited by nucleoside triphosphates

(Wiegand et al., 1975). Unlike nuclease B, S 1 nuclease is

more sensitive to phosphate than to deoxyadenosine.

Mode of Hydrolysis

Exonuclease Activity

Does nuclease 8 initiate hydrolysis from the ends of

DNA molecules? Linear, uniformly labeled[ H] DNA was label-
32
ed at the 5' or 3' end with 32P (see Chapter II for prepara-
32
tion of substrate). A selective release of the 32P-label to

an acid soluble form would indicate terminal directed hydro-

lysis. When [ H] DNA labeled at the 5'-end with 32P was the
32
the substrate for nuclease B the terminal 32P became acid sol-

uble faster than the 3H label (figure 10A). When the nuclease




























Figure 9

Effect of small molecules on nuclease 8 activity. Re-
action mixtures (0.1 ml) containing 0.1 M potassium ace-
tate, 2 nmole of denatured [3H] P22 DNA and the indicat-
ed concentration of inhibitor were incubated for 10 min.
at 370C. The amount of DNA acid soluble was determined
as described in Chapter II. (o) ATP or dATP; (e) AMP;
(A) deoxyadenosine; (A) phosphate; (m) pyrophosphate.




49




100 -

A




o




E20 0 ,
480-








e A
4 20-



t ( o A

10-6 10-4 10-2

Concentration (Molar)


























Figure 10

The action of nuclease a on terminally labelled DNA.
A. A reaction mixture (1.0 ml) containing 16 nmol heat
denatured $XRFIII[5'-32p 3H] DNA (3.8 X 103 cpm 32p-
label per nmol, 1.7 X 104 cpm 3H-label per nmol) in
0.1 M potassium acetate pH 6.0 was incubated with 10
units of nuclease 0. At the indicated times aliquots
(0.1 ml) were removed and the amount of label made acid
soluble was determined. B. A reaction mixture (0.6 ml)
containing 36 nmol heatadenatured zXRFIII[3'-32p, 3H]
DNA (1.2 X 102 cpm 32P-label per nmol, 4.2 X 104 cpm
3H-label per nmol) was incubated with 60 units of
nuclease a and 0.1 ml aliquots processed as above.
(0, 3H), (e, P).





















50


a 25-




1 75-
a B

50 / [3'-3" 'H DNA


25 /


O I I I 1
10 20 30 40 50
Time (min)







substrate was [3H] DNA 32P-labeled at the 3'-end, both 32p

H became acid soluble at the same rate (figure 10B). Pref-

erential release of a terminal label suggests that nuclease 8

can hydrolyze DNA in an exonucleolytic fashion. This terminal

hydrolysis is directed from the 5'-end of the polynucleotide.

Distributive Mode of Hydrolysis

I wanted to determine if nuclease 8 hydrolyzed a DNA

molecule processively or if the enzyme dissociated from the

substrate after each nucleolytic cleavage. Nuclease 6 was

incubated with denatured PX174 [ H] DNA at a 20-fold molar

excess of DNA molecules to enzyme molecules. The moles of

nuclease were determined by using a molecular weight of

68,000 and assuming the preparation was 70% pure nuclease.

After 10 minutes an equal amount of unlabeled DNA was added

to the reaction mixture (figure 11). Immediately the rate

at which [ H] DNA became acid soluble decreased 2-fold which

was the same rate seen if the unlabeled DNA was present from

the start of the reaction. Therefore nuclease B appears to

hydrolyze DNA in a random or distributive fashion.

Activity on Circular DNA

Linear DNA appears to be hydrolyzed by nuclease 8 in an

exonucleolytic manner. Does nuclease 8 require a free end

to hydrolyze DNA? The enzyme was incubated with >X174 phage

[3H] DNA (a single stranded circular molecule) and the reac-

tion products were sedimented through an alkaline sucrose

gradient (figure 12A). The DNA sedimented more slowly after

enzyme treatment suggesting extensive digestion of the circular




















M-k -H + r-
0 d


4-1 t I .Q 0 0
X0 ~~~) a),- (d
*.- ( 4 3 r H -4
4J ) >iu ) 1 0 4JI
0(3 0 3 0 c! t i :3
H ,~ C0 om 0 44

0l U O - rC a)0 0)4-Z
0 H 4 4 (0 0 0
) 4 u) 0 r OO



4 40 -C M 0 0C
n a) -o E -(
0* 0) 0 0. 2--


H f- 0 0 4J 4
O H 4- 4. 1 l '.

S r-,-I O .. -
: 3 44- 4 >i (-H ()






SH --0 0 o) E>


M W W -,. 4J C> 4J>
> i0- O 0 3
H 0 -HO 0H O0



A -- 0) -O )

a ---4 m4

C. 'O O 0 0 o m
14) 0 0 -I 0 *0
,4 t I4- 4 -o a -

S0 z1 w zC O a)
O l G 4 4O '3l O
-r ( d l- En r 41 0)









> 0 0 m C --4 C:
4C- !:JO0 Hl mn r




Z ZnW 0* H (U3 0



Sr-i ) 0 M-C4 Q a-0a
N C 3 ( 4 O Z 0 r0
r-q Z 4(1 z m r-I a)




O *q F 0 M O ( 4
-1 r-i -4 0C 0 Co >
4) 4J 1 4 -1 VO C)
q Z 4 41 z m r- a)











0



c'J

O -
(\


E


(salouu) alqnlos


VNaG [HJ


P!OV

























Eo o Q) E
BO 0 -
4J r u) M Z 0
ScEmm Q) 4O
) -H 4 >1 M 4



(1)4 o
O m n o a un O

O- oH o or- C
S-4 *H a -r a4
O 0 4- 4 O- H
S I-r-l 4- 3 4-I
() 4J 0 4 J- -,
H X X *ri
tN r- -4 'a -r- 4 r,

MQ. 4 r-A Q) m
*N H H-HI 0
r-4 ,Z CJ2 I) e E- r0 *
(U C) r0 (v g r Zw (0
X z04 o 4-
> ux WaOn +
a )' U 0
H ) H q a) 9- o

C O1044 (o c)
0r-r I ID 04 4 a) HLn
-r-I 0gi U )

J- 0 C) N ) 0 51
Of -r r-4 Z 0 o



OJ C-I 4 -
0 0 Y (U
a -, 4- J
z-r1 0 < E = C rd
o uj 0- 0 -H 4-1
M0 -, a a040 0
(1 Q) 04 o E-0 e -i
Nr ) aC oZ 4
(V 4 rO r-l (0 >4 0
S0 a) 0 C0)4


c -l X *_Q (M Q.-
n i0 0- O u4















(o)(.OI x Uwd3) VNG [d,]

wC) d Io) VNG


o0 t 0 to


(--)(_Ol x wdo) VNG [H,]


Z-

C
O 0
4-

LL








substrate. This experiment suggests nuclease $ can cleave

DNA in an endonucleolytic manner.

Nuclease B can also nick form I DNA. Endonuclease

activity was followed using an assay that measures the con-

version of covalently closed, circular duplex DNA to a nicked

form which can be selectively denatured at pH 12.3 and then

retained on nitrocellulose filters (Kuhnlein et al., 1976).

A comparison was made of the rate that nuclease 8 cleaves

single or double strand DNA substrates. The endonuclease

assay measured cleavage of form I DNA. Cleavage of single

stranded [ H] DNA was monitored by.measuring the amount of
32
P susceptible to bacterial alkaline phosphatase (Kushner

and Grossman, 1971). Nuclease 8 cleaves single stranded DNA

1,200-fold faster than form I DNA (table II). I believe

both exonucleolytic and endonucleolytic hydrolysis are cat-

alyzed by the same enzyme because these activities coelectro-

phoresed (figure 1) and cosedimented (figure 3).

Products of Hydrolysis

Size of Nucleotide Products

Denatured [ H] DNA was made totally acid soluble by

nuclease 8. The products of hydrolysis were analyzed by

chromotography on DEAE-cellulose in the presence of 7 M urea

(Tomlinson and Tener, 1963). About 95% of the [3H] label

eluted at the size of mono- or dinucleotides (figure 13).

When only 5% of the input [ H] DNA was acid soluble 30% of

the product was mononucleotide. Short oligonucleotides

(chain length 2-7) contained between 5 and 15% of the acid

soluble radioactivity. The formation of mononucleotide

















TABLE II. Reaction Rates of Nuclease 8


DNA Average chain % RF bound Bonds
length to filters broken


pmol
Single stranded 8.7 180
Superhelical 26.7 0.15


A reaction mixture of 0.1 ml containing 1.6 nmol of zX174
RFI[3H] DNA (4.5 X 103 cpm/nmol) was incubated at 370C with
1 unit of nuclease a. After 10 min, the number of molecules
nicked was determined as describe in Chapter II. In an
identical reaction, 1.6 nmol of single-stranded OX174[3H] DNA
(7.6 X 104 -cpm/nmol) were substituted for the RFI DNA. After
incubation for 10 min, the reaction was stopped by raising
the pH to 8.0 with Tris-HCl and MgC12 was added to 10 mM.
Alkaline phsophatase (0.25 unit) was added and the solution
incubated at 450C for an additional 10 min. Radioactivity
unable to adsorb to norite was determined as described in
Chapter II. The reciprocal of the fraction of 32P label not
adsorbing to norite was taken as the average chain length.























(I ri
-O(d rd 0 ,
0 Q) O 90 4- *4
N | 0 ) -*H a)
CN C $ 4
c4 $4 r-W-HU)
PA r4a) a >i 01 44
0 QE-Q ( O 0 04
(U r 4+J 44
I-dP EC a) M3
C4-0 C) 0 a) 0( e
(a r r-4 l '
C 0) O0
Q a)0 M4 0 4 4C *H
Q) r- 0 0
*O OLO n u E
Cd u 0 ( *r
0 C r- D) ,C r-4
H (U a) 4k E
ll 44- U X E U
U 0 0 104 ra to
4 CD r-4Z *

-4 r-4i E-4 0
Hm Z ) i r $


0 0g 0 O 0C
t) E Z 0 W 0 r k.0
S O4 un Mw rd4-)
0 00Qgc 4 04C)
O E o<(fl
0 0- o r-
0Q40 OrQ0r-4OWOO0
U < 4J a 1 0
al) O m 4-1 oJ.Q
4)OC H a H +J C H
r-1 *r P z (U 0
4-4 r-I a 0 00
OX 0 4 r- 2 -H Q

S *) 4- r4o T F
SCN 0 + 4 -4J e -q c 6
*H4O) otol*
4 --I (1 ,Q (0
r 4)J 4) 3 -r-H 4-4 4 o
C td(0 Hd 0 fo >O
-r4 Q ) --H) 4 0) 4-14)

r r-4 o 4 o r- 0 -



IQ 4 0O
a) r- 0 O 0 U-H
N 4 a) o-i 0 4JU-)n M ra
-rl Z r--i 0 o -O *H m
(n )Q > 4 3 (d 4 $4
















(o--o)uWuo09Z ) aOUDqjosqV


(*-.) (,.-o x Jwdo)


[H_]


sap!loalonN








residues as the major product of hydrolysis suggests nuclease

8 cleaves a single stranded substrate exonucleolytically.

Phosphoryl Ester Species of Digestion Products

Mononucleotides produced by nuclease might contain

either 5' or 3' phosphates. Two methods were used to analyze

32
the mononucleotide products of digestion. [32 P] DNA was made

87% acid soluble by nuclease B. The products of digestion

were treated with snake venom 5'-Nucleotidase, alkaline

phosphatase, or nuclease P1 (an enzyme with 3'-phosphomono-

esterase activity). Phosphatase activity was measured by
32
determining the amount of 32P-label-not adsorbed to norite
32
(table III). Noritenonadsorbable 32P was formed by alkaline

phosphatase and nuclease Pl but not by treatment with 5'-

Nucleotidase. This indicates that nuclease 8 produces nucleo-

side 3'-monophosphates. Another way to analyze the phosphoryl

ester species of mononucleotides is by chromatography on a

Dowex column (Cohn and Volkin, 1951). This column can chro-

matographically separate 3' and 5' isomers of mononucleotides.

In an experiment performed in collaboration with Dr. William

Holloman, [32 P mononucleotides produced by nuclease 8 co-

chromatographed with nucleoside 3'-monophosphates included

as standards (figure 14). Although the isomers of dTMP and

dGMP used as standards did not totally separate it is clear
32
the 32P-label is skewed toward the trailing edge of the A260

peaks as would be expected for the 3' isomer.

The experiments described above show that mononucleotides

produced by nuclease 8 contained 3' phosphates. Do
















TABLE III. Mononucleotide Isomers
of Digestion Products


nmol
[32p]-
Initial digest Second digest norite
nonad-
sorbable

Nuclease 8 (87% acid soluble) 5'-Nucleotidase 0.019
Alkaline phosphatase 1.19
Nuclease P1 3.04
None 0.06


A solution (0.4 ml) containing 50 mM potassium: acetate, pH
5.5, and 18.4 nmol of ZXl74[32P] DNA (3.2 X 104 cmp/nmol) was
incubated at 370C with 40 units of nuclease 8. After 30 min,
the solution was made 10 mM in MgCl2. Aliquots of 0.1 ml
removed for further digestion with 5'-nucleotidase, alkaline
phosphatase, or nuclease Pl were treated as follows: (a)
20 pl of 1.0 M sodium glycine, pH 9.0 and 1 unit of 5'-
nucleotidase; (b) 20 ul of 1.0 M Tris-HCl, pH 8.0, and 0.05
unit of alkaline phosphatase; and (c) 10 units of nuclease
PI. Reaction mixutres were incubated for 20 min.at 37C and
32p label unable to adsorb to norite was determined (see
Chapter II).






















Q)
0)
Ord
0 (0 NT



M 4-14 00 E
U)* 0 4 -I (
H-r En 0)0 0 4O

I n -*r4 E-0 -H M

& n*H OH O M
Q1- QO l C t
o 0 0 ) (a4
r4i 4:c3 0 0





0 1 Z > *H
*M tn a 0 0 -
0l rin, (u U-4 -q




H Or 44 -JLm A5
r O O 4l 03



r n 0 *4H 4m J cr-L *A



-- 44 4 0 r C)O-4
o 0 -> r a)


g Or- Oc 0 4 *0 4


I o l ,3 >-o
0 0 -0 34 4->

J 44-1 0 5 M Z -H *
fo Mn 00 C
0 ro N S a-H
1 -4 4u 04
gn r- 0 ( 0 d>i



0 r-4 ) 4 3 0 4
1 (t i o C) O *-rH S*


4 .r- r (dO '
us* 3 0 0 1 O 0

0 ( %Jr-l Q) c
r4 -r >iZ 0 03 f r 0-4
,C:Q >1 4J Q U a)4J
04 C0 0 : *H4J fa
Mi E r-4-i4a rOU-H
4 (3 a >.ir m 0 0 (a r-i
MN 0 4- X0 *O-d r-


r= 4: r j C) C.)
O& r-I < O ai 0

01r- 04 04 4J qT 44 )u
4 E X r-4- 44 .4

,C X O -il u a)
UQ U a 4-1 X ,4 3: Q


J 0 q 3 *+ a
,Cn 0 0P X 0 *d -a





U`Q. U mid E- X A 3 rQ





64






(o--o) (wdo) AIH!^IODO!PDo


W o
o o
(,-_.) "'uOSZ


0
D aouoqjosqv








oligonucleotides produced by nuclease B contain 3' phosphates

and 5' hydroxyls at the chain termini? In collaboration

with Dr. William Holloman, acid precipitable oligonucleotides

produced by nuclease 6 were examined as substrates for snake

venom phosphodiesterase (see Chapter II). The snake venom

enzyme is a 3'-directed exonuclease that is more active upon

oligonucleotides bearing 3' hydroxyls (Turner and Khorana,

1959). As seen in table IV, prior treatment of nuclease 8

generated oligonucleotides with alkaline phosphatase increased

the activity of snake venom phosphodiesterase ten-fold. This

experiment suggests the oligonuclsotides produced by nuclease

8 are terminated by 3' phosphates.

Other Activities

5'-Nucleotidase Activity

The most purified preparations of nuclease S could remove
32
a phosphate from 5' mononucleotides. 32P-labeled mononucleo-
32
tides prepared by digestion of [ P] DNA with nuclease P1

was a substrate for nuclease B preparations and snake venom

5'-nucleotidase (table V). Routine 5' nucleotidase assays

were performed using mononucleotides generated by sequential
32
digestion of [ P] DNA with pancreatic DNAse followed by

snake venom phosphodiesterase (see Chapter II). The 5' nucleo-

tidase activity coelectrophoresed (figure 1) and cosedimented

(figure 3) with the activity hydrolyzing single stranded DNA.

The reaction optima (eg. pH, ionic strength and activity in

the presence of EDTA) for nucleotidase activity were identical

to those for hydrolysis of denatured DNA. Nuclease 6 does not





















TABLE IV. Activity of Snake Venom Phosphodiesterase
on DNA Partially Digested by Nuclease 5



Nuclease 8 DNA digest Rate of hydrolysis by
phosphodiesterase


nmol/min
Untreated 0.095
Dephosphorylated 1.04


ZX174 [3H] DNA was made 23% acid soluble by treatment with
nuclease B. The acid precipitable DNA was recovered and
half the DNA was treated with alkaline phosphatase.
Subsequent digestion with phosphodiesterase was performed
as described in Chapter II.

















TABLE V. 5'-Nucleotidase Activity of Nuclease a


nmol
[32p]-
Initial digest Second digest norite
nonad-
sorbable


Nuclease Pl (97% acid soluble) 5'vNucleotidase 3.44
Alakaline phasphatase 3.58
Nuclease 8 2.72
None 0.12


A 0.4-ml solution containing 50 mM potasssium acetate, pH
5.5, 10 mM MgCl2 and 18.4 nmol of [32p] DNA (3.2 X 104 cpm/
nmol) was incubated for 30 min with 40 units of nuclease Pl.
Aliquots of 0.1 ml removed for further digestion with
5'-nucleotidase, alkaline phosphatase, or nuclease 8 were
treated as follows: (a) 20 pl of 1.0 M sodium glycine, pH
9.0, and 1 unit of 5'-nucleotidase; (b) 20 pl of 1.0 M Tris-
HC1, pH 8.0, and 0.05 unit of alkaline phosphatase; and (c)
10 units of nuclease 8. These aliquots were incubated for
20 min at 370C and 32P label unable to adsorb to norite
was determined.









produce free phosphate from p-nitrophenol phosphate, a syn-

thetic compound that is a substate for alkaline phosphatase.

I could not detect any ATP hydrolysis (less than 5 pmol)

when incubating [y-32P]ATP with enough nuclease 8 to hydro-

lyze 25 nmol of denatured DNA.

I suggested earlier that nuclease 8 hydrolyzed linear DNA

in a 5'-directed manner because 32P-label was preferentially

released from [3H] DNA labeled at its 5'-end with [32P]PO4.

Perhaps nuclease a is not an exonuclease and the release of 32P

was an artifact of 5' nucleotidase activity. To distinguish

between these possibilities 5'-[32P] DNA was digested with

nuclease 8 and the amount of label made acid soluble or norite
32
nonadsorbable was determined (figure 15). 32P-label was made

acid soluble at a higher rate than the formation of free 32P

suggesting that nuclease B is indeed a 5'-directed exonuclease.

Activity on RNA

Hela cell ribosomal RNA was used to determine if nuclease

8 can hydrolyze RNA. Electrophoresis of the reaction products

in acrylamide tube gels showed the RNA migrated farther in the

gel than untreated RNA. The smaller molecular weight RNA pro-

duced was of a discrete size suggesting either site specific

cleavage of the RNA or some secondary structure in ribosomal

RNA prevented further digestion. It cannot be ruled out that

RNAse activity is due to a contaminant in nuclease 0 fractions.

Summary

Nuclease 8, isolated from wild type cells of Ustilago

maydis, is a single polypeptide of 68,000 d that is highly
























I 4-1 r-I
H OE 4
(a ( 0

43 t .-
*H H -4 4-
G r 0 Or-i
) Cr- 3 0 C- d a)
-1 C 0 4J 1
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71



active on single stranded DNA. The enzyme digests linear DNA

in a 5'-directed exonucleolytic manner producing mostly mono-

nucleotide products. The nuclease can endonucleolytically

cleave circular DNA and both mononucleotide and oligonucleotide

products contain a 3' phosphate. The enzyme also possesses

5' nucleotidase activity and may hydrolyze RNA.














CHAPTER IV
PURIFICATION AND CHARACTERIZATION OF DBP III


DNA is thought to exist in a cell as a highly folded,

topologically constrained molecule (Stonington and Pettijohn,

1971; Worcel and Burgi, 1972; Ide et al., 1975; Laemmli et al.,

1977). Proteins and RNA appear to be responsible for holding

eucaryotic DNA folded in supercoiled loops (Georgiev et al.,

1978; Benyajata and Worcel, 1976; Cook et al., 1976). Super-

helicity alters the secondary structure of DNA and this

change can be recognized by some proteins that bind DNA. For

example, histone HI demonstrates a binding preference for DNA

that is supercoiled (Vogel and Singer, 1975; Singer and Singer,

1978). In the experiments described below a search was design-

ed to examine extracts of U. maydis for proteins binding to

superhelical DNA. Three chromatographically separable activ-

ities were found that bind to superhelical DNA. The first

part of this chapter discusses the purification of DNA bind-

ing activities and presents some properties of a nuclease

activity copurifying with one of the DNA binding activities.

The latter part of this chapter describes the characterization

of a nuclease free DNA binding activity, termed DBP -III.

Purification

The assay used to purify DNA binding activity is a

modification of the filter assay originally developed by

Riggs et al. (1970) (see Chapter II). Protein and radioactive








form I DNA are mixed and filtered through a nitrocellulose

filter which traps DNA-protein complexes but does not retain

protein-free DNA. The reaction sample (0.1 ml) was diluted

to 1 ml with 5 X SSC and filtered. The filters were washed

with two 1 ml aliquots of 5 X SSC, dried and counted for

radioactivity. Approximately 5% of the DNA bound without

protein addition and this amount could not be reduced by

further washing of the filter.

A detailed description of the steps used in purification

of DNA binding activities is given in chapter II. Nucleic

acid free extracts were found to be difficult to assay

reproducibly until the protein was further fractionated by

DEAE-cellulose chromatography. It was necessary to include

EDTA in all assays until the final step in purification to

prevent nicking of the substrate by endogenous nucleases.

Nucleic acid free extracts were applied to a DEAE-cellulose

column and absorbed proteins were eluted with an increasing

gradient of NaC1. Fractions containing binding activity were

dialyzed to remove the NaCl and applied to a phosphocellulose

column and eluted with a NaC1 gradient. The activity that

bound to the phosphocellulose column was concentrated by

precipitation with ammonium sulfate and layered onto a

Sephacryl S-300 column. Two peaks of activity were observed

to elute from this column. The first peak eluted after 120 ml

of buffer was collected and the second peak eluted after 180

ml of buffer had passed through the column.








Most (90%) of the activity in Fraction III was not re-

tained when the extract was passed through a phosphocellulose

column (figure 16A, peak 1). The DNA-binding activity that

did not absorb to phosphocellulose was applied to a Sephacryl

S-300 column. DNA binding activity eluted slightly ahead of

BSA which was run as a standard. Fractions demonstrating DNA

binding activity from this Sephacryl column contained at

least ten polypeptides when electrophoresed in a SDS-gel

(see chapter II). The major staining band migrated as a

60,000 d polypeptide when compared to the mobility of stand-

ard proteins. Since the DNA binding activity not absorbing

to phosphocellulose was found not to bind to a variety of

other columns (eg. hydroxylapatite, carboxymethyl cellulose

and Affigel-Blue), the characterization of DNA binding was

not pursued.

The DNA binding activity absorbing to phosphocellulose

separated into two distinct peaks when applied to a Sephacryl

S-300 column. Approximately 70% of the binding activity

applied to the Sephacryl column eluted at 180 ml slightly

behind a BSA standard (figure 16B, peak 2). The fractions
++
containing DNA binding activity also possessed a Mg stimu-

lated nuclease activity (Table VI). The nuclease, active on

single stranded DNA, had a pH optimum of 8.0 (figure 17).

Several factors suggested to me that this nuclease activity

may be due to the presence of U. maydis nuclease a (Holloman

et al., 1981). Both nuclease a and the nuclease eluting from

Sephacryl are highly active on single stranded DNA, have a pH




















I
>1 4-I I
to >i HO > "
tn z< C >3 > 4-3 r c 4 Ca
C Z -H ( *H 4-E r i -.-H
-H Q *- (n 4) Ma0i0
rO (a 0 M M 0U) rl "U r! (1 >l C! Q)
C 0 m M r- o d 0 o d O- -l Qa
Scr- (0 ti 0n Uo F F r: te
0) 3 ^ 5 r.: a) a) (1) S cl 4rO 0)
ri H > U -4 r0 m 0 4 0) Z 4J
>i Z a)r-iH >r -J1Hd lO u 4 i a- Cf m) c0 L
4- Q Q ) -)-PZ rE > >r r-4
,--H 0 ( 0 H a) 0 a U) o a)
>M H ) 0 0 > 4-4Q1 rq >1 r-q l *ri 0 0
H 4 -H- HO ro 4 41 U
>HOOO> o > J,.QDir-- -HO
*HH -'--HO c- ,J 4i -o
4-H 4-P4 4-) UrO-HOrZ >1
o rE m U U Z-Q-i C > rCQ M 4-
(rd r O Q0 JC d M -H l iM (75 +4J nM
o X 4 -H c 4-) M > -H
Mo-r (0 4 M 4 > -r. U 0) 4- CO -H > 4J-
*HUj 0 -- m H M 4. (~ cc-l-.
rO (0 4 >o' 0 = -' 4-l 44 0 -H ( U 0
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(d r U 0 (d Q) rO 0a -Hrr r Cj 4-) r-l
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UC L (d n Q r-L4 4-l) (n M3 0 M (1) V)c-'ri Q H LO 4-J











olqnloS P!oV selouW
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TABLE VI. Effect of Small Molecules
on Nuclease Activity



% Activity
Standard* 100
- Mg 10
++
- Mg+ + 2 mM CaC12 10
++
- Mg + 0.1 mM CoC12 90
+ 10 mM g-mercaptoethanol 80
+ 1 mM ATP 100
+ 10 mM EDTA < 5


*Reaction mixtures (0.1 ml) containing 2 nmol of denatured
P22 [3H]DNA (2.5 X 104 cpm/nmol), 0.1 M Tris-HC1, pH 8.0,
5 mM MgC12 and 10 pl of the Sephacryl S-300 eluate (frac-
tion 58) were incubated 15 min at 370. Acid soluble radio-
activity was measured as described in Chapter II. 100%
activity represents 0.2 nmole of DNA acid soluble.




























Figure 17 ,

PH optimum of nuclease activity. Reaction mixtures
(0.1 ml) containing 2 noml denatured P22 DNA (2.5 X
104 cpm/nmol), 5 mM MgCl and 0.1 M of either potas-
sium acetate (*), potassium phosphate (A) or Tris-
HC1 (o) were incubated with 10 41 of the Sephacryl
eluate (fraction 59). After 15 min.at 370Cthe amount
of DNA made acid soluble was determined (see chapter
II).













I 1 I I I


5 6 7 8 9


pH


1.0


0.8


0.6

0.4


0.2


-o

0

0



E
C-








optimum of 8.0 and are inhibited by EDTA. Polypeptides from

the Sephacryl column fractions containing nuclease activity

were analyzed by SDS-gel electrophoresis. The major band

had a mobility corresponding to a molecular weight of 55,000.

The polypeptide in nuclease a preparations responsible for

nuclease activity has molecular weight of 55,000 (Holloman

et al., 1981). Another way to compare nuclease a and the

nuclease activity from the Sephacryl column was to assay

purified nuclease a for DNA binding using the filter assay.

I found that, in the presence of EDTA, purified nuclease a

preparations retain form I DNA on nitrocellulose filters.

In addition, the ratio of DNA binding activity to single

strand nuclease activity was the same for two different

preparations of nuclease a (Table VII). These data suggest to

me that the fractions eluting from Sephacryl at 180 ml contain

nuclease a.

The DNA binding activity eluting from the Sephacryl S-300
0
column at 120 ml (Fraction V) could be stored at 4 C for 2

months without loss of activity. The DNA binding activity,

termed DBP III, was further characterized as described below.

Fraction V was subjected to SDS-gel electrophoresis

(see chapter II). The major band had a molecular weight of

47,000 relative to protein standards. The amount of 47,000 d

protein in the gel was approximated by comparing the intensity

of Coomassie stain with standard proteins. Twenty percent of

the protein applied to the gel ( 5 ug) could be visualized as

a 47,000 d band. Why would a polypeptide of 47,000 d elute






















TABLE VII.


DNA Binding Activity in Purified
Nuclease a Preparations


Nuclease a Nuclease Activity DNA Binding Activity

Preparation units/ml nmol DNA bound/ml

1 200 300

2 40 60


Two different preparations of Ustilago nuclease a were assayed
for the ability to retain form I DNA to nitrocellulose filers.
Purification of nuclease a and the determination of nuclease
activity were performed by Dr. William Holloman as described
previously (Holloman et al., 1981). Retention of form I DNA
to nitrocellulose filters in the presence of 1.5 M NaCl was
measured as described in Chapter II.







from Sephacryl columns at the position of blue dextran?

Small polypeptides can elute from gel filtration columns as

apparently large molecules for various reasons. For example,

the 47,000 d polypeptide could be the reduced subunit of a

larger protein. Perhaps protein aggregation by ionic or

hydrophobic interactions could result in the observed elution

profile from Sephacryl. Renaturation of the polypeptides

separated by SDS-gel electrophoresis and reconstitution of DNA

binding activity would be required to definitively state that

the 47,000 d polypeptide is the DNA binding protein.

DBP III (Fraction V) was assayed for nuclease and

topoisomerase activity. DBP III sufficient to bind 1 nmole of

RFI DNA did not nick RFI DNA (less than 25 pmol) or

hydrolyze single stranded DNA (less than 10 pmol). Although

1 nmole of RFI DNA was bound by the protein no detectable

covalently closed relaxed DNA (RFIV) was observed when the

sample was deproteinized and analyzed by agarose gel electro-

phoresis (less than 0.2 nmol).

Purified DBP III was free of nuclease activity and bound

to form I DNA. I observed that DBP III did not retain form II

DNA on nitrocellulose filters. This apparent specificity for

form I DNA led me to characterize the interaction of DBP III

with DNA.

Reaction Conditions for DBP III

Maximal binding activity of DBP III was observed at pH

6.0 in acetate buffer. At pH 7.5 in Tris-HCl buffer 20-fold

less binding activity was seen as compared to pH 6.0 in








acetate (figure 18). Binding was not affected by including
++ ++ ++
either divalent cations (eg. Mg Ca or Co ) or EDTA in

the reaction mixture (Table VIII). Addition of reducing

agents such as B-mercaptoethanol and dithiothreitol or

addition of the sulfhydryl blocking reagent N-ethylmaleimide

had no effect. High energy compounds such as ATP and NAD did

not change the amount of DNA bound by DBP III. Addition of

SDS or proteinase K to reaction mixure (0.5%) ablated DNA

binding activity. Binding was very fast but showed a thres-
0
hold temperature of 26 C (figure 19).

When reactions were performed in buffer containing 0.2 M

NaC1 a 50% reduction was seen in DNA binding activity. If

the NaCl was added after incubating the reaction for 5 min. at
0
37 C there was no decrease in binding (Table VIII). The same

amount of DNA binding activity was observed if the reaction

mixture (0.1 ml) was diluted and filtered in 0.5 X SSC or in

10 X SSC. These results suggest that protein binding to DNA

is sensitive to high ionic strength but once formed the

protein DNA complex is resistant to dissociation by salt.

Although many nuclear proteins are removed from DNA by 2 M

salt extraction some protein material remainsbound to the

chromosome and maintains the highly folded structure of DNA

(Benyajata and Worcel, 1976; Adolf et al., 1977). Histones

(Spelsberg and Hnilica, 1971) and helix destabilizing proteins

(Herrick and Alberts, 1976) are examples of DNA binding

proteins which are dissociated from DNA by 2 M salt.


























Figure 18

Optimal pH of DBP III activity. DNA binidng activity
was assayed as described in Chapter II. Reaction mix-
tures (total volume of 0.1) containing 2 nmol of form I
(X174 [3H] DNA and 0.1 M of either potassium acetate (e),
sodium piperazine-N,N'-bis[2 ethane sulfonic acid] (o) or
Tris-HCl (A) at the indicated pH were incubated with
25 pl of Fraction V DBP III. Incubation was for 10 min.
at 370C.


















a --------

_0
I L 1.0- *
1.0


zE o
0


E 0
C

4.0 5.0 6.0 70

pH
















TABLE VIII. Effect of Small Molecules
on DNA Binding



Reaction Conditions % Activity


Standard* 100
+ 10 mM MgCl2 100
+ 2 mM CaC12 100
+ 0.2 mM CoCl2 100
+ 2 mM EDTA 100
+ 10 mM 8-mercaptoethanol 100
+ 10 mM N-ethylmaleimide 100
+ 1 mM ATP 100

+ 0.2 M NaCl 50
+ 0.5% SDS 0
370 for 5 min then:
+ 2 M NaC1 100
+ 0.5% SDS 0



*Reaction mixtures (0.1 ml) containing 0.1 M potassium
acetate, 1 nmol [3H]-labeled ZX174 form I DNA and sufficient
DBP III to bind 0.5 nmole of DNA were incubated at 370 for
10 min except where indicated. DNA binding activity was
measured as described in Chapter II.




























Figure 19

Effect of temperature on DBP III activity. Reaction mix-
tures (total volume of 0.1 ml) containing 1 nmole of
0X174 [3H] form I DNA and 0.1 M potassium acetate were
incubated at the indicated temperatures. 20 ul of Frac-
tion V DBP III was added and incubation was continued
for 10 min. The reaction was terminated by addition of
1 ml of 5 X SSC and DNA binding activity was measured as
described in Chapter II.




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