Characterization of a topoisomerase from Ustilago Maydis

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Title:
Characterization of a topoisomerase from Ustilago Maydis
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Topoisomerase from Ustilago Maydis
Ustilago Maydis
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xi, 121 leaves : ill. ; 29 cm.
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Rowe, Thomas Cardon, 1951-
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DNA Recombinant   ( mesh )
DNA Replication   ( mesh )
Immunology and Medical Microbiology thesis Ph.D   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida.
Bibliography:
Bibliography: leaves 115-120.
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Photocopy of typescript.
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Vita.

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CHARACTERIZATION OF A TOPOISOMERASE FROM
Ustilago maydis










BY

THOMAS CARDON ROWE


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


1982




































To my father
Digitized by the Internet Archive
in 2011 with funding from
University of Florida, George A. Smathers Libraries with support from LYRASIS and the Sloan Foundation


http://www.archive.org/details/characterization00rowe















ACKNOWLEDGMENTS

I would like to extend my sincerest thanks to Dr. William Holloman

for his guidance and genuine friendship. He will always be an inspira-

tion to my work. In addition I would like to thank Drs. Kenneth Berns,

Bert Flanegan, George Gifford, Nicholas Muzyzcka, and Gary Stein for

their helpful suggestions and encouragement during the course of these

studies.

Special thanks also go to the crew in D4-42. In particular, I

would like to thank Drs. James Rusche and Michael Brougham for their

support, friendship and great discussions (scientific and otherwise).

Finally, I would like to warmly thank the remaining students,

faculty and secretarial staff in the Immunology and Medical Microbiology

Department for making my stay here such an enjoyable one.
















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS. . . . iii

LIST OF TABLES . . .. . vi

LIST OF FIGURES. . . . vii

KEY TO ABBREVIATIONS . . ix

ABSTRACT . . . .. x

CHAPTER
I. INTRODUCTION. . . 1

Introduction . . 1
Mechanism of Topoisomerase Action. . 1
Role of Topoisomerases in Genetic Recombination. 4
General Aim of the Studies . 6

II. PURIFICATION AND PROPERTIES OF THE TOPOISOMERASE. 8

Development of Novel Filter Assays to Detect
Topoisomerase Activity . .. 8
Isolation of a Topoisomerase with a High Molecular
Weight . . 12
Examination of Parameters Affecting Enzymatic
Activity . . 18
Antibiotic Inhibitors. . ... 30
Comparison of Superhelical Substrates. .. 32
Activity is Stimulated by Hl Histone . 37

III. PURIFICATION AND PROPERTIES OF HI-LIKE PROTEINS
FROM Ustilago maydis . . 47

An Endogenous Factor in the Topoisomerase
Preparation Stimulates Relaxing Activity 47
Purification of an Hl-like Protein from Ustilago 48
A 25,000 Dalton Acid-Soluble Protein also
Stimulates Topoisomerase Activity. . 60
Conclusions. . ... 63












Page


IV. CHARACTERIZATION OF THE COVALENT TOPOISOMERASE-
DNA LINKAGE. . . .

Detection of the Covalent Complex. . .
Identification of the Protein Complexed to
the DNA. . . .
Topoisomerase Links to the 3' End of the DNA Break .
Identification of the Covalent Linkage .
Discussion . . .

V. INVOLVEMENT OF TOPOISOMERASE IN GENETIC RECOMBINATION

The Ustilago Topoisomerase Recombines
Complementary Single-Stranded Rings of DNA .
Relaxing Activity Reduced in the rec 2 Mutant.
Discussion . ... . .


APPENDICES
A.


PHAGE, BACTERIAL AND FUNGAL STRAINS . .


B. PREPARATION OF DNA. . . .

C. ASSAYS. . . .


Topoisomerase. .
DNA-Binding Protein. .
Exonuclease. .
Protein. .


. 104
. 105
. 106
. 106

. 107

. 107
. 108
. 109


D. PROTEIN PURIFICATION. . .


Topoisomerase. .
25,000 Dalton Acid-Soluble
DBP IV . .


Protein .


E. ELECTROPHORESIS . .


. 112


F. REAGENTS AND MATERIALS. . .


Chromatographic Media. .
Enzymes . .
Miscellaneous. .


113
113
113


REFERENCES . . . .

BIOGRAPHICAL SKETCH. . . .


) I I ) I


I ) I I j



















LIST OF TABLES


Table Page

I. PURIFICATION OF THE ENZYME. . .... 13

II. ESTIMATION OF SIZE OF THE TOPOISOMERASE ... 17

III. INHIBITION BY NUCLEOTIDES AND POLYNUCLEOTIDES 29

IV. INHIBITION BY ANTIBIOTICS . ... 31

V. SUSCEPTIBILITY OF TOPOISOMERASE-DNA COMPLEXES TO
EXONUCLEASE I AND EXONUCLEASE VII . 82

VI. PURIFICATION OF DBP IV. . .... 110
















LIST OF FIGURES


Figure Page

1. Topoisomerase assays. . . 11

2. Polyacrylamide gel electrophoresis of the topoisomerase
preparation . . .. 16

3. Centrifugation of topoisomerase in a glycerol gradient. 20

4. pH optima of the topoisomerase. . 22

5. Salt optima of the topoisomerase. . .... 24

6. Temperature optima of the topoisomerase . 27

7. Removal of positive superhelical turns-gel assay. 34

8. Removal of positive superhelical turns. . 36

9. Stimulation of topoisomerase activity by histone HI .. 39

10. Stimulation of topoisomerase activity by histones
and polyamines. . . ... 41

11. Stimulation of topoisomerase by electrophoretically
purified HI histone . .... 43

12. An acid-soluble protein from the topoisomerase
preparation stimulates relaxing activity. 50

13. Procedure for isolation of HI histone from Ustilago 52

14. SDS polyacrylamide gel of HI histone from Ustilago .. 55

15. pH dependent binding of the Ustilago HI to single-
stranded DNA. . . 57

16. Ability of the Ustilago Hi histone to bind super-
helical DNA . . .. 59

17. Stimulation of topoisomerase activity by Ustilago
HI histone. . . 62

18. Electrophoresis of a 25,000 dalton acid-soluble
protein and DBP IV on an SDS polyacrylamide gel 65















Page

19. Acid-solubility of DBP IV . 67

20. Stimulation of the topoisomerase by DBP IV. 69

21. Effect of pH on the binding of DBP IV to DNA. 71

22. Covalent attachment of the topoisomerase to DNA 77

23. Analysis of the enzyme-oligonucleotide complex by SDS
gel electrophoresis . .... 80

24. Proteinase K treatment of topoisomerase completed
with DNA labeled at the 3' or 5' end. . 85

25. Paper electrophoresis of the [32P] labeled
topoisomerase oligonucleotide complex following
acid hydrolysis . . ... 88

26. Interlocking of complementary single-stranded rings
of DNA by the topoisomerase . .... 94

27. Topoisomerase activity in the rec 2 mutant. 97


viii
















KEY TO ABBREVIATIONS

ATP adenosine-5'-triphosphate

BSA bovine serum albumin

CTP cytosine-5'-triphosphate

DBP DNA binding protein

DNA deoxyribonucleic acid

EDTA ethylenediamine tetra-acetic acid

GTP guanosine-5'-triphosphate

PMSF phenylmethylsulfonylfluoride

poly(dA) polydeoxyadenylic acid

poly(dC) polydeoxycytidylic acid

poly(dG) polydeoxyguanylic acid

poly(dT) polydeoxythymidylic acid

RF I form I, superhelical DNA

RF II form II, nicked circular DNA

RF III form III, linear DNA

RF IV form IV, relaxed closed circular DNA

RNA ribonucleic acid

rpm revolutions per minute

SDS sodium dodecyl sulfate

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
















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


CHARACTERIZATION OF A TOPOISOMERASE FROM
Ustilago maydis

By

Thomas Cardon Rowe

December 1982

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

A topoisomerase from the lower eukaryote Ustilago maydis was

isolated and characterized. By gel filtration, the topoisomerase has a

native molecular weight of 270,000. The enzyme can remove both positive

and negative superhelical turns from DNA in an ATP-independent reaction.

Activity is stimulated by 10 mM Mg2+, but is totally inhibited by

Zn2+ at concentrations as low as 20 pM. The topoisomerase is active

over a broad pH range from 5.5 8.0. The optimal temperature for

activity is 20" 30"C.

ATP inhibits topoisomerase activity and may reflect a means by which

this enzyme can be regulated. Inhibition does not appear to require

hydrolysis of ATP since several structural analogs of ATP also inhibit

activity.

Histone HI from calf thymus stimulated the topoisomerase greater

than 25-fold. An Hi-like protein has been isolated from Ustilago which

also markedly stimulates the topoisomerase. This interaction may be

important in regulating topoisomerase in cellular processes.













The topoisomerase breaks single-stranded DNA and forms a covalent

complex with the 3' end of the DNA break. The covalent linkage appears to

involve a phosphotyrosine bond.

The rec 2 mutant of Ustilago has decreased levels of topoisomerase

activity possibly implicating this enzyme in eukaryotic genetic

recombination.

















CHAPTER I
INTRODUCTION

Introduction

Topoisomerases are enzymes which catalyze the interconversion of

topological isomers of DNA by a concerted breakage and reunion of the

DNA phosphodiester bonds (1,2). By altering the topological structure

of DNA, topoisomerases are believed to play a key role in regulating the

biological activity of DNA (3).

Although topoisomerases were initially identified by their ability

to change the superhelical density of DNA, they have since demonstrated

an ability to carry out other enzymatic gymnastics leading to an

interconversion between knotted and unknotted forms of single- and

double-stranded DNA as well as the catenation and decatenation of duplex

DNA rings (1-6). Because of their adeptness at solving toplogical

puzzles, topoisomerases are thought to play an important role in DNA

replication, transcription and genetic recombination (1,3,5,7).

Mechanism of Topoisomerase Action

Topoisomerases derive their name from an ability to change the

topological linking number, L, of covalently closed circular duplex DNA.

The linking number is defined as the number of times one strand of the

DNA duplex winds about the other strand when the DNA is forced to lie in

a plane (7) and can be related to the number of Watson-Crick turns, R,

and the number of superhelical turns, T, by the following simple

equation L = 5 + T (8). The linking number for a closed circular













molecule remains constant unless the DNA backbone becomes broken. There

are two basic ways topoisomerases can alter the topological linking

number of DNA and they are designated as being type I or type II enzymes

depending upon which mechanism they employ (1-3,5).

Type I Topoisomerases

A type I enzyme alters the topological linking number of steps of

one by introducing transient single-strand breaks into the DNA resulting

in a covalently linked enzyme-DNA intermediate involving a phosphotyro-

sine bond (9,10). Resealing of the break requires no energy cofactor

but instead is thought to be driven by the energy stored in the covalent

enzyme-DNA bond (11). With the exception of the lambda integrase topoi-

somerase, all prokaryotic type I enzymes tested form a covalent complex

with the 5' end of the DNA (12). This is in contrast to eukaryotic type

I topoisomerases and the lambda integrase topoisomerase which form a

covalent complex with the 3' end of the DNA (13,14). Prokaryotic and

eukaryotic enzymes also differ in their ability to relax positively and

negatively supercoiled DNA. Although eukaryotic topoisomerases relax

both positively and negatively supercoiled DNA (15,16), prokaryotic type

I enzymes, excluding the lambda integrase topoisomerase (17), can remove

only negative superhelical turns from DNA (11,18,19). Both eukaryotic

and prokaryotic enzymes are able to catenate duplex rings of DNA if one

of the rings of DNA involved in the reaction contains a preexisting nick

(20,21). The prokaryotic topoisomerase from E. coli called omega

protein also displays an ability to knot and unknot both single-stranded

and duplex DNA circles (21). However, like the catenation reaction, the











introduction and removal of knots from duplex DNA circles requires that

the DNA has a preexisting nick in one of the strands (21). Another

reaction which is catalyzed by both eukaryotic and prokaryotic type I

topoisomerases is the intertwining of complementary single-stranded

rings of DNA to form covalently closed circular duplex DNA (22,23).

Type II Topoisomerases

Type II topoisomerases introduce transient double-strand breaks

into DNA. Passing a strand through the break type II topoisomerases can

change the topological linking number in steps of two, a process which

is called sign inversion (24). Similar to type I enzymes, type II

topoisomerases form a covalent enzyme-DNA complex involving a phospho-

tyrosine bond (9). In both eukaryotes and prokaryotes this linkage is

with the 5' end of the DNA (9). The first enzyme discovered in this

group, DNA gyrase, has the unique ability to catalyze the conversion of

relaxed closed circular duplex DNA to a negatively supercoiled form, a

process which requires hydrolysis of ATP (25). Gyrase is inhibited

by the antibiotics nalidixic acid and novobiocin. The sensitivity of

gyrase to these two antibiotics has been effectively used to establish

the biological role of this topoisomerase in DNA replication, transcrip-

tion and recombination (3).

Type II topoisomerases have been isolated from a variety of

prokaryotic and eukaryotic organisms (26-28). They all share an ability

to remove positive as well as negative supercoils from DNA (27,3). In

addition, these enzymes can introduce and remove knots from double-

stranded DNA as well as catenate and decatenate duplex DNA rings











(27,28). All of these reactions proceed in the absence of preexisting

nicks in the DNA which is consistent with an enzyme mechanism involving

double-stranded breaks in the DNA (24,27). Although all of the topolog-

ical reactions catalyzed by eukarotyic and T4 type II topoisomerases

require ATP, there are examples of type II activities which can occur in

the absence of ATP, such as the relaxation of negatively supercoiled DNA

by gyrase (29).

Role of Topoisomerases in Genetic Recombination

Having discussed some of the salient features of topoisomerases,

how might those enzymes be involved in DNA recombination? One possible

way topoisomerases might influence genetic recombination is by altering

the superhelix density of DNA. Interestingly, it was studies on the

role of superhelicity in lambda site-specific recombination that led to

the discovery of DNA gyrase (26). Mizuuchi et al. found that super-

helical lambda DNA served as a good in vitro substrate for recombination

when it was incubated with E. coli extracts containing the lambda

integrase protein (30). However, no recombination took place if

superhelical lambda DNA was replaced by relaxed, nonsupercoiled lambda

DNA, unless ATP was included in the reaction mixture. This was somewhat

puzzling until it was discovered that ATP was required by an enzyme in

the extract, DNA gyrase, to convert relaxed lambda DNA to a superhelical

form which could then undergo recombination (26).

Site-specific recombination involving the transposition element

y6 is also dependent on the superhelical state of the DNA (31).

Resolvase, an enzyme coded for by y6 carries out the final step in

Y6 transposition only if the DNA is negatively supercoiled.








5



Superhelicity is also believed to play an important role in homol-

ogous genetic recombination (32,33). The ability of duplex DNA to take

up homologous single-stranded DNA, forming a recombination intermediate

called a heteroduplex joint, is strongly influenced by the superhelical

state of the DNA (34). Because of their ability to change the topologi-

cal linking number of DNA, topoisomerases may also promote homologous

recombination between DNA molecules containing no free ends. The abil-

ity of type I topoisomerases to catalyze the intertwining of complement-

ary single-stranded rings suggests that they are capable of playing such

a role (22,23).

A more direct way in which topoisomerases might promote recombina-

tion involves their inherent ability to break and rejoin the DNA back-

bone, two essential steps in the process of recombination. The first

evidence suggesting this type of functional role for topoisomerases came

when the lambda integrase protein was discovered to have a type I topoi-

somerase activity (17). A tantalyzing mechanism by which a nicking-

sealing enzyme might promote lambda site-specific recombination has been

proposed. The initial step is the formation of a four-stranded DNA

structure between specific sites on the lambda and bacterial DNAs. Two

molecules of topoisomerase (integrase protein) then bind to and create

single-strand breaks at identical sites on strands of the lambda and

bacterial DNAs having the same polarity. By a simple rotation of the

DNA around these breaks, ends of the lambda DNA can be aligned with the

ends of the bacterial DNA. A subsequent topoisomerase mediated reseal-

ing of the breaks produces a Holliday structure. The resulting Holliday

structure can then be resolved into a fully recombinant molecule by a











final round of breakage and reunion involving the remaining virgin pair

of strands (17,35).

Resolvase, the enzyme which catalyzes a site-specific recombina-

tion event in yS transposition, may act by a similar mechanism. It is

interesting that resolvase has recently been shown to form covalent

complexes with DNA (36) suggesting that it might use a topoisomerase-

like mechanism to recombine DNA. A role for topoisomerases in trans-

position gains additional support from genetic studies. The transposi-

tion frequencies of the transposable elements Tn 5, Tn 9, and Tn 10 are

dramatically reduced in strains of E. coli deficient in the type I

topoisomerase omega protein (37).

Topoisomerases may also promote illegitimate recombination of DNA.

Treatment of E. coli with oxolinic acid, a drug which causes DNA gyrase

to create double-strand breaks in DNA, results in over a 10-fold

increase in the level of illegitimate recombination (38). If cells

containing a drug resistant form of gyrase are treated with oxolinic

acid, no rise in the level of illegitimate recombination occurs.

Eukaryotic type I topoisomerases may also play a role in illegitimate

recombination. Evidence for this has come from in vitro studies showing

the ability of eukaryotic type I topoisomerases to recombine nonhomol-

ogous DNAs (14,39).

General Aim of the Studies

Although the role of topoisomerases in prokaryotic DNA metabolism

is becoming clearer, information concerning their cellular role in

eukaryotes is almost nonexistent (3). The studies described in the

following chapters represent initial efforts to biochemically and








7



genetically characterize a topoisomerase from the lower eukaryote

Ustilago maydis. Such studies will hopefully provide a foundation for

probing the role of these enzymes in eukaryotic DNA recombination.

For the sake of continuity, as well as to make the reading easier,

the detailed procedures (materials and methods) used in these studies

are found in Appendices A F.
















CHAPTER II
PURIFICATION OF PROPERTIES OF THE TOPOISOMERASE

There are a variety of approaches which can be taken in studying

recombination. One approach is to reconstruct the events of recombin-

ation in vitro using purified cellular proteins. Developing an in vitro

system required actively engaging in purifying proteins which might play

an important role in recombination. The purification and characteriza-

tion of one of these proteins, DNA topoisomerase, are described here.

Development of Novel Filter Assays to
Detect Topoisomerase Activity

Due to differences in their tertiary configurations, superhelical

and relaxed DNAs can be easily separated by electrophoresis through

agarose gels (40). This commonly used technique has provided a simple

means for monitoring the conversion of superhelical to relaxed DNA by

DNA topoisomerase. However, this assay is time consuming and only

semiquantitative. As an alternative, two filter assays were developed

which provide a quantitative and rapid determination of topoisomerase

activity.

The D-loop filter assay discriminates between superhelical and

relaxed DNA by their differing abilities to form stable triple-standard

structures called D-loops (41). Superhelical, but not relaxed DNA, can

take up homologous single-stranded fragments to form D-loop complexes

which are selectively retained on a nitrocellulose filter. Relaxation

of superhelical DNA by topoisomerase prevents subsequent D-loop











formation. Therefore a loss of DNA bound to a nitrocellulose filter is

considered as a measure of topoisomerase activity.

Superhelical and relaxed DNA can also be distinguished by filtration

through nitrocellulose under conditions of high ionic strength and this

forms the basis of the second filter assay for topoisomerase activity (42).

At 3.5 M salt superhelical, but not relaxed DNA, will bind to a nitrocellu-

lose filter. This selective binding may be a result of a structural change

in the DNA from a right-handed to a left-handed helix, a transition known

to be strongly affected by the superhelical density of DNA as well as the

ionic environment (43,44). Superhelical DNA treated with topoisomerase is

converted to a relaxed form which is unable to bind to a nitrocellulose

filter. Therefore, a loss in DNA binding to a nitrocellulose filter is a

measure of topoisomerase activity.

One drawback to both of the filter assays is that neither can differ-

entiate between relaxation activity and endonuclease activity. Fortunate-

ly, these two possibilities can be easily distinguished using a nicking

assay described by Kuhnlein et al. (45). By using this assay in conjunc-

tion with the filter assays, topoisomerase activity can be readily

measured.

A comparative measurement of topoisomerase activity using the two

filter assays and the gel assay is shown in Figure 1. The loss in DNA

binding to a nitrocellulose filter in the two filter assays is seen to be

in good agreement with the appearance of a slower moving, relaxed form of

DNA present in lanes f and g of the agarose gel. Evident in all three

assays is the sigmoidal character of topoisomerase activity, a property

exhibited throughout every step in the purification of this enzyme. For

















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this reason, an effort was made to use concentrations of the enzyme

within the linear portion of the assay when characterizing a particular

reaction parameter.

Isolation of a Topoisomerase with a High Molecular Weight

Due to an instability of the enzyme in low salt buffers and large

losses of activity incurred during dialysis, efforts were made during

the purification to maintain the enzyme in concentrations of salt above

0.1 M and to avoid dialysis. PMSF was also routinely included in the

purification buffers to minimize degradation of the topoisomerase by

serine proteases.

The scheme used to purify the topoisomerase is outlined in Table

I. Following breakage of the cells in I M KC1, polyethylene glycol was

added to the extract and the resulting precipitate removed. The super-

nantant, still containing 1 M salt, was directly applied to a hydroxyl-

apatite column. Activity was eluted using an increasing potassium phos-

phate gradient in 1 M KCI and loaded directly on to an octyl-Sepharose

column equilibrated in 1.0 M KC1. The column was then equilibrated in

buffer containing 0.1 M KC1 before eluting activity with an increasing

gradient of Triton X-100. Relaxing activity was loaded directly onto a

phosphocellulose column and subsequently recovered in an increasing KC1

gradient. At this stage the enzyme was found to be very stable if

stored at -200C in the presence of 0.1 M KC1 and 50% glycerol. Loss of

activity under these conditions was less than 10% over a two-year

period.

Despite all the precautions taken, the final yield of activity was

a mere 0.4%. Although initially disconcerting, this may in fact












TABLE I
PURIFICATION OF THE ENZYME



Specific
Volume Activity Protein Activity
Fraction ml units x 10"4 mg units/mg


I. Crude Extract 240 920. 2,400. 3,800

II. Polyethyleneglycol 315 315. 583. 5,400

III. Hydroxylapatite 280 560. 15. 37,300

IV. Octyl-Sepharosea 85 227. -

V. Phosphocellulose 18 19.2 4.5 42,700

VI. Sephacryl S200b 8 3.4 0.40 85,000


protein determination was not performed since Triton interfered with the
assay.

bTotal amounts indicated are corrected for the fact that only a portion of
Fraction V was taken through this step.

NOTE: See Appendix D for details of purification.











represent a loss of an activator of the topoisomerase during the course

of purification, a subject which will be returned to later.

The purity of fraction V was tested by electrophoresis through

polyacrylamide gels under nondenaturing conditions. Staining of the gel

with Coomassie blue revealed two major bands (Fig. 2). The faster

mobility band, representing about one-third of the total stained

protein, contained the topoisomerase activity. Electrophoresis of this

band in a second dimension in a polyacrylamide gel containing SDS

revealed a number of bands ranging in size from 2 x 104 to 1.2 x 105

daltons. Not knowing if -these multiple bands were degradation products

or if they were components of a multi-subunited enzyme, efforts were

undertaken to do additional studies designed to determine the native

molecular weight of the enzyme. When a portion of fraction V was gel

filtered through Sephacryl S-200 in 0.5 M KC1, 45% of the activity was

recovered in a peak corresponding to a molecular weight of 270,000 while

a small amount, less than 1%, emerged with an apparent size of 30,000

daltons (Table II). This is considerably larger than previously

described eukaryotic type I topoisomerases which have molecular weights

ranging from 60 100,000 (3). Gel filtration in lower salt solutions

resulted in a drastic reduction or total loss in activity. The

recoverable activity had molecular weights corresponding to 85,000 and

30,000 daltons. No 270,000 dalton form was observed. If the

topoisomerase does indeed consist of multiple subunits, low salt

conditions may lead to their dissociation.














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TABLE II
ESTIMATION OF SIZE OF THE TOPOISOMERASE


Activity Molecular Recovery
KC1 Peak Kd1/3 Weight Percent


0.5 M 1 0.54 270,000 45
2 0.91 30,000 1

0.1 M 1 0.69 85,000 5
2 0.91 30,000 5

0.05 None 0


NOTE: A column of Sephacryl S-200 (1.4 x 56 cm) was equilibrated with
20 mM potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM 2-mercaptoethanol,
10% glycerol, and the indicated salt concentrations. The excluded and
included volumes were determined with the use of 3H-labeled X174
phage and [32P] orthophosphate, respectively. Marker proteins used
to calibrate the column included ferritin, catalase, bovine serum
albumin, ovalbumin, pancreatic deoxyribonuclease, and cytochrome c. The
hydrodynamic distribution coefficent Kd was determined (47) and
linearly related to molecular weight (48) by assuming the topoisomerase
is a globular protein. Topoisomerase (7000 units) was loaded, and the
column was run at 10 ml/hr. Fractions of 1 ml each were collected and
assayed for activity by the agarose gel assay. Recovery of activity was
estimated by visual inspection of the gels.











Sedimentation analysis was also used to determine the size of the

native enzyme. As shown in Figure 3, activity was present in a single

peak with a sedimentation coefficient of 2.5 S corresponding to a

spherical shaped protein of 29,000 daltons. Although a variety of

buffers and salt conditions were used, recovery of activity was poor,

less than 1%. Even presoaking the centrifuge tubes in solutions of BSA

did not improve recovery. This loss of activity is puzzling especially

since the topoisomerase should be stable in the solutions that were

used. Possibly the enzyme was affected by the high pressures generated

during the centrifugation (49).

A variety of other purification schemes were also attempted in an

effort to improve upon the specific activity of the enzyme but the

specific activities obtained were never much higher than the value of

8.5 x 104 for fraction VI.

Examination of Parameters Affecting Enzymatic Activity

As is illustrated in Figure 4, relaxation activity spans a pH

range from 5.5 8.0 being acutely sensitive to the buffer used. While

high activity was observed at pH 7.5 in phosphate buffer, activity was

halved in Tris-HC1l and not measurable in either 4-(2-hydroxyethyl)-1-

piperazine ethane sulfonic acid or imidizole buffer. Salt also affected

activity as is shown in Figure 5. There was a sharp response in

activity as the salt concentration increased with optimal stimulation

occurring around 0.2 M KC1. Although as much as a 5-fold increase in

activity could be seen with KC1, Mg2+ proved to be much more

effective in promoting activity. In the presence of 10 mM MgC12,














e a




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Figure 5. Salt optima of the topoisomerase.

Reaction mixtures (25 pl) containing 25 mM Tris-HC1, pH 7.5, 10%
glycerol, 0.4 mM 2-mercaptoethanol, 3 nmol PXRF I [3H] DNA (1.5 x
104 cpm/nmol), 100 ng topoisomerase and various concentrations of KC1
were incubated at 25"C for 30 min. Reactions were terminated by adding 1
ml of buffer containing 3.5 M NaCI and topoisomerase activity determined
using the high salt filter assay as described in Appendix C.






































E 50


0.
.4
C.


KCI (M)












relaxation of the DNA was enhanced 25-fold. The effect of other dival-

ent cations on topoisomerase activity was also tested with curious

results. In contrast to Mg2+, Cu2+ and Zn2+ were strong

inhibitors of the topoisomerase. The presence of as little as 20 JM

CuC12 or ZnC12 totally inhibited activity. Whether this is due to a

direct interaction of these metal ions with the topoisomerase or if it

is a result of an interaction with the DNA is unclear. Topoisomerase

was active in the presence of the chelating agents EDTA, 8-hydroxyquino-

line, diethyldithiocarbamic acid and isoniazed but was potently

inhibited by 1,10 phenanthroline. The inhibition does not apparently

involve the chelation of an enzyme-bound metal since the nonchelating

analog 4,7 phenanthroline also inhibited activity. The optimal

temperature for topoisomerase activity was found to be between 20 30C

with no detectable activity at 420C (Fig. 6). Surprisingly, the enzyme

is fairly active at low temperatures with 70% relaxation occurring at

O0C. A reducing agent is necessary to maintain activity. Storage of

the enzyme in the absence of 2-mercaptoethanol resulted in a loss of

activity. However, restoration of activity could be seen upon the

addition of fresh reducing agent to the reaction mixture. In addition,

the inclusion of 2 mM N-ethylmaleimide to a reaction containing 0.1 mM

2-mercaptoethanol inactivated the enzyme.

As was discussed in Chapter I, eukaryotic type I topoisomerases

catalytically remove superhelical turns in the absence of an added

energy cofactor (7). This is in contrast to eukaryotic type II enzymes

whose catalytic action on DNA is coupled with the hydrolysis of ATP

































Figure 6. Temperature optima of the topoisomerase.

Reactions (50 111) containing 75 mM potassium phosphate, pH 7.5, 10%
glycerol, 3 nmol OXRF I [3H] DNA (1.5 x 104 cpm/nmol) and 40 ng of
topoisomerase were incubated for 30 min at the indicated temperatures.
Reactions were terminated by adding 10 Pl 3 M KCI. Topoisomerase
activity was then determined using the D-loop assay as described in
Appendix C.





































100 o-




4 50-




0 10 20 30 40

Temperature (OC)












(3). Similar to type I enzymes, the Ustilago topoisomerase relaxes DNA

in the absence of an energy cofactor. However, curiosity led us to

investigate if ATP had any effect on the Ustilago topoisomerase. At 5

mM, ATP totally blocked activity (Table III). This inhibition was not

simply due to chelation of an endogenous metal essential to topoisomer-

ase activity since EDTA at the same concentration exhibited no effect on

the action of the enzyme.

Is hydrolysis of ATP necessary for inhibition? Several different

nonhydrolyzable analogs of ATP including a,$-methylene ATP, g,y-methy-

lene ATP, adenyl-5'-imidodiphosphate, and adenosine-5'-0-(3-thiotriphos-

phate) were tested and all of them totally inhibited the topoisomerase

at 5 mM suggesting that hydrolysis was not required for inhibition

(Table III). In addition, attempts were made to measure ATPase activity

and no significant hydrolysis was detected in standard reaction mixtures

containing [Y-32p] ATP and superhelical DNA. Inhibition is nucleo-

tide specific. None of the other nucleotide triphosphates tested inhib-

ited the topoisomerase at 5 mM. Although slight inhibition of activity

was caused by ADP, AMP was without effect confining the inhibition to

the triphosphate form of adenosine.

Inhibition is probably the result of a direct interaction of ATP

with a site on the topoisomerase. Efforts to dissociate the ATP inhibi-

tion from the topoisomerase by electrophoresis of the enzyme through a

polyacrylamide gel under nondenaturing conditions were unsuccessful.

The topoisomerase activity associated with the faster moving band in

Figure 2 was still found to be blocked by ATP. In addition, topoisomer-

ase isolated using a number of different purification schemes was always











TABLE III
INHIBITION BY NUCLEOTIDES AND POLYNUCLEOTIDES


Concentration Activity
Compound mM Percent


Control 100
ATP 5.0 0
ADP 5.0 70
AMP 5.0 100
dATP 5.0 90
a,8-methylene ATP 1.6 50
a,B-methylene ATP 3.2 0
0,y-methylene ATP 1.5 100
,y-methylene ATP 3.0 0
AMP-PNPa 5.0 0
ATP-y-Sa 5.0 0
CTP 5.0 100
GTP 5.0 100
TTP 5.0 100
UTP 5.0 100
dCTP 5.0 100
dGTP 5.0 100
Poly(rA) 0.02 100
Poly(dA) 0.02 100
Poly(dC) 0.02 100
Poly(dT) 0.02 100
Poly(dG) 0.01 0
Poly(d(G-C)) 0.02 100
jX174 DNA 0.2 50
P22 DNA 0.16 100


aAMP-PNP, adenyl-5'-imidodiphosphate.

bATP-Y-S, adenosine-5'-0-(3-thiotriphosphate).

NOTE: Reaction mixtures containing the indicated concentrations of
compounds were run at standard conditions and activity was estimated by
the agarose gel assay. Because of the narrow range where activity
increases with added enzyme, several enzyme concentrations were used in
testing each compound to ensure that the response was in the linear
range of the assay. All the results presented are relative to a control
where approximately 90% of the substrate DNA was relaxed. It should be
emphasized that these results are only approximate since activity was
estimated by visual inspection of the gels.











found to be sensitive to ATP, again implying the inhibition results from

an interaction of ATP with part of the topoisomerase. Whether this

inhibition involves a blockage of the enzyme's active site, or is a

result of an allosteric change mediated by the binding of ATP to a

region other than the active site, is unclear. Regardless, inhibition

of the topoisomerase by ATP may be a key to understanding how this

enzyme is regulated in the cell.

Several type I topoisomerases are sensitive to polynucleotides.

Single-stranded DNA effectively inhibits the E. coli omega protein (11),

calf thymus nicking-closing enzyme (51), and the bacteriophage lambda

integrase gene topoisomerase (17). The Ustilago topoisomerase is also

inhibited by single-stranded DNA but is more acutely sensitive to the

homopolymer poly(dG) (Table III). Other polymers tested, including

poly(d(G-C)), were ineffective inhibitors at comparable concentrations.

Possibly the unusual secondary structure of poly(dG) is responsible for

this selective inhibition (52).

Antibiotic Inhibitors

Antibiotics can serve as powerful tools for elucidating the cellu-

lar role of proteins. The sensitivity of DNA gyrase to the drugs nalid-

ixic acid and novobiocin was utilized to establish the regulatory role

of this topoisomerase in prokaryotic replication, transcription and

recombination (3). The Ustilago enzyme is also inhibited by nalidixic

acid and novobiocin. The rate of relaxation by the Ustilago topoisomer-

ase is reduced approximately 2-fold in reactions containing 20 ug/ml of

nalidixic acid (Table IV). This is a 10-fold lower concentration













TABLE IV
INHIBITION BY ANTIBIOTICS


Concentration Activity
Drug pg/ml Percent


Control 100


Novobiocin 100.0 30
200.0 0

Nalidixic acid 20.0 50

Berenil 2.8 50
5.6 10


NOTE: Reaction mixtures containing the indicated concentrations of
antibiotic were assayed under standard conditions as described in Table
III.












than is required to cause a similar inhibition of DNA gyrase (53).

While 200 pg/ml of novobiocin are required to totally inhibit the

Ustilago enzyme, significant loss of activity is seen at 100 yg/ml.

Berenil, an antitrypanosomal drug previously shown to inhibit a

mitochondrial topoisomerase, also inhibits the Ustilago enzyme (54).

Half-inhibition of topoisomerase activity occurs at 5.6 pg/ml.

Comparison of Superhelical Substrates

Like other eukaryotic topoisomerases, the Ustilago enzyme can

remove positive as well as negative supercoils from DNA. To create a

positively supercoiled substrate, ethidium bromide was added to a

reaction containing closed circular relaxed DNA. If the topoisomerase

acts upon this substrate, subsequent removal of both the enzyme and the

ethidium bromide should result in a negatively superhelical molecule of

DNA which can be easily distinguished from the starting material by

electrophoresis through an agarose gel. The presence of a faster moving

band in Figure 7 indicates an ability of the topoisomerase to remove

positive superhelical turns from DNA.

Are positive and negative supercoils equally good substrates for

the topoisomerase? A comparison was made using the high salt filter

assay. Although positive superhelical DNA was relaxed at a much slower

rate than negatively superhelical DNA, there was no longer a sigmoidal

dependence of activity on the enzyme concentration (Fig. 8). Although

this difference in reaction kinetics may simply be an artifact of using

ethidium bromide to generate positive superhelical DNA, it might

alternatively represent the action of topoisomerase on two different

types of structural elements in the DNA.































Figure 7. Removal of positive superhelical turns--gel assay.

Reaction mixtures (50 P1) containing 20 mM Tris-HCL, pH 7.5, 0.1 M
KC1, 5% glycerol, 0.8 Pg/ml ethidium bromide and 3 moles PXRF I or
RF IV DNA were incubated in the presence or absence of topoisomerase at
25'C. After 30 min the ethidium bromide and protein were removed by
extracting with 1 volume of phenol. The DNA was then loaded onto a 1.2%
agarose gel. (Lane A) kXRF I DNA; (Lane B) 4XRF IV DNA; (Lane C) (XRF
IV DNA and 100 ng topoisomerase.












ABC













4-4
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0 0 1 c X 0 a -
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0 4IJ > 0) ( 0 C 4 C 3 0 wc


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Activity is Stimulated by Hi Histone

Histone HI shows a specific interaction with superhelical DNA

(55). This tantalyzing property led to a search for a possible inter-

relationship between HI histone and the Ustilago topoisomerase. The

dramatic effect of Hi on topoisomerase activity is illustrated in Figure

9. In the presence of Hi, topoisomerase activity is stimulated more

than 25-fold. Although the ratio of HI to DNA used in Figure 9 was

0.025 (w/w), a ratio of 0.01 (w/w) or 1.4 HI molecules per molecule of

superhelical DNA was equally as effective in stimulating the

topoisomerase. The ratio of Hi to DNA is important; a 10-fold increase

or decrease relative to 0.01 (w/w) abolishes the stimulatory effect. In

agreement with Bina-Stein and Singer, high ratios of Hi to DNA, above

0.2 (w/w), inhibit topoisomerase activity (56). In contrast to Hi, none

of the other histones, poly L-lysine, or spermidine proved to be as

effective in stimulating topoisomerase activity (Fig. 10).

Although the HI histone used in these studies was greater than 90%

pure, as judged from SDS polyacrylamide gels, the possibility existed

that stimulation of the topoisomerase was due to a contaminating protein

present in the HI preparation. To free Hi of contaminants it was

electrophoresed through a 15% SDS polyacrylamide gel. The gel was

stained with Coomassie blue and the two major staining bands, containing

different structural species of Hi (57,58) were separately cut out and

the protein extracted. Addition of protein from either of these bands

to reaction mixtures stimulated the topoisomerase greater than 10-fold

(Fig. 11), providing additional evidence that HI histone is responsible

for stimulation of the topoisomerase. This result also implies that































Figure 9. Stimulation of topoisomerase activity by histone Hi.

Reaction mixtures (25 Pl) containing 40 mM Tris-HC1l pH 7.5, 10 mM
MgC12, 10% glycerol, 3 nmol of 4XRF I [3H] DNA (1.5 x 104
cpm/nmol), the indicated amounts of topoisomerase, and either 1 pg/ml of
histone HI (o) or 1 Pg/ml of bovine serum albumin (*) were incubated at
25'C. After 30 min EDTA was added to a final concentration of 15 mM, and
SDS was added to 0.5%. The mixture was diluted with 3 ml of 3.5 M NaCI
buffer and filtered through nitrocellulose as described in Appendix C.
It was important to dilute the sample 100-fold before filtering to
prevent interference by the SDS.
















-o
j 1.5
0






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0 0 0 a (0 0 0
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I- jO- O0 *oi
-4 t- (0 0 -4 :czo
0 rCu- w co to 00




4- 44 .00 ca Q. 0
0 O -4 0 a C
-4 44 -4 0C U Ei 0



*ci a B- o c c
u 0 N t0 0 C- 4 -
V )0 l> 0 l **C 0 0
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o C cOO 0 O
)o sa % 0 0) w W N

$4- o O S w o' e

* S 0) aB .4 0 o
0 C- to 0 03 *r- 0
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Oj o 0 w 3

0 N 'U >1 0 O C
) H -.0 Cuo -0 Ia M =

0 c o o o) CO ) 0 "0 C
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m- 60 O 0 0 -4 0"




0 w-4 C4 "4 -4 0 3L w








41

















Figure 11. Stimulation of topoisomerase by electrophoretically
purified HI histone.

HI histone (501g) was separated from contaminating protein by
electrophoresis through a 15% SDS polyacrylamide gel as described in
Appendix E. Following electrophoresis the gel was stained with Coomassie
blue to visualize the protein. Hi histone was present as a doublet and
represented greater than 90% of the stained protein. The fast and slow
moving bands of the doubletwhich represent different structural forms of
HI (57,58), stained with approximately equal intensity. Each of these
bands was excised from the gel and separately dounce homogenized in 500
pi of 20 mM Tris-HCI, pH 7.5, 1 mM EDTA, 50 mM NaCl and 0.1% SDS. After
the suspensions were allowed to stand at room temperature for 1 hr they
were briefly centrifuged to remove the gel debris. To the resulting
supernatants was added 125 p1 of 100% TCA (final concentration 20%).
After standing for 1 hr at -20CC, the protein was pelleted by
centrifuging in an Eppendorf Microfuge for 5 min at 4*C. The resulting
pellets were washed 1 time with 500 pl acid acetone (1/100 dilution of
concentrated HC1 in acetone) and then 2 times with 500 Pl of acetone.
After removing the residual acetone under vacuum, the fast and slow
mobility forms of Hi were each resuspended in 100 11 of 20 mM Tris-HCl,
pH 7.5, 0.5 mM EDTA. To determine stimulation of the topoisomerase a
1:2.5 or 1:10 dilution of the gel extracted histone was made into
reactions (25 1I containing 25 mM Tris-HCl, pH 7.5, 10 mM MgC1 0.4 mM
2-mercaptoethanol, 5% glycerol, 10 Pg/ml BSA, 3 nmol PX RF I [ H1 DNA,
+ topoisomerase. After 30 min at 25 "C, the reactions were terminated
by adding EDTA to 15 mM and SDS to 0.5%. The products of the reactions
were then analyzed by electrophoresis as described in Appendix C. A, 1.7
ng topoisomerase; B, 8.5 ng topoisomerase; C, 1.7 ng topoisomerase, fast
mobility form of HI histone (1:10); D, 1.7 ng topoisomerase, fast
mobility form of HI histone (1:2.5); E, 1.7 ng topoisomerase, slow
mobility form of Hi histone (1:10); F, 1.7 ng topoisomerase, slow
mobility form of Hi histone (1:2.5); G, fast mobility form of Hi histone
(1:2.5); H, slow mobility form of HI histone (1:2.5); I, no topoisomerase
or Hi histone.








43











more than one structural form of Hi has an ability to stimulate topoiso-

merase activity.

The effect of Hi on topoisomerase activity is not entirely

surprising. Histone HI has been previously reported to copurify with

type I topoisomerases from calf thymus and mouse L cells (59,60). Other

studies have shown that HI histone can substitute for a cellular protein

in promoting catenation of duplex DNA by type II topoisomerases from

Drosophila and yeast (61,62). Possibly Hi, or an Hi-like protein, makes

up an integral part of the Ustilago topoisomerase. Separation of this

Hi-like component from the nicking-closing component of the topoisomer-

ase during purification might explain the low yields of activity as well

as the inability to completely purify the enzyme. If Hi is indeed

complementing a component normally present in the topoisomerase, this

may provide clues about the mechanism and regulation of topoisomerase

activity in the cell.

Although a direct interaction of Hi with the topoisomerase may be

responsible for stimulating relaxation of the DNA, other possibilities

should be considered. As was mentioned earlier, Hi histone has a

specific ability to interact with superhelical DNA (55,63). Bina-Stein

and Singer have suggested this specificity is related to the binding of

HI at crossover points in the DNA (56). These crossover points occur at

a much higher frequency in a DNA molecule with superhelical character

(64). Because of its cationic character, HI may act to neutralize the

high negative charge density which would occur at a DNA crossover and

thus serve to stabilize an association between two duplex strands of

DNA. Perhaps the topoisomerase acts at a crossover structure in the












DNA. An ability to stabilize this structure may account for the ability

of Hi to stimulate topoisomerase activity.

Binding of HI at crossover points in the DNA has received addit-

ional support from chromatin studies. Based on electron microscopy and

nuclease digestion data, binding of HI to chromatin causes the DNA to

enter and exit a nucleosome at the same site resulting in a crossover

structure (64,65). This result is interesting in light of recent

studies which show the ability of DNA gyrase to form a nucleosome-like

structure with DNA (66). The ability to form similar types of struc-

tural complexes with DNA maybe a property common to all topoisomerases.

If so, Hi or Hl-like proteins may play an important role in regulating

topoisomerase activity because of their ability to interact with such

complexes.

Structures in the DNA other than a crossover, such as a left-

handed Z-helix (43,67), might also be stabilized by HI and serve as a

site for topoisomerase action. The formation of a Z-configuration in

alternating d(pCpG) sequences has been shown to be facilitated by salt

or by the DNA being negatively supercoiled (44,68). Because of its high

charge, Hi may be able to substitute for the high concentrations of salt

ordinarily required to induce DNA into a Z-form. This idea is supported

by the observation that a large change in conformation, as monitored by

a partial inversion of the circular dichroism spectrum, accompanies the

binding of HI to DNA (69) at the moderate salt concentrations (0.14 M)

where topoisomerase activity is high. When the alternating sequence

d(pCpG) undergoes a salt-induced transition from a B-form to a Z-form,

an even more dramatic change takes place as is evidenced by a full







46



inversion of the spectrum. Possibly binding of HI to superhelical DNA

helps to stabilize short stretches of DNA in the Z-form providing a

target for topoisomerase action.
















CHAPTER III
PURIFICATION AND PROPERTIES OF Hi-LIKE PROTEINS FROM

Ustilago maydis

The effect of HI histone on the ability of the Ustilago

topoisomerase to relax superhelical DNA may represent an important

mechanism for controlling DNA structure. However, because the HI

histone used to stimulate the topoisomerase had been obtained from calf

thymus, some doubt remained as to whether a protein with properties

similar to HI histone existed in Ustilago. The results of the

investigator's efforts to identify an Hl-like protein in Ustilago are

presented in this chapter.

An Endogenous Factor in the Topoisomerase
Preparation Stimulates Relaxing Activity

Greater than 99% of the topoisomerase activity present in crude

extracts of Ustilago was lost during the course of enzyme purification

(refer to Table I). In contrast, Vosberg and Vinograd found they could

obtain good yields of highly purified topoisomerase from LA9 cells, rat

liver and calf thymus (70). Interestingly, the major polypeptide present

in their highly purified enzyme was HI histone. If HI histone was neces-

sary for good topoisomerase activity, a separation of HI or an Hi-like

protein from the Ustilago topoisomerase during the purification pro-

cedure might have accounted for the high losses in activity. Such a

separation may have been responsible for the large loss in activity which

resulted when the topoisomerase was chromatographed on phosphocellulose.

An effort was therefore made to determine if HI histone was present in the











enzyme fraction prior to phosphocellulose chromatography. Because of

its high lysine content, HI histone can be easily purified from most

other proteins by its ability to remain soluble in 5% perchloric acid

(71). When an aliquot from fraction IV of the topoisomerase purifica-

tion was extracted with 5% perchloric acid, the acid soluble portion was

found to contain a component which stimulated topoisomerase activity

(Fig. 12). By itself, the acid-soluble fraction contains no topoisomer-

ase activity. Although maximal stimulation of the topoisomerase by the

acid-soluble fraction occurred at pH 6.0 in sodium acetate buffer, good

stimulation was also observed at pH 7.5 in Tris-HC1. Electrophoresis of

this fraction through an SDS polyacrylamide gel revealed a faintly

staining band with a corresponding molecular weight of 20,000 (Fig. 12

B). However, the low amount of protein loaded onto the gel made it

difficult to detect minor protein species. Therefore, it was unclear if

the 20,000 dalton protein was responsible for stimulating the topoisom-

erase.

Purification of an HI-like Protein from Ustilago

The stimulation of the Ustilago topoisomerase by an endogenous acid-

soluble component suggested the presence of an Hl-like protein in

Ustilago. Acid extraction procedures have been successfully used to

isolate Hl-like proteins from several lower eukaryotes (72-76). Several

laboratories have isolated an Hl-like protein from Saccharomyces by

extracting with 5% perchloric acid (72,73). Using a modification of this

procedure, it was possible to isolate an HI-like protein from Ustilago.

The scheme used to isolate these proteins is outlined in Figure 13. The

sulfhydral reagents p-chloromercuriphenyl sulfonate and 5,5'-dithiobis

(2-nitrobenzoate) were initially included in the extraction buffers














S-4 *

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4-1 0 W '0 n'I) U) 0 4.J0 W B S 0
0 0)0 MON bO 0)@
cac Q) k Q0 c*c4 C C Oo (o
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u 4-i = 0 Wd 0 C) -)W 0-4 0 0 o00V
ca 0)J w u 0 v c 0 I v -4

r 9O 0 Cn r-4 CO (L) 0 0 E 0 u
4 cO r- -k 0 W m Q 4J M :) ca < 0
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SO04UO j

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Figure 13. Procedure for isolation of HI histone from Ustilago.

All steps of the purification were carried out between 0-4C.
Cells freshly harvested (100 g) were washed 1 time in 200 ml 10 mM Tris,
pH 7.5, 1 mM EDTA, 0.35 M NaCl. The cells were then resuspended in 300
ml of the same buffer and crushed at 20,000 psi by passage through a
French pressure cell. The broken cells were then centrifuged in the JA
20 rotor of a Beckman J21-C centrifuge at 3500 rpm for 30 min. The
supernatant was discarded and the pellet resuspended and washed 3 times
in the above buffer. The pellet was then resuspended in 150 ml of 5%
perchloric acid. After standing 10 min the suspension was centrifuged
and the supernatant saved. This was repeated one more time and the
pellet discarded. To the combined supernatants (= 300 ml) was then added
65 ml of 100% trichloroacetic acid (final concentration = 18%). After
standing for 10 min the resulting white precipitate was pelleted by
centrifugation. The supernatant was discarded and the pellet washed 1
time with acid-acetone and 2 times with acetone. Residual acetone was
removed by placing the pellet under vacuum. The yield of protein was
approximately 1 mg.



















Cells


Breakage in
0.35M salt


Supernatant


Sediment


Supernatant


Sediment


5% Perchloric acid
extraction



Supernatant
18% Trichloroacetic acid




Sediment
= 30K protein











to prevent proteolysis (77). However, since the yields of the Hl-like

protein did not appear to be dramatically improved, these reagents were

subsequently left out. An average of 1 mg of pure protein could be

obtained from 100 g of cells.

The purity and size of the acid-extracted Hi-like protein was deter-

mined by electrophoresis through a SDS polyacrylamide gel (Fig. 14). By

this criterion the Ustilago protein is greater than 95% pure and has a

molecular weight of 30,000. Under these electrophoretic conditions it

comigrates with calf thymus Hi histone. The two heavy staining bands for

calf thymus Hi represent several different structural forms of HI histone

(57,58). The single staining band for the Ustilago Hi-like protein

suggests that lower eukaryotes lack some of these structural forms. Other

alternatives are that structural forms of HI were lost or degraded during

the purification of the Hi-like protein or that the gel system used was

unable to resolve the different structural forms which might be present.

The ability of the Hl-like protein from Ustilago to bind single-

stranded DNA was tested and found to be strongly dependent upon pH (Fig.

15). Binding at pH 4 or 6 was half that observed at pH 5 where binding was

found to be optimal. Essentially no binding of DNA occurred at pH 8.

One of the hallmarks of HI histone is its special ability to bind

superhelical DNA (55). The ability of the Ustilago protein to bind super-

helical and relaxed forms of DNA was compared. As illustrated in Figure

16, binding of the Hl-like protein to superhelical DNA was as much as

5-fold higher than to relaxed DNA which was in good agreement with the

value reported for calf thymus Hi histone (78).































Figure 14. SDS polyacrylamide gel of HI histone from Ustilago.

A 15% SDS polyacrylamide gel was prepared as described in Appendix
E. A, Ustilago Hi histone (5pg); B, calf thymus HI histone (10 pg).














B

+- 94
68
c43


-*-25.7


A

































Figure 15. pH dependent binding of the Ustilago Hi to
single-stranded DNA.

Reactions (50 pl) containing either 20 mM potassium phosphate (e)
or sodium acetate (0) and 1 mM EDTA, 2 mM 2-mercaptoethanol, 5% glycerol,
50 mM NaCl, 1.5 nmol 4X phage [3H] DNA and 1 Pg of Ustilago HI histone
were incubated at 25C. After 10 min reactions were terminated by adding
1 ml of the reaction buffer. The samples were then filtered through
nitrocellulose filters and processed as described in Appendix C.
Activity was determined relative to the value obtained at pH 5. At this
pH approximately 80% of the input DNA was retained on the filter.





















100


75
u.
50


S25


4.0 5.0 6.0 7.0 8.0


pH

































Figure 16. Ability of the Ustilago H1 histone to bind superhelical
DNA.

Reaction mixtures (50 pl) containing 50 mM sodium acetate, pH 6.0,
0.1 M, NaC1, 5 mM 2-mercaptoethanol, 5% glycerol and either fa RF I
[3H] DNA (*) or fa RF IV [3H] DNA (o), were incubated with Ustilago
H1 histone at 25C. Reactions were stopped by the addition of 1 ml of
reaction buffer and processed as described in Appendix C. Under these
conditions, 1 Ug of Ustilago Hl retained 0.1 nmol of fa RF I DNA to a
nitrocellulose filter.


















0Ioo

CD
m
C


Q0)

0


0.4 0.6 0.8
Protein (jg)












The Hl-like protein stimulates topoisomerase activity! However,

stimulation is pH-dependent (Fig. 17). At pH 7.5, where the Hi-like

protein exhibits little binding to DNA, only slight stimulation of

topoisomerase activity is observed. However, at pH 6, where its binding

to DNA is high, the Hl-like protein stimulated topoisomerase activity

greater than 5-fold. Although the stimulatory protein displayed no

relaxation activity in the absence of added topoisomerase, low levels of

endonuclease activity were detectable at pH 6. The ratio of Ustilago

Hi-like protein to DNA required to obtain significant stimulation of the

topoisomerase was 0.25 (w/w). This is 5-fold higher than the ratio of

calf thymus Hi to DNA necessary to induce a similar level of stimulation

at pH 6. Unlike the Ustilago protein, calf thymus HI effectively binds

to DNA over the entire pH range from 6 8 (unpublished results, 78).

It is also able to stimulate the Ustilago topoisomerase at both pH 6 and

7.5. This suggests that stimulation of the topoisomerase by either calf

thymus Hi or the Ustilago Hi-like protein requires their ability to bind

DNA.

A 25,000 Dalton Acid-Soluble Protein also
Stimulates Topoisomerase Activity

Another acid-soluble protein has been isolated from Ustilago which

also is able to stimulate topoisomerase activity. This protein was

purified from the 0.35 M salt supernatant normally discarded during the

purification of the Hl-like protein (refer to Fig. 2). Extraction of

the 0.35 M salt supernatant with acid and subsequent chromatography of

the acid-soluble fraction on carboxymethylcellulose yielded a homogenous

preparation of this protein (refer to Appendix D for details of the






























Figure 17. Stimulation of topoisomerase activity by Ustilago Hi
histone.

Reactions (25 1i) containing either 25 mM Tris-HC1, pH 7.5 (lanes
A, B, E, F, I) or 25 mM sodium acetate, pH 6.0 (lanes C, D, G, H, J), and
10 mM MgC12, 0.5 mM 2-mercaptoethanol, 5% glycerol, 10 pg/ml BSA and the
following additions were incubated at 25C. After 30 min the reactions
were stopped by adding EDTA to 15 mM and SDS to 0.5%. The products of
the reactions were then analyzed by electrophoresis through a 1.2%
agarose gel as described in Appendix C. A and C, 2 ng topoisomerase; B
and D, 10 ng topoisomerase; E, 2 ng topoisomerase, 0.25 yg Ustilago HI
histone; F, 2 ng topoisomerase, 1.0 pg Ustilago HI histone; G, 2ng
topoisomerase, 0.25 pg Ustilago HI histone; H, 2 ng topoisomerase, 1.0 ig
Ustilago HI histone; I and J, 2 ng topoisomerase, 50 ng calf thymus HI
histone.



















O u, O O
in o 0 X 0 O 0 0
a a" o ( o o
CL o I r L I + +
I- 0. 0. + 0-. + + .+


AB CD E F G H


I J











purification). As determined from electrophoresis through a 15% SDS

polyacrylamide gel, it has a molecular weight of 25,000. This is very

similar to the molecular weight of one of the Ustilago DNA binding

proteins, DBP IV (refer to Appendix D). A subsequent comparison of the

electrophoretic mobilities of DBP IV and the 25,000 dalton acid-soluble

protein showed them to be nearly identical (Fig. 18). If DBP IV is

structurally related to the 25,000 dalton protein it should be soluble

in acid. To test this, DBP IV was extracted with 5% perchloric acid and

the acid-soluble and acid-insoluble fractions subsequently electrophor-

esed through a SDS polyacrylamide gel. As is evident from Figure 19,

DBP IV appears only in the acid-soluble fraction.

DBP IV strongly stimulated the Ustilago topoisomerase. Similar to

calf thymus Hl, but unlike the Ustilago HI-like protein, DBP IV stimu-

lated topoisomerase activity at both pH 6 and 7.5 (Fig. 20). DBP IV

also displayed an ability to bind to DNA equally well at both of these

pHs (Fig. 21). At a protein to DNA ratio of 0.05 (w/w) DBP IV was

equally as effective as calf thymus HI histone in stimulating topoisom-

erase activity.

Conclusions

An Hl-like protein purified from Ustilago stimulates relaxation of

superhelical DNA by the topoisomerase. On SDS polyacrylamide gels it

comigrates with calf thymus histone HI. Like calf thymus HI the

Ustilago protein is soluble in 5% perchloric acid and shows a preferen-

tial binding to superhelical DNA (55). In contrast to calf thymus H1,

the Ustilago Hl-like protein binds DNA in a pH-dependent fashion (78).

The ability of the Hi-like protein to stimulate topoisomerase activity
































Figure 18. Electrophoresis of a 25,000 dalton acid-soluble protein
and DBP IV on a SDS polyacrylamide gel.

A 15% SDS polyacrylamide gel was prepared and run as described in
Appendix E. Protein was visualized using the silver staining technique.
Molecular weight markers included BSA, ovalbumin, carbonic anhydrase and
soybean trypsin inhibitor. A, DBP IV (1.0 Pg); B, acid-soluble protein
(3 Pg).










AB

68 K
c-43 K
30 K

-- 21 K
































Figure 19. Acid-solubility of DBP IV.

Approximately 5 yg of DBP IV from fraction II of the purification
was extracted with perchloric acid and analyzed as described in the
legend to Figure 12. Protein was visualized using the silver staining
technique as described in Appendix E. Molecular weight markers included
ovalbumin, a -chymotrypsinogen and B-lactoglobulin. A, Perchloric acid
precipitate; B, Perchloric acid supernatant.










AB

43 K
e--25.7 K
8.4 K

































Figure 20. Stimulation of the topoisomerase by DBP IV.

Reactions (25 pl) containing 25 mM Tris-HC1, pH 7.5 (lanes A, B, C,
G, H) or 25 mM sodium acetate, pH 6.0 (lanes D, E, F, I), and 10 mM
MgC12, 0.4 mM 2-mercaptoethanol, 5% glycerol, 10 ig/ml BSA and the
following additions were incubated at 25C. After 30 min the reactions
were terminated and the products analyzed as described in the legend to
Figure 17. A and D, 2.5 ng topoisomerase; B and E, 12.5 ng topoisomer-
ase; C and F, 2.5 ng topoisomerase, 100 ng DBP IV; G and H, 100 ng DBP
IV; I, no additions.






69









AB C DEFG H I

































Figure 21. Effect of pH on the ability of DBP IV to bind
single-stranded DNA.

Reaction (50 1i1) containing either 20 mM potassium phosphate (e) or
sodium acetate (o) and 1 mM EDTA, 2 mM 2-mercaptoethanol, 5% glycerol, 50
mM NaCI, 1.5 nmol 4Xphage [3H] DNA (2x 107 cpm/pmol) and 250 ng DBP
IV were incubated at 250C. After 10 min, the reactions were terminated
and analyzed as was described in the legend to Figure 15. Activity was
determined relative to the value obtained at pH 7.5. At this pH, DBP IV
caused the retention of approximately 92% of the DNA to the nitrocellu-
lose filter.

























100-


75


50


25


4.0 5.0 6.0 7.0 8.0















is also pH dependent and directly correlates with its ability to bind

DNA. This suggests an interaction between the Ustilago HI-like protein

and DNA is necessary for stimulation of the topoisomerase.

HI-like proteins have been isolated from several other fungi and

the amino acid contents of these proteins are known (72-76). Therefore,

an amino acid analysis of the Ustilago H1-like protein should provide

important information concerning its structure and identity.

During the course of purifying the Hl-like protein, a second

acid-soluble protein was discovered which also showed an ability to

stimulate topoisomerase activity. This 25,000 molecular weight protein

was present in the 0.35 M salt supernatant which was normally discarded

during the purification of the Hl-like protein. The purification

scheme for the 25,000 dalton protein was very similar to a procedure

used by Johns to isolate high-mobility group (HMG) proteins (79-81).

HMGs are highly charged, low molecular weight proteins which are thought

to play an important role in controlling DNA structure (82-84).

Additional studies have been undertaken to determine if the 25,000

dalton Ustilago protein is an RMG. These studies have been encouraged

by recent work in another laboratory which showed that HMG 17 strongly

stimulated relaxation of superhelical DNA by a type I topoisomerase from

HeLa cells. There has also been a report that topoisomerase activity

has been found in association with HMG 14 and HMG 17 in actively

transcribing regions of chromatin (84).

The 25,000 dalton protein may be structurally related to a

Ustilago DNA binding protein, DBP IV. Their molecular weights were

found to be identical when determined by electrophoresis through SDS







73



polyacrylamide gels. Like the 25,000 dalton protein, DBP IV was soluble

in 5% perchloric acid. In addition, DBP IV showed a similar ability to

stimulate the Ustilago topoisomerase. Studies aimed at elucidating the

primary structure of these two proteins are in progress and should

reveal if they are indeed related.















CHAPTER IV
CHARACTERIZATION OF THE COVALENT TOPOISOMERASE-DNA LINKAGE

One of the trademarks of topoisomerases is their ability to form a

transient covalent complex with DNA (1-3). Breakage of DNA by prokary-

otic topoisomerases and eukaryotic type II topoisomerases results in the

formation of a covalent complex between the enzyme and the 5' end of the

DNA break (6,12). Eukaryotic type I topoisomerases, on the other hand,

form a covalent complex with the 3' end of the DNA break (13,14). Both

eukaryotic and prokaryotic enzymes are linked to the DNA through a phos-

photyrosine bond (9,10). The energy conserved in the phosphotyrosine

linkage is thought to drive the reclosing of the transient break in the

DNA.

Although the mechanism of action for topoisomerases isolated from

prokaryotic as well as higher eukaryotic organisms has been well studied,

no such studies have been undertaken in lower eukaryotic organisms. This

is unfortunate since lower eukaryotic organisms provide such an ideal

genetic setting for testing the relevance of biochemical studies. The

following work represents an initial effort to bridge this present gap in

biochemical knowledge.

Detection of the Covalent Complex

Under conditions of low salt ((50 mM), rat liver type I topoisomer-

ase breaks circular single-stranded phage DNA to form a linear DNA

molecule with enzyme covalently attached at the 3' end. Subsequent

incubation of the DNA-protein complex in the presence of 0.25 M salt or

10 mM MgC12 leads to the recircularization of the DNA (39).










The Ustilago topoisomerase also breaks circular single-stranded

phage DNA to form linear DNA molecules with protein tightly attached.

Conversion of circular single-stranded DNA to a linear form can be easily

assessed by electrophoresis of the DNA through agarose gels containing 30

mM NaOH. Topoisomerase was incubated with circular single-stranded f3

DNA at pH 9.0 in reactions containing low concentrations of salt (<50

mM). Analysis of the products of this reaction on an alkaline agarose

gel revealed a significant decrease in the amount of circular DNA present

with a corresponding increase in a DNA species which migrated slightly

slower than unit-length linear single-stranded fa DNA (Fig. 22 A).

Treatment of the reaction products with 0.5% SDS prior to electrophoresis

did not alter the gel pattern (data not shown). However, if the reaction

products were treated with proteinase K prior to electrophoresis, the DNA

band present in lane A vanished and a new band appeared having an

electrophoretic mobility identical to linear single-stranded fa DNA (Fig.

22 B).

Attempts to recircularize the fa DNA which had been broken by the

Ustilago topoisomerase were unsuccessful. Reactions containing circular

single-stranded f a DNA previously broken by the topoisomerase were

incubated in the presence of 0.2 M salt or 10 mM MgC12 for 2 hr at

25C. Subsequent electrophoresis of the reactions on alkaline agarose

gels revealed no significant recircularization of the DNA (data not

shown).

Identification of the Protein Complex to the DNA

The inability to disrupt the complex formed between the Ustilago

topoisomerase and single-stranded DNA, by treatment with 30 mM NaOH or































Figure 22. Covalent attachment of the topoisomerase to DNA.

Reactions (40 Pl) containing 10 mM Tris-HCl, pH 9.0, 1 mM EDTA, 0.2
mM dithiothreitol, 50 lg/ml BSA, 5% glycerol, 2.4 nmol fa phage DNA, and
either 1 Pg of topoisomerase (lanes A and B) or no topoisomerase (lane C)
were incubated at 25"C for 20 min. Reactions A and C were terminated by
adding 4 1I of 0.3 M NaOH. Reaction B was treated with proteinase K (100
Pg/ml) for 30 min at 370C before adding 4 pl of 0.3 M NaOH. Fifteen pl
of each reaction was loaded onto a 1.2% agarose gel containing 30 mM
NaOH, 0.5 mM EDTA. Electrophoresis was at 5.5 v/cm for 16 hr at 5'C.
Following electrophoresis the gel was stained with 1 pg/ml eithidium
bromide before photographing under ultraviolet light.










ABC











0.5% SDS suggested that a covalent protein-DNA linkage was involved. To

determine the identity of the protein linked to the DNA, the Ustilago

topoisomerase was incubated with denatured 4X174 DNA which had been

labeled by nick translation with [a32P] dCTP. The resulting DNA-

protein complex was digested with Micrococcal nuclease and then extracted

with phenol. The protein in the phenol phase was recovered by acetone

precipitation and electrophoresed through an SDS polyacrylamide gel. The

gel was then dried down on Whatman 3 MM paper and autoradiographed. The

three radiolabeled protein bands present in Figure 23 have molecular

weights of 100,000, 110,000 and 130,000. When the radiolabeled protein

was treated with proteinase K prior to electrophoresis, no radioactive

bands were detectable on the autoradiograph (data not shown) confirming

that the bands present in Figure 23 represented proteins tightly coupled

to short pieces of DNA.

Eukaryotic type I topoisomerases are highly susceptible to proteo-

lytic degradation during enzyme purification and this may explain why

there is more than one radiolabeled protein present in the autoradiograph

in Figure 23. Eukaryotic type I topoisomerases isolated from HeLa cells

and Drosophila melanogaster, under conditions which minimized proteolytic

degradation, yielded single polypeptides with molecular weights of

120,000 and 110,000 respectively (2). Perhaps greater precautions

against proteolytic degradation during the purification of the Ustilago

topoisomerase will yield a single polypeptide.























Figure 23. Analysis of the enzyme-oligonucleotide complex by SDS
gel electrophoresis.

The procedures used to label and denature the DNA are described in
Appndix B. A 150 pl reaction containing 6 mM Tris-HC1, pH 9.0, 0.2 mM
dithiothreitol, 50 pg/ml BSA, 150 pmol denatured nick translated [3H]
PBR DNA (3.3 x 107 cpm/nmol) and 1 pg topoisomerase was incubated at
25"C for 20 min. The reaction was stopped by adding NaOH to 50 mM and
incubating for 5 min at 370C. The reaction was then made 30 mM in
Tris-HC1 and the pH titrated to 9.0 with HC1. After adding CaC12 to a
final concentration of 2 mM, 0.1-0.2 units of Micrococcal nuclease were
added. After incubating at 370C for 1 hr the reaction was extracted with
1 volume of phenol. The phenol phase was saved and extracted 6 times
with equal volumes of 20 mM Tris-HC1, pH 7.5, 1 mM EDTA. The protein-
oligonucleotide complex was precipitated from the phenol phase by adding
10 volumes ice-cold acetone and placing the sample in a dry ice-ethanol
bath. After 20 min the sample was centrifuged and the resulting pellet
resuspended in sample buffer. After heating at 100%C for 3 min, the
sample was analyzed by electrophoresis through a 7.5% SDS polyacrylamide
gel as described in Appendix E. Following electrophoresis the gel was
stained with Coomassie blue to visualize the marker protein. The gel was
then dried down on Whatman 3 mm paper and autoradiographed using Kodak
X-Omat AR 5 film with a Dupont lightning-plus intensifying screen at
-70"C.












205 K
1I 16K
94 K
68 K


43 K

M-- 30 K


S--- tracking dye












Topoisomerase Links to the 3' End of the DNA Break

Two approaches were taken to determine whether the Ustilago topoi-

somerase was linked to the 3' or 5' end of the broken strand of DNA. In

the first approach [3H] labeled circular single-stranded fa DNA, which

had been broken by the topoisomerase, was treated with exonuclease I or

exonuclease VII. If breakage of the DNA by the topoisomerase produces a

free 3' hydroxyl terminus the broken DNA should be susceptible to diges-

tion by both exonuclease I and exonuclease VII. However, if breakage

produces a free 5' hydroxyl terminus the broken fragment of DNA should

only be susceptible to digestion by exonuclease VII. The results of this

experiment are presented in Table V and show that breakage of the DNA by

the Ustilago topoisomerase produces a free 5' terminus suggesting that

the topoisomerase is linked to the 3' side of the DNA break. This result

also points out that cleavage of DNA by the topoisomerase occurs under

normal reaction conditions and is not simply an artifact of stopping the

reaction with alkali or detergent.

To determine whether the topoisomerase was linked to DNA at the 3'

or 5' end, complexes formed between f phage [3H] DNA (2 x 104

cpm/nmol) and topoisomerase were treated with exonuclease I (exo I) or

exonuclease VII (exo VII). A 75 pl reaction containing 10 mM Tris-HC1,

pH 9.0, 0.2 mM dithiothreitol, 50 pg/ml BSA, 5 nmol f0 phage [3H] DNA

and 1 pg topoisomerase was incubated at 250C. After 10 min three 20 pl

aliquots were removed and analyzed as described in Appendix C. One ali-

quot was diluted into a 200 pl reaction containing 0.8 units of exonucle-

ase I. Another aliquot was diluted into a 200 p1 reaction containing 0.6

units of exonuclease VII. The third aliquot was electrophoresed through a















TABLE V
DIGESTION OF TOPOISOMERASE-DNA COMPLEXES WITH EXONUCLEASE I AND
EXONUCLEASE VII



Acid Solubility
Percent Circular DNA
Exo I Exo VII Percent


Control 5 9 100

Topoisomerase 6 58 41











1.2% alkaline agarose gel as described in the legend to Figure 22. A

control reaction containing no topoisomerase was processed in the same

manner. The percent circular DNA present was determined from densitom-

eter tracings of the photographic negative of the agarose gel.

The attachment of the topoisomerase to the 3' end of a broken DNA

strand was shown in another way. Form III fa DNA, produced by digesting

f RFI DNA with the restriction endonuclease Sau 96, was labeled with

[Y-32p] ATP at its 5' ends using T4 polynucleotide kinase. The DNA

was denatured and incubated with the Ustilago topoisomerase. Electro-

phoresis of the reaction products on an alkaline agarose gel revealed

several major bands (Fig. 24 B). If the reaction products were treated

with proteinase K prior to electrophoresing the mobilities of these bands

were shifted (Fig. 24 C). This is what would be expected if the topoi-

somerase was linked to the 3' end of the broken DNA strand. When the

same experiment was carried out with DNA labeled with [y-32P] dCTP at

its 3' ends, using the large Klenow fragment of E. coli DNA polymerase,

no shift in the mobility of the reaction products was observed following

treatment with proteinase K (Figs. 24 E and F). The results from this

study in combination with the exonuclease digestion results provide

strong evidence for the tight attachment of the Ustilago topoisomerase to

the 3' and of the broken DNA strand.

Breakage of end-labeled DNA by the topoisomerase produces discrete

bands (Figs. 24 B, C, E, F) inferring that cleavage of the DNA is site-

specific. Type I topoisomerases from HeLa cells, calf thymus and rat

liver also cleave DNA in a site-specific fashion (85,86). As determined

from DNA sequencing, the cut sites on SV40 DNA for the HeLa cell and calf






























Figure 24. Proteinase K treatment of topoisdmerase completed with
DNA labeled at the 3' or 5' end.

Form III fa DNA, prepared by digesting form I fa DNA with the
restriction endonuclease Sau 96, was labelled with [32P] at the 3' or
5' end as described in Appendix B. Denaturation of the DNA prior to
reacting with the topoisomerase was carried out as described in Appendix
B. Reactions (40 pl) containing 10 mM Tris-HCl pH 9.0, 0.2 mM dithio-
threitol, 50 pg/ml BSA, and 0.3 nmol of denatured 5' (lanes A-C) or 3'
(lanes D-F) [3P] end labeled DNA were incubated with (lanes B, C, E,
F) or without (lanes A and D) 1 ig of topoisomerase at 250C for 30 min.
Reactions were terminated by adding 4 pl of 0.3 M NaOH. Prior to adding
NaOH, samples in lanes C and F were treated with proteinase K (100 pg/ml)
for 30 min at 37"C. Twenty pl of each sample was analyzed by electro-
phoresis through a 1.6% agarose gel containing 30 mM NaOH, 1 mM EDTA at
10 v/cm for 5 hr at 5"C. Following electrophoresis the gel was soaked
for 10 min in 500 ml of 0.1 M Tris-HC.1 pH 7.5 before it was dried down on
Whatman 3 mm paper. Autoradiography was carried out as described in the
legend to Figure 23.














5'
ABC


3'
DE F












thymus topoisomerases are identical. However, there does not appear to

be a consensus sequence for determining cleavage (87). It has been

suggested that secondary structure may be a major factor contributing to

the site-specific cleavage of DNA by type I topoisomerase (86).

Identification of the Covalent Linkage

Both eukaryotic and prokaryotic topoisomerases form covalent com-

plexes with DNA involving a phosphotyrosine bond (9,10). To determine if

the Ustilago topoisomerase was covalently linked to DNA through a

phosphotyrosine bond, enzyme was complexed to denatured
which had been labeled by nick translation with [Y-32P] dCTP.

Following digestion with Micrococcal nuclease, the complexes were

purified by phenol extraction and acetone precipitation as described in

the legend to Figure 23. The purified protein-oligonucleotide complexes

were then hydrolyzed in 5.6 M HC1 at 1100C for 2 hr and the products of

hydrolysis analyzed by high voltage paper electrophoresis (Fig. 25).

Under these conditions of hydrolysis both DNA and protein should be

broken down to produce inorganic phosphate and amino acids respectively.

However, a phosphotyrosine bond, if present, should be stable. Almost

all of the radioactivity present in the autoradiograph in Figure 25 is

contained in two spots. The upper spot comigrated with inorganic

phosphate while the lower remaining spot of radioactivity comigrated with

the phosphotyrosine marker suggesting that the covalent linkage between

the Ustilago topoisomerase and DNA involves a phosphotyrosine bond.

Discussion

In summary, the Ustilago topoisomerase breaks single-stranded DNA

in low salt forming a tight complex with the 3' side of the DNA break.





























Figure 25. Paper electrophoresis of the [32p] labeled
topoisomerase-oligonucleotide complex following acid hydrolysis.

The topoisomerase-oligonucleotide complex was obtained as described
in the legend to Figure 23. The complex which had been precipitated from
the phenol phase was resuspended in 40 tl of 5.6 M constant boiling HC1.
The sample was sealed in a 1.4 ml Eppendorf tube under a nitrogen
atmosphere before heating in a 110%C oil bath. After 2 hr the sample was
cooled and the HCl removed in vacuo in the presence of NaOH pellets. The
resulting residue was resuspended in 30 1l of H20 containing 100 nmol
each of phosphotyrosine, Tyr(P), phosphoserine, Ser(P), and phosphothre-
onine, Thr(P), and 500 nmol of sodium phosphate pH 7.0, Pi. The sample
was analyzed by paper ionophoresis as described in Appendix E.
Autoradiography was carried out as described in the legend to Figure 23.
The dashed lines represent the locations of the marker compounds which
were visualized as described in Appendix E.







88












This complex probably involves a covalent linkage since neither alkali or

detergent were able to disrupt it. In addition, acid hydrolysis of the

radiolabeled complex yielded a radioactive product which coelectrophor-

esed with authentic phosphotyrosine implying that the linkage was not

only covalent, but that it involved a phosphotyrosine bond.

The Ustilago enzyme, in contrast to the rat liver type I topoisom-

erase, appears to be unable to reseal the break which it introduces into

single-stranded DNA. Circular single-stranded f DNA broken by the

Ustilago topoisomerase does not recircularize in the presence of either

0.2 M salt or 10 mM MgC12, conditions which are normally optimal for

the nicking-closing activity of this enzyme on duplex superhelical DNA.

Perhaps the problem of reclosure is not an enzymatic one but reflects the

low frequency with which the two ends of the broken strand come into

close enough alignment for religation to occur. This would not be a

problem in a duplex DNA molecule where the stable secondary structure of

the DNA would be expected to maintain the ends of a break in close align-

ment ensuring efficient reclosure of the transient break created by the

topoisomerase.

Topoisomerases join a host of other proteins in being able to form

a covalent protein-nucleic acid linkage. The poliovirus encoded protein

VPg forms a covalent linkage with the 5' end of poliovirus RNA. Like

topoisomerases, this covalent linkage involves a phosphotyrosine bond

(87,88). Covalent protein-nucleic acid complexes have also been

described for a number of other viruses (89-91). These covalent com-

plexes are thought to play an important role in the initiation of repli-

cation. Whether topoisomerases can function in a similar manner is at




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