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Characterization of a topoisomerase from Ustilago Maydis

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Title:
Characterization of a topoisomerase from Ustilago Maydis
Added title page title:
Topoisomerase from Ustilago Maydis
Added title page title:
Ustilago Maydis
Creator:
Rowe, Thomas Cardon, 1951-
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Language:
English
Physical Description:
xi, 121 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Circles ( jstor )
DNA ( jstor )
Duplexes ( jstor )
Electrophoresis ( jstor )
Enzymes ( jstor )
Gels ( jstor )
Histones ( jstor )
pH ( jstor )
Purification ( jstor )
Ustilago ( jstor )
DNA Recombinant ( mesh )
DNA Replication ( mesh )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( 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.
General Note:
Photocopy of typescript.
General Note:
Vita.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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09301196 ( OCLC )
ABY0600 ( NOTIS )

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Full Text



















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




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
FOR THE
FULFILLMENT OF THE REQUIREMENTS
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


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/characterizationOOrowe


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.
111


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 HI Histone 37
III. PURIFICATION AND PROPERTIES OF Hl-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
iv


Page
IV. CHARACTERIZATION OF THE COVALENT TOPOISOMERASE-
DNA LINKAGE 74
Detection of the Covalent Complex 74
Identification of the Protein Complexed to
the DNA 75
Topoisomerase Links to the 3' End of the DNA Break 81
Identification of the Covalent Linkage 86
Discussion 86
V. INVOLVEMENT OF TOPOISOMERASE IN GENETIC RECOMBINATION 91
The Ustilago Topoisomerase Recombines
Complementary Single-Stranded Rings of DNA .... 91
Relaxing Activity Reduced in the rec 2 Mutant. ... 92
Discussion y95
APPENDICES
A. PHAGE, BACTERIAL AND FUNGAL STRAINS 100
B. PREPARATION OF DNA 101
C. ASSAYS 104
Topoisomerase 104
DNA-Binding Protein 105
Exonuclease 106
Protein 106
D. PROTEIN PURIFICATION 107
Topoisomerase 107
25,000 Dalton Acid-Soluble Protein 108
DBP IV 109
E. ELECTROPHORESIS 112
F. REAGENTS AND MATERIALS 113

Chromatographic Media 113
Enzymes 113
Miscellaneous 113
REFERENCES 115
BIOGRAPHICAL SKETCH 121
v


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
vi


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
vi i


Page
19. Acid-solubility of DBP IV 67
20. Stimulation of the topoi some rase 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 complexed
with DNA labeled at the 3' or 5' end 85
25. Paper electrophoresis of the [^P] 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
vi i i


KEY TO ABBREVIATIONS
ATP
adenosine-5 1-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 mil sodium citrate)
lx


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 Cardn Rowe
December 1982
\
x
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 Mg^+, but is totally inhibited by
Zn^+ at concentrations as low as 20 yM. The topoisomerase is active
over a broad pH range from 5.5 8.0. The optimal temperature for
activity is 20 30C.
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 Hl-like protein has been isolated from Ustilago which
also markedly stimulates the topoisomerase. This interaction may be
important in regulating topoisomerase in cellular processes.
x


The topoisomerase breaks single-stranded DNA and
complex with the 3' end of the DNA break. The covalent
involve a phosphotyrosine bond.
The rec 2 mutant of Ustilago has decreased levels
activity possibly implicating this enzyme in eukaryotic
forms a covalent
linkage appears to
of topoisomerase
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, B,
and the number of superhelical turns, x, by the following simple
equation L = B + x (8). The linking number for a closed circular
1


2
molecule remains constant unless the DNA backbone becomes broken. There
are two basic ways topoisoinerases 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


3
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 y
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


4
(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_L 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
is also dependent on the superhelical state of the DNA (31).
Resolvase, an enzyme coded for by y6 carries out the final step in
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


6
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 J^n _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
8


9
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


Figure 1.
Topoisomerase assays.
Reaction mixtures (100 yl) containing 75 mM potassium phosphate, pH 7.5, 1 mM EDTA, 5% glycerol,
and 5 nmol of RF I [%] DNA (1.5 x 10^ cpra/nmol), and the indicated amounts of topoisomerase were
incubated at 25C. At the end of 30 min three 25 yl aliquots were removed from each reaction mixture
and processed as described in Appendix C. One aliquot was loaded onto a 1.2% agarose gel, the second
aliquot was mixed with 1 ml of buffer containing 3.5 M NaCl () and the third aliquot was combined
with 6.6 nmol of single-stranded DNA fragments and incubated at 75C for 2 1/2 hr to promote D-loop
formation (). The following amounts of enzyme were used in the lanes of the agarose gel: a, no
enzyme; b, 1.25 ng; c, 2.5 ng; d, 3.75 ng; e, 5 ng; f, 10 ng; g, 20 ng.


RF DNA Released from Filter (nmol)
O p o
fo ai m
o O cn
IT


12
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 1 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 KC1 and loaded directly on to an octy1-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 -20C 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


13
TABLE I
PURIFICATION OF THE ENZYME
Fraction
Volume
ml
Activity
units x 10-4
Protein
mg
Specific
Activity
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 S200^
8
3.4
0.40
85,000
aProtein determination was not performed since Triton interfered with the
assay.
^Total 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.


14
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 10^ to 1.2 x 10^
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.


Figure 2. Polyacrylamide gel electrophoresis of the topoisomerase preparation.
Polyacrylamide gels containing 5% acrylamide, 0.14% N,N'-methylenebisacrylamide, 0.1 M potassium
phosphate, pH 7.5, 10% glycerol, were cast in tubes (5 x 80 mm) with ammonium persulfate as catalyst.
The gels were prerun overnight with electrode buffer composed of 0.1 M potassium phosphate, pH 7.5, 10%
glycerol in order to remove ammonium persulfate. Electrode buffer was replaced, and 100 yl aliquots of
the topoisomerase preparation (500 units) containing 50% glycerol and 5 yg of fluorescamine-labeled
pancreatic ribonuclease (46) were layered on parallel gels. The samples were overlaid with 100 yl of
20% glycerol and then electrophoresed at 5 mA/tube. Cooling was provided by circulating ice water
around the tubes during the course of the run. After 12 h when the ribonuclease marker, located under
ultraviolet light, had migrated approximately halfway down the tube, the run was ended. One gel was
stained for protein with Coomassie brilliant blue. A parallel gel was cut into 2 mm slices, and each
slice was soaked in 100 hi of 50 mM potassium phosphate, pH 7.5, 1 mM EDTA, 5 mM 2-mercaptoethanol, 10%
glycerol, 0.1 M KC1. After 18 h each slice was assayed for topoisomerase activity by the agarose gel
method. Approximately 10% of the activity was recovered after electrophoresis. The rapidly migrating
material near slice number 15 is the ribonuclease marker.


O'
5 10
Slice Number
15


17
TABLE II
ESTIMATION OF SIZE OF THE TOPOISOMERASE
Activity
Kdl/3
Molecular
Recovery
KC1
Peak
We ight
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 -^H-labeled xl74
phage and [^2p] 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 K 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.


18
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 10^ 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-HCl and not measurable in either 4-(2-hydroxyethyl)-l-
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, Mg^+ proved to be much more
effective in promoting activity. In the presence of 10 mM MgCl2,


Figure 3. Centrifugation of topoisoraerase in a glycerol gradient.
Topoisomerase (500 units) together with 1 mg of bovine serum albumin (BSA) and 10 yg of catalase
in 0.1 ml was layered on top of a 5 ml 20 40% gradient of glycerol containing 20 mM potassium
phosphate, pH 7.0, 5 mM 2-mercaptoethanol, 0.5 M KC1. Centrifugation was at 4C for 22 h at 50,000 rpm
in the Beckman SW 50.1 rotor. At the end of the run fractions were collected from the bottom of the
gradient and assayed for topoisomerase activity by the agarose gel assay. Activity was determined by
visual inspection, and recovery was estimated at less than 1%. Catalase activity was located by assay
and bovine serum albumin by protein determination; peaks are indicated in arbitrary units. The
sedimentation coefficient and molecular weight of the topoisomerase were determined according to Martin
and Ames (50). Sedimentation is from right to left.


5 10 15 20
Fraction Number
ro
o


Figure 4. pH optima of the topoisomerase.
Reaction mixtures (50 pi) containing 1 mM EDTA, 1 mM 2-mercaptoethanol, 3 nmol RF I [^H] DNA
(1.5 x 10^ cpm/nmol), 1 unit of topoisomerase and 20 mM sodium citrate (D), Tris-HCl (A), potassium
phosphate (), HEPES ( ), or Imidizole (x) were incubated at 37C for 30 min. Reactions were stopped
by adding 2.5 pi 10% SDS and 10 pi 3 M NaCl. Each reaction was then split into two 30 pi aliquots and
processed as described in Appendix C. One aliquot was used to assess nicking activity. The pH of the
second aliquot was adjusted by adding 3 pi of 0.5 M Tris-HCl, pH 7.5 before adding 5 nmol of
single-stranded DNA fragments. After heating at 75C for 3 h, the amount of D-loop formation was
determined as described in Appendix C. >


N>
N)


Figure 5. Salt optima of the topoisomerase.
Reaction mixtures (25 pi) containing 25 mM Tris-HCl, pH 7.5, 10%
glycerol, 0.4 mM 2-mercaptoethanol, 3 nmol x RF I ^H] DNA (1.5 x
10^ cpm/nmol), 100 ng topoisomerase and various concentrations of KC1
were incubated at 25C for 30 min. Reactions were terminated by adding 1
ml of buffer containing 3.5 M NaCl and topoisomerase activity determined
using the high salt filter assay as described in Appendix C.


24
KCI (M)


25
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 Mg^+, Cu^+ and Zn^+ were strong
inhibitors of the topoisomerase. The presence of as little as 20 pM
CuCl2 or ZnCl2 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 42C (Fig. 6). Surprisingly, the enzyme
is fairly active at low temperatures with 70% relaxation occurring at
0C. 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 Vl) containing 75 mM potassium phosphate, pH 7.5, 10%
glycerol, 3 nmol XRF I [%] DNA (1.5 x 10^ cpm/nmol) and 40 ng of
topoisomerase were incubated for 30 min at the indicated temperatures.
Reactions were terminated by adding 10 pi 3 M KC1. Topoisomerase
activity was then determined using the D-loop assay as described in
Appendix C.


27


28
(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 ,8-methylene ATP, g,y-raethy-
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 ancj 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


29
TABLE III
INHIBITION BY NUCLEOTIDES AND POLYNUCLEOTIDES
Compound
-
Concentration
mM
Activity
Percent
Control
100
ATP
5.0
0
ADP
5.0
70
AMP
5.0
100
dATP
5.0
90
a,g-methylene ATP
1.6
50
a, 6-methylene ATP
3.2
0
B,y-raethylene ATP
1.5
100
B,y-methylene ATP
3.0
0
AMP-PNP3
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
X 174 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.


30
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 yg/ml of
nalidixic acid (Table IV). This is a 10-fold lower concentration


TABLE IV
INHIBITION BY ANTIBIOTICS
Drug
Concentration
yg/ml
Activity
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.


32
than is required to cause a similar inhibition of DNA gyrase (53).
While 200 Mg/ml of novobiocin are required to totally inhibit the
Ustilago enzyme, significant loss of activity is seen at 100 pg/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 yg/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 turnsgel assay.
Reaction mixtures (50 hi) containing 20 mM Tris-HCl, pH 7.5, 0.1 M
KC1, 5% glycerol, 0.8 yg/ml ethidium bromide and 3 nmoles <|>x RF I or
RF IV DNA were incubated in the presence or absence of topoisomerase at
25C. 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) XRF I DNA; (Lane B) <)>XRF IV DNA; (Lane C) xRF
IV DNA and 100 ng topoisomerase.


34
ABC
M
U


Fig. 8. Removal of positive superhelical turns.
The high salt binding assay was used for measuring removal of both negative and positive
superhelical turns. However, in the latter case an increase, rather than decrease, in the amount of
DNA bound to filters is a measure of removal of positive supercoils. When form I DNA alone was washed
through a nitrocellulose filter, as described, approximately 70% of the input radioactivity bound to
the filter. When form IV DNA was tested, 2% bound. If ethidium bromide was present there was no need
to extract it from the DNA as long as the solution was diluted sufficiently before filtering. Again
less than 2% of the radioactivity bound to a filter if a solution containing forra IV DNA and 1 yg/ml of
ethidium bromide was diluted 1 to AO in 3.5 M NaCl before filtering. A, rate of reaction. Two
reaction mixtures (150 yl) were set up under the standard condition, one containing 7.5 nmol of <¡>XRF
I[3H] DNA (2.2 x 10^ cpm/nmol) and the other containing 7.5 nmol of 4>XRF IV [3H] DNA (2.2 x
10^ cpm/nmol) and 1 yg/ml of ethidium bromide. Topoisomerase (300 ng) was added to each reaction,
and the mixtures were incubated at 25C. AT the indicated times aliquots of 25 yl were removed,
diluted into 1 ml of the 3.5 M NaCl buffer for binding, and processed as described. After 30 min of
reaction the amount of DNA bound to a filter after removal of positive superhelical turns reached a
maximum of 65% of the input form IV DNA. B, enzyme concentration curve. Reaction mixtures (25 yl)
containing 1.25 nmol of <|>XRF I [3H] DNA or XRF IV [3H] DNA plus 1 yg/ml of ethidium bromide and
the indicated amounts of topoisomerase were incubated at 25C for 30 min and then filtered as above.
(), negative superhelical DNA; (), positive superhelical DNA.


Time (min) Enzyme (ng)
Topoisomerase Activity
o o
ro CJ1
9£


37
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 yl) containing 40 mM Tris-HCl. pH 7.5, 10 mM
MgCl2> 10% glycerol, 3 nmol of X RF I [^H] DNA (1.5 x 10^
cpm/nraol), the indicated amounts of topoisomerase, and either 1 yg/ml of
histone HI () or 1 yg/ml of bovine serum albumin () were incubated at
25C. 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 NaCl
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.


Topoisomerase
o
en
(nmol
5
DNA released)
en
04


Figure 10. Stimulation of topoisomerase activity by histones and polyamines.
Reaction mixtures (25 yl) containing 25 mM Tris-HCl, pH 7.5, 10 mM MgCl^, 10% glycerol, 3 nmol
of <}>x RF I [3H] DNA, and the following additions were incubated at 25 C for 30 min. Reactions were
terminated by adding 5 yl of 2% SDS in 0.12 M EDTA and proteinase K to 100 yg/ml. After 30 min at 37C
samples were loaded and run on an agarose gel as described in Appendix C. (a) 10 ng of topoisomerase;
(b) 20 ng of topoisomerase; (c) 50 ng of topoisomerase; (d) 10 ng of topoisomerase, 1 yg/ml of Hi; (e)
20 ng of topoisomerase, 1 yg/ml of HI; (f) 10 ng of topoisomerase, 1 yg/ml of H2a; (g) 20 ng of
topoisomerase, 1 yg/ml of H2a; (h) 10 ng of topoisomerase, 1 yg/pl of H2b; (i) 20 ng of topoisomerase,
1 yg/ml of H2b; (j) 10 ng of topoisomerase, 1 yg/ml of H3 and H4; (k) 20 ng of topoisomerase, 1 yg/ml
of H3 and H4; (1), 10 ng of topoisomerase, 1 yg/ml of poly-L-lysine; (m) 20 ng of topoisomerase, 1
yg/ml of poly-L-lysine; (n) 10 ng of topoisomerase, 2 mM spermidine; (o) 20 ng of topoisomerase, 2 mM
spermidine; (p) no enzyme.


m n
1
. 1
1
1
|Y
1
1

1 till I
t
1
1
a
i
i mi
i
a 1
I Mill
sxmr
I XIII
1 III!
<
a
-


Figure 11. Stimulation of topoisomerase by electrophoretically
purified HI histone.
Hi histone (50yg) 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 doublet^ which 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
y 1 of 20 mM Tris-HCl, 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 yl of 100% TCA (final concentration 20%).
After standing for 1 hr at -20C, the protein was pelleted by
centrifuging in an Eppendorf Microfuge for 5 min at 4C. The resulting
pellets were washed 1 time with 500 yl acid acetone (1/100 dilution of
concentrated HC1 in acetone) and then 2 times with 500 yl of acetone.
After removing the residual acetone under vacuumn, the fast and slow
mobility forms of HI were each resuspended in 100 yl 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 PI containing 25 mM Tris-HCl, pH 7.5, 10 mM MgClo, 0.4 mM
2-mercaptoethanol, 5% glycerol, 10 yg/ml BSA, 3 nmol <¡>X RF I [^H] 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.


ABC DEFGH I
| Topo
I HI
) lower band
l HI
I upper band
Controls


44
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 Hl-like protein, makes
up an integral part of the Ustilago topoisomerase. Separation of this
Hl-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


45
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 6-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 topoisome rase action.


CHAPTER III
PURIFICATION AND PROPERTIES OF Hl-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 Hl-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
47


48
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-HCl. 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 Hl-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 Hl-like protein from Ustilago.
The scheme used to isolate these proteins is outlined in Figure 13. The
sulfhydral reagents p-chloromercuriphenyl sulfonate and 5,51-dithiobis
(2-nitrobenzoate) were initially included in the extraction buffers


Figure 12. An acid-soluble protein from the topoisoinerase preparation stimulates relaxing activity.
To obtain the acid-soluble protein, 38.5 pi of 70% perchloric acid were added to 500 yl of
fraction IV of the topoisomerase purification. After standing for 10 min the sample was centrifuged
for 5 min in an Eppendorf microfuge. The pellet was discarded and to the supernatant was added 167 yl
of 100% trichloroacetic acid. After standing 10 min the sample was centrifuged and the resulting
supernatant discarded. The pellet was washed 1 time with 500 yl,acid-acetone (0.1 ml concentrated HC1
in 10 ml acetone) and 2 times with 500 yl of acetone. After removing the residual acetone under
vacuum, the pellet was resuspended in 50 yl of 25 mM Tris-HCl, pH 7.5. Part A: Stimulation of the
topoisomerase by the acid-soluble protein. Reaction mixtures (25 yl) containing 10 mM MgCl2> 0.4 mM
2-mercaptoethanol 0.4 mM potassium phosphate, pH 7.5, 5% glycerol, 50 yg/ml BSA, 2.1 nmol (|>x RF I DNA,
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%. Samples were then analyzed by electrophoresis through a 1.2% agarose
gel as described in Appendix C. A, 30 mM Tris-HCl, pH 7.5, 2.5 ng topoisomerase; B, 30 mM Tris-HCl, pH
7.5, 12.5 ng topoisomerase; C, 30 mM Tris-HCl, pH 7.5, 2.5 ng topoisomerase, 200 ng acid-soluble
protein; D, 30 mM sodium acetate, pH 6.0, 2.5 ng topoisomerase; E, 30 mM sodium acetate, pH 6.0, 12.5
ng topoisomerase; F, 30 mM sodium acetate, pH 6.0, 2.5 ng topoisomerase, 200 ng acid-soluble protein;
G, 30 mM sodium acetate, pH 6.0, 200 ng acid-soluble protein; H, 30 mM sodium acetate, pH 6.0. Part B:
Electrophoresis of the acid-soluble protein through a 15% SDS-polyacrylamide gel was carried out as
described in Appendix E. A, molecular weight markers from top to bottom are: phosphorylase b
(94,000), BSA (68,000), ovalbumin (43,000), carbonic anhydrase (29,000), soybean trypsin inhibition
(21,000); B, acid-soluble protein (25 yl).


) Topo
i pH 7.5
| + Acid soluble
' fraction
) Topo
) pH 6.0
/ + Acid soluble
fraction
! Controls
os


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.


52
I
Supernatant
Sediment
Supernatant
Cells
Breakage in
0.35M salt
Sediment
5% Perchloric acid
extraction
Supernatant
18% Trichloroacetic acid
Sediment
= 30 K protein


53
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 Hl-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 Hl-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 Hl-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 yg).


55


Figure 15. pH dependent binding of the Ustilago HI to
single-stranded DNA.
Reactions (50 yl) containing either 20 mM potassium phosphate ()
or sodium acetate (o) and 1 mM EDTA, 2 mM 2-mercaptoethanol, 5% glycerol,
50 mM NaCl, 1.5 nmol cf>x phage [^H] DNA and 1 yg 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.


-O
X
Relative Activity (%)
rv) ai -si
CJl o CJl
100
On
"'4


Figure 16. Ability of the Ustilago Hi histone to bind superhelical
DNA.
Reaction mixtures (50 yl) containing 50 mM sodium acetate, pH 6.0,
0.1 M, NaCl, 5 mM 2-mercaptoethanol, 5% glycerol and either f8 RF I
[^H] DNA () or f8 RF IV [^H] DNA (), were incubated with Ustilago
Hi 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 Pg of Ustilago HI retained 0.1 nmol of f9 RF I DNA to a
nitrocellulose filter.


Relative DNA Binding (%)
CJ1
o
o
o
on
<0


60
The Hl-like protein stimulates topoisomerase activity! However,
stimulation is pH-dependent (Fig. 17). At pH 7.5, where the Hl-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
Hl-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 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 Hl-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 carboxyraethylcellulose 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 yl) containing either 25 mM Tris-HCl, 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 MgCl2, 0.5 mM 2-mercaptoethanol, 5% glycerol, 10 yg/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 yg Ustilago Hi histone; G, 2ng
topoisomerase, 0.25 yg Ustilago HI histone; H, 2 ng topoisomerase, 1.0 yg
Ustilago HI histone; I and J, 2 ng topoisomerase, 50 ng calf thymus HI
histone.


as
N)


63
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, DBF 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 HI, but unlike the Ustilago Hl-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 HI,
the Ustilago Hl-like protein binds DNA in a pH-dependent fashion (78).
The ability of the Hl-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).


65


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.


43 K
25.7 K
18.4 K


Figure 20. Stimulation of the topoisomerase by DBP IV.
Reactions (25 yl) containing 25 mM Tris-HCl, 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
MgCl2, 0.4 mM 2-mercaptoethanol, 5% glycerol, 10 yg/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 DBF IV; G and H, 100 ng DBP
IV; I, no additions.


69
ABC DEFG H I


Figure 21. Effect of pH on the ability of DBP IV to bind
single-stranded DNA.
Reaction (50 Pi) containing either 20 mM potassium phosphate () or
sodium acetate ( o) and 1 mM EDTA, 2 mM 2-mercaptoethanol, 5% glycerol, 50
mM NaCl, 1.5 nmol ^yphage [%] DNA (2x 10^ cpm/ymol) and 250 ng DBP
IV were incubated at 25C. 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.


Relative Activity (%)
no cn ->J
cn o cn
100


72
is also pH dependent and directly correlates with its ability to bind
DNA. This suggests an interaction between the Ustilago Hl-like protein
and DNA is necessary for stimulation of the topoisomerase.
Hl-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 Hl-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 HMG. 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 MgCl2 leads to the recircularization of the DNA (39).
74


75
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 f9
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 f8 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 f9 DNA (Fig.
22 B).
Attempts to recircularize the f 9 DNA which had been broken by the
Ustilago topoisomerase were unsuccessful. Reactions containing circular
single-stranded f 9 DNA previously broken by the topoisomerase were
incubated in the presence of 0.2 M salt or 10 mM MgCl2 for 2 hr at
25 C. 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 yl) containing 10 mM Tris-HCl, pH 9.0, 1 mM EDTA, 0.2
mM dithiothreitol, 50 yg/ml BSA, 5% glycerol, 2.4 nmol f3 phage DNA, and
either 1 yg of topoisomerase (lanes A and B) or no topoisomerase (lane C)
were incubated at 25C for 20 min. Reactions A and C were terminated by
adding 4 yl of 0.3 M NaOH. Reaction B was treated with proteinase K (100
yg/ml) for 30 min at 37C before adding 4 yl of 0.3 M NaOH. Fifteen yl
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 5C.
Following electrophoresis the gel was stained with 1 yg/ml eithidium
bromide before photographing under ultraviolet light.


77
ABC
i
circles
linears


78
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 <{>xl74 DNA which had been
labeled by nick translation with [a-^P] 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 yl reaction containing 6 mM Tris-HCl, pH 9.0, 0.2 mM
dithiothreitol, 50 yg/ml BSA, 150 pmol denatured nick translated [^H]
PBR DNA (3.3 x 10? cpm/nmol) and 1 yg topoisomerase was incubated at
25C for 20 min. The reaction was stopped by adding NaOH to 50 mM and
incubating for 5 min at 37C. The reaction was then made 30 mM in
Tris-HCl and the pH titrated to 9.0 with HC1. After adding CaCl2 to a
final concentration of 2 mM, 0.1-0.2 units of Micrococcal nuclease were
added. After incubating at 37C 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-HCl, 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 100C 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-0mat AR 5 film with a Dupont lightning-plus intensifying screen at
-70C.


80
205 K
I 16 K
94 K
68 K
43 K
30 K
tracking dye


81
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 [^H] labeled circular single-stranded f9 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 f9 phage [%] DNA (2 x 104
cpm/nmol) and topoisomerase were treated with exonuclease I (exo I) or
exonuclease VII (exo VII). A 75 pi reaction containing 10 mM Tris-HCl,
pH 9.0, 0.2 mM dithiothreitol, 50 yg/ml BSA, 5 nmol f3 phage [^Hj DNA
and 1 yg topoisomerase was incubated at 25C. After 10 min three 20 yl
aliquots were removed and analyzed as described in Appendix C. One ali
quot was diluted into a 200 yl reaction containing 0.8 units of exonucle
ase I. Another aliquot was diluted into a 200 yl reaction containing 0.6
units of exonuclease VII. The third aliquot was electrophoresed through a


82
DIGESTION
TABLE V
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


83
1.2% alkaline agarose gel as described in the legend to Figure 22. A
control reaction containing no topoisoraerase 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 f3 DNA, produced by digesting
f3 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] exp 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 topoismerase complexed with
DNA labeled at the 3' or 5' end.
Form III f9 DNA, prepared by digesting form I f0 DNA with the
restriction endonuclease Sau 96, was labelled with [^p] 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 yl) containing 10 mM Tris-HCl pH 9.0, 0.2 mM dithio-
threitol, 50 yg/ml BSA, and 0.3 nmol of denatured 5' (lanes A-C) or 3'
(lanes D-F) [^P] end labeled DNA were incubated with (lanes B, C, E,
F) or without (lanes A and D) 1 yg of topoisomerase at 25C for 30 rain.
Reactions were terminated by adding 4 yl of 0.3 M NaOH. Prior to adding
NaOH, samples in lanes C and F were treated with proteinase K (100 yg/ml)
for 30 min at 37C. Twenty yl of each sample was analyzed by electro
phoresis through a 1.6% agarose gel containing 30 mid NaOH, 1 mM EDTA at
10 v/cm for 5 hr at 5C. Following electrophoresis the gel was soaked
for 10 rain in 500 ml of 0.1 M Tris-HC.l 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.


85


86
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 Xl74 RF DNA
which had been labeled by nick translation with [y-^^P] 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 HCl at 110C 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 [^P] 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 yl 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 110C oil bath. After 2 hr the sample was
cooled and the HC1 removed in vacuo in the presence of NaOH pellets. The
resulting residue was resuspended in 30 y 1 of H2O 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, P. 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


Full Text
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
FOR THE
FULFILLMENT OF THE REQUIREMENTS
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA

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/characterizationOOrowe

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.
111

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 HI Histone 37
III. PURIFICATION AND PROPERTIES OF Hl-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
iv

Page
IV. CHARACTERIZATION OF THE COVALENT TOPOISOMERASE-
DNA LINKAGE 74
Detection of the Covalent Complex 74
Identification of the Protein Complexed to
the DNA 75
Topoisomerase Links to the 3' End of the DNA Break . 81
Identification of the Covalent Linkage 86
Discussion 86
V. INVOLVEMENT OF TOPOISOMERASE IN GENETIC RECOMBINATION . 91
The Ustilago Topoisomerase Recombines
Complementary Single-Stranded Rings of DNA .... 91
Relaxing Activity Reduced in the rec 2 Mutant. ... 92
Discussion y95
APPENDICES
A. PHAGE, BACTERIAL AND FUNGAL STRAINS 100
B. PREPARATION OF DNA 101
C. ASSAYS 104
Topoisomerase 104
DNA-Binding Protein 105
Exonuclease 106
Protein 106
D. PROTEIN PURIFICATION 107
Topoisomerase 107
25,000 Dalton Acid-Soluble Protein 108
DBP IV 109
E. ELECTROPHORESIS 112
F. REAGENTS AND MATERIALS 113
«
Chromatographic Media 113
Enzymes 113
Miscellaneous 113
REFERENCES 115
BIOGRAPHICAL SKETCH 121
v

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
vi

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
vi i

Page
19. Acid-solubility of DBP IV 67
20. Stimulation of the topoi some rase 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 complexed
with DNA labeled at the 3' or 5' end 85
25. Paper electrophoresis of the [^P] 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
vi i i

KEY TO ABBREVIATIONS
ATP
adenosine-5 1-triphosphate
BSA
bovine serum albumin
CTP
cytosine-5 1-triphosphate
DBP
DNA binding protein
DNA
deoxyribonucleic acid
EDTA
ethylenediamine tetra-acetic acid
GTP
guanosine-5'-triphosphate
PMSF
phenylmethylsulfonylfluoride
poly(dA)
polydeoxyadenyIic 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)
IX

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 Cardón Rowe
December 1982
v
\
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 Mg^+, but is totally inhibited by
Zn^+ at concentrations as low as 20 yM. 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 Hl-like protein has been isolated from Ustilago which
also markedly stimulates the topoisomerase. This interaction may be
important in regulating topoisomerase in cellular processes.
x

The topoisomerase breaks single-stranded DNA and
complex with the 3' end of the DNA break. The covalent
involve a phosphotyrosine bond.
The rec 2 mutant of Ustilago has decreased levels
activity possibly implicating this enzyme in eukaryotic
forms a covalent
linkage appears to
of topoisomerase
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, B,
and the number of superhelical turns, x, by the following simple
equation L = B + x (8). The linking number for a closed circular
1

2
molecule remains constant unless the DNA backbone becomes broken. There
are two basic ways topoisoinerases 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

3
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 y
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

4
(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_L 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
is also dependent on the superhelical state of the DNA (31).
Resolvase, an enzyme coded for by y6 , carries out the final step in
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

6
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 J^n _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
8

9
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

Figure 1.
Topoisomerase assays.
Reaction mixtures (100 yl) containing 75 mM potassium phosphate, pH 7.5, 1 mM EDTA, 5% glycerol,
and 5 nmol of RF I [%] DNA (1.5 x 10^ cpra/nmol), and the indicated amounts of topoisomerase were
incubated at 25°C. At the end of 30 min three 25 yl aliquots were removed from each reaction mixture
and processed as described in Appendix C. One aliquot was loaded onto a 1.2% agarose gel, the second
aliquot was mixed with 1 ml of buffer containing 3.5 M NaCl (°) and the third aliquot was combined
with 6.6 nmol of single-stranded DNA fragments and incubated at 75°C for 2 1/2 hr to promote D-loop
formation (®). The following amounts of enzyme were used in the lanes of the agarose gel: a, no
enzyme; b, 1.25 ng; c, 2.5 ng; d, 3.75 ng; e, 5 ng; f, 10 ng; g, 20 ng.

RF DNA Released from Filter (nmol)
O p o
no m m
ai O cn
IT

12
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 1 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 KC1 and loaded directly on to an octy1-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 -20°C 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

13
TABLE I
PURIFICATION OF THE ENZYME
Fraction
Volume
ml
Activity
units x 10-4
Protein
mg
Specific
Activity
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 S200^
8
3.4
0.40
85,000
aProtein determination was not performed since Triton interfered with the
assay.
^Total 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.

14
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 10^ to 1.2 x 10^
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.

Figure 2. Polyacrylamide gel electrophoresis of the topoisomerase preparation.
Polyacrylamide gels containing 5% acrylamide, 0.14% N,N'-methylenebisacrylamide, 0.1 M potassium
phosphate, pH 7.5, 10% glycerol, were cast in tubes (5 x 80 mm) with ammonium persulfate as catalyst.
The gels were prerun overnight with electrode buffer composed of 0.1 M potassium phosphate, pH 7.5, 10%
glycerol in order to remove ammonium persulfate. Electrode buffer was replaced, and 100 yl aliquots of
the topoisomerase preparation (500 units) containing 50% glycerol and 5 yg of fluorescamine-labeled
pancreatic ribonuclease (46) were layered on parallel gels. The samples were overlaid with 100 yl of
20% glycerol and then electrophoresed at 5 mA/tube. Cooling was provided by circulating ice water
around the tubes during the course of the run. After 12 h when the ribonuclease marker, located under
ultraviolet light, had migrated approximately halfway down the tube, the run was ended. One gel was
stained for protein with Coomassie brilliant blue. A parallel gel was cut into 2 mm slices, and each
slice was soaked in 100 hi of 50 mM potassium phosphate, pH 7.5, 1 mM EDTA, 5 mM 2-mercaptoethanol, 10%
glycerol, 0.1 M KC1. After 18 h each slice was assayed for topoisomerase activity by the agarose gel
method. Approximately 10% of the activity was recovered after electrophoresis. The rapidly migrating
material near slice number 15 is the ribonuclease marker.

ON

17
TABLE II
ESTIMATION OF SIZE OF THE TOPOISOMERASE
Activity
Kdl/3
Molecular
Recovery
KC1
Peak
We ight
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 -^H-labeled xl74
phage and [^2p] 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 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.

18
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 10^ 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-HCl and not measurable in either 4-(2-hydroxyethyl)-l-
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, Mg^+ proved to be much more
effective in promoting activity. In the presence of 10 mM MgCl2,

Figure 3. Centrifugation of topoisoraerase in a glycerol gradient.
Topoisomerase (500 units) together with 1 mg of bovine serum albumin (BSA) and 10 yg of catalase
in 0.1 ml was layered on top of a 5 ml 20 - 40% gradient of glycerol containing 20 mM potassium
phosphate, pH 7.0, 5 mM 2-mercaptoethanol, 0.5 M KC1. Centrifugation was at 4°C for 22 h at 50,000 rpm
in the Beckman SW 50.1 rotor. At the end of the run fractions were collected from the bottom of the
gradient and assayed for topoisomerase activity by the agarose gel assay. Activity was determined by
visual inspection, and recovery was estimated at less than 1%. Catalase activity was located by assay
and bovine serum albumin by protein determination; peaks are indicated in arbitrary units. The
sedimentation coefficient and molecular weight of the topoisomerase were determined according to Martin
and Ames (50). Sedimentation is from right to left.

5 10 15 20
Fraction Number
N>
O

Figure 4. pH optima of the topoisomerase.
Reaction mixtures (50 pi) containing 1 mM EDTA, 1 mM 2-mercaptoethanol, 3 nmol RF I [^H] DNA
(1.5 x 10^ cpm/nmol), 1 unit of topoisomerase and 20 mM sodium citrate (D), Tris-HCl (A), potassium
phosphate (°), HEPES ( ®), or Imidizole (x) were incubated at 37°C for 30 min. Reactions were stopped
by adding 2.5 pi 10% SDS and 10 pi 3 M NaCl. Each reaction was then split into two 30 pi aliquots and
processed as described in Appendix C. One aliquot was used to assess nicking activity. The pH of the
second aliquot was adjusted by adding 3 pi of 0.5 M Tris-HCl, pH 7.5 before adding 5 nmol of
single-stranded DNA fragments. After heating at 75°C for 3 h, the amount of D-loop formation was
determined as described in Appendix C. >

N)
N)

Figure 5. Salt optima of the topoisomerase.
Reaction mixtures (25 pi) containing 25 mM Tris-HCl, pH 7.5, 10%
glycerol, 0.4 mM 2-mercaptoethanol, 3 nmol x RF I [^H] DNA (1.5 x
10^ 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 NaCl and topoisomerase activity determined
using the high salt filter assay as described in Appendix C.

24
KCI (M)

25
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 Mg^+, Cu^+ and Zn^+ were strong
inhibitors of the topoisomerase. The presence of as little as 20 pM
CuCl2 or ZnCl2 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 - 30°C
with no detectable activity at 42°C (Fig. 6). Surprisingly, the enzyme
is fairly active at low temperatures with 70% relaxation occurring at
0°C. 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 Vl) containing 75 mM potassium phosphate, pH 7.5, 10%
glycerol, 3 nmol XRF I [%] DNA (1.5 x 10^ cpm/nmol) and 40 ng of
topoisomerase were incubated for 30 min at the indicated temperatures.
Reactions were terminated by adding 10 pi 3 M KC1. Topoisomerase
activity was then determined using the D-loop assay as described in
Appendix C.

27

28
(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,g-methylene ATP, g,y-raethy-
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 ancj 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

29
TABLE III
INHIBITION BY NUCLEOTIDES AND POLYNUCLEOTIDES
Compound
-
Concentration
mM
Activity
Percent
Control
100
ATP
5.0
0
ADP
5.0
70
AMP
5.0
100
dATP
5.0
90
a,g-methylene ATP
1.6
50
a, 6-methylene ATP
3.2
0
B,y-raethylene ATP
1.5
100
6,y-methylene ATP
3.0
0
AMP-PNP3
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
X 174 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.

30
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 yg/ml of
nalidixic acid (Table IV). This is a 10-fold lower concentration

TABLE IV
INHIBITION BY ANTIBIOTICS
Drug
Concentration
yg/ml
Activity
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.

32
than is required to cause a similar inhibition of DNA gyrase (53).
While 200 Mg/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 yg/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 hi) containing 20 mM Tris-HCl, pH 7.5, 0.1 M
KC1, 5% glycerol, 0.8 yg/ml ethidium bromide and 3 nmoles <|>x RF 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) 'Í’XRF I DNA; (Lane B) <)>XRF IV DNA; (Lane C) xRF
IV DNA and 100 ng topoisomerase.

34
ABC
M

Fig. 8. Removal of positive superhelical turns.
The high salt binding assay was used for measuring removal of both negative and positive
superhelical turns. However, in the latter case an increase, rather than decrease, in the amount of
DNA bound to filters is a measure of removal of positive supercoils. When form I DNA alone was washed
through a nitrocellulose filter, as described, approximately 70% of the input radioactivity bound to
the filter. When form IV DNA was tested, 2% bound. If ethidium bromide was present there was no need
to extract it from the DNA as long as the solution was diluted sufficiently before filtering. Again
less than 2% of the radioactivity bound to a filter if a solution containing forra IV DNA and 1 yg/ml of
ethidium bromide was diluted 1 to 40 in 3.5 M NaCl before filtering. A, rate of reaction. Two
reaction mixtures (150 yl) were set up under the standard condition, one containing 7.5 nmol of <¡>XRF
I[3H] DNA (2.2 x 10^ cpra/nmol) and the other containing 7.5 nmol of 4>XRF IV [3H] DNA (2.2 x
10^ cpm/nmol) and 1 yg/ml of ethidium bromide. Topoisomerase (300 ng) was added to each reaction,
and the mixtures were incubated at 25°C. AT the indicated times aliquots of 25 yl were removed,
diluted into 1 ml of the 3.5 M NaCl buffer for binding, and processed as described. After 30 min of
reaction the amount of DNA bound to a filter after removal of positive superhelical turns reached a
maximum of 65% of the input form IV DNA. B, enzyme concentration curve. Reaction mixtures (25 yl)
containing 1.25 nmol of XRF I [3H] DNA or X RF IV [3H] DNA plus 1 yg/ml of ethidium bromide and
the indicated amounts of topoisomerase were incubated at 25°C for 30 min and then filtered as above.
(°), negative superhelical DNA; (•), positive superhelical DNA.

Time (min) Enzyme (ng)
Topoisomerase Activity
o o
ro bi
9£

37
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 yl) containing 40 mM Tris-HCl. pH 7.5, 10 mM
MgCl2> 10% glycerol, 3 nmol of X RF I [^H] DNA (1.5 x 10^
cpm/nraol), the indicated amounts of topoisomerase, and either 1 yg/ml of
histone HI (°) or 1 yg/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 NaCl
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.

Topoisomerase
o
en
(nmol
5
DNA released)
en
04

Figure 10. Stimulation of topoisomerase activity by histones and polyamines.
Reaction mixtures (25 yl) containing 25 mM Tris-HCl, pH 7.5, 10 mM MgCl^, 10% glycerol, 3 nmol
of <}>x RF I [3H] DNA, and the following additions were incubated at 25 °C for 30 min. Reactions were
terminated by adding 5 yl of 2% SDS in 0.12 M EDTA and proteinase K to 100 yg/ml. After 30 min at 37°C
samples were loaded and run on an agarose gel as described in Appendix C. (a) 10 ng of topoisomerase;
(b) 20 ng of topoisomerase; (c) 50 ng of topoisomerase; (d) 10 ng of topoisomerase, 1 yg/ml of Hi; (e)
20 ng of topoisomerase, 1 yg/ml of HI; (f) 10 ng of topoisomerase, 1 yg/ml of H2a; (g) 20 ng of
topoisomerase, 1 yg/ml of H2a; (h) 10 ng of topoisomerase, 1 yg/pl of H2b; (i) 20 ng of topoisomerase,
1 yg/ml of H2b; (j) 10 ng of topoisomerase, 1 yg/ml of H3 and H4; (k) 20 ng of topoisomerase, 1 yg/ml
of H3 and H4; (1), 10 ng of topoisomerase, 1 yg/ml of poly-L-lysine; (m) 20 ng of topoisomerase, 1
yg/ml of poly-L-lysine; (n) 10 ng of topoisomerase, 2 mM spermidine; (o) 20 ng of topoisomerase, 2 mM
spermidine; (p) no enzyme.

abcdefghi jklmnop
M M M
MM M*
• M M M M
mm»*"*
•Ml
mm M
«• m
m
Ml **
«* to* to
111 *• M
■ m *" »•
9 to^ * to ^ • Mm
É>
to
mw m
f
t
s

Figure 11. Stimulation of topoisomerase by electrophoretically
purified HI histone.
Hi histone (50yg) 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 doublet^ which 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
y 1 of 20 mM Tris-HCl, 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 yl of 100% TCA (final concentration 20%).
After standing for 1 hr at -20°C, 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 yl acid acetone (1/100 dilution of
concentrated HC1 in acetone) and then 2 times with 500 yl of acetone.
After removing the residual acetone under vacuumn, the fast and slow
mobility forms of Hi were each resuspended in 100 yl 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 PI containing 25 mM Tris-HCl, pH 7.5, 10 mM MgClo, 0.4 mM
2-mercaptoethanol, 5% glycerol, 10 yg/ml BSA, 3 nmol <¡>X RF I [^H] 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.

ABC DEFGH I
| Topo
I HI
) lower band
l HI
I upper band
£ Controls

44
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 Hl-like protein, makes
up an integral part of the Ustilago topoisomerase. Separation of this
Hl-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

45
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 6-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 topoisome rase action.

CHAPTER III
PURIFICATION AND PROPERTIES OF Hl-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 Hl-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
47

48
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-HCl. 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 Hl-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 Hl-like protein from Ustilago.
The scheme used to isolate these proteins is outlined in Figure 13. The
sulfhydral reagents p-chloromercuriphenyl sulfonate and 5,51-dithiobis
(2-nitrobenzoate) were initially included in the extraction buffers

Figure 12. An acid-soluble protein from the topoisoinerase preparation stimulates relaxing activity.
To obtain the acid-soluble protein, 38.5 pi of 70% perchloric acid were added to 500 yl of
fraction IV of the topoisomerase purification. After standing for 10 min the sample was centrifuged
for 5 min in an Eppendorf microfuge. The pellet was discarded and to the supernatant was added 167 yl
of 100% trichloroacetic acid. After standing 10 min the sample was centrifuged and the resulting
supernatant discarded. The pellet was washed 1 time with 500 yl,acid-acetone (0.1 ml concentrated HC1
in 10 ml acetone) and 2 times with 500 yl of acetone. After removing the residual acetone under
vacuum, the pellet was resuspended in 50 yl of 25 mM Tris-HCl, pH 7.5. Part A: Stimulation of the
topoisomerase by the acid-soluble protein. Reaction mixtures (25 yl) containing 10 mM MgCl2> 0.4 mM
2-mercaptoethanol , 0.4 mM potassium phosphate, pH 7.5, 5% glycerol, 50 yg/ml BSA, 2.1 nmol (|>x RF I DNA,
and the following additions were incubated at 25°C. After 30 min the reactions were stopped by adding
EDTA to 15 mM and SDS to 0.5%. Samples were then analyzed by electrophoresis through a 1.2% agarose
gel as described in Appendix C. A, 30 mM Tris-HCl, pH 7.5, 2.5 ng topoisomerase; B, 30 mM Tris-HCl, pH
7.5, 12.5 ng topoisomerase; C, 30 mM Tris-HCl, pH 7.5, 2.5 ng topoisomerase, 200 ng acid-soluble
protein; D, 30 mM sodium acetate, pH 6.0, 2.5 ng topoisomerase; E, 30 mM sodium acetate, pH 6.0, 12.5
ng topoisomerase; F, 30 mM sodium acetate, pH 6.0, 2.5 ng topoisomerase, 200 ng acid-soluble protein;
G, 30 mM sodium acetate, pH 6.0, 200 ng acid-soluble protein; H, 30 mM sodium acetate, pH 6.0. Part B:
Electrophoresis of the acid-soluble protein through a 15% SDS-polyacrylamide gel was carried out as
described in Appendix E. A, molecular weight markers from top to bottom are: phosphorylase b
(94,000), BSA (68,000), ovalbumin (43,000), carbonic anhydrase (29,000), soybean trypsin inhibition
(21,000); B, acid-soluble protein (25 yl).

>
) Topo
DO
) pH 7.5
O
| + Acid soluble
’ fraction
O
) Topo
m
i pH 6.0
~n
/ + Acid soluble
’ fraction
CD
X !
J Controls
os

Figure 13. Procedure for isolation of HI histone from Ustilago.
All steps of the purification were carried out between 0-4°C.
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.

52
I
Supernatant
I
Sediment
Supernatant
Cells
Breakage in
0.35M salt
Sediment
5% Perchloric acid
extraction
Supernatant
18% Trichloroacetic acid
Sediment
= 30 K protein

53
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 Hl-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 Hl-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 Hl-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 yg).

55

Figure 15. pH dependent binding of the Ustilago HI to
single-stranded DNA.
Reactions (50 yl) containing either 20 mM potassium phosphate (•)
or sodium acetate (o) and 1 mM EDTA, 2 mM 2-mercaptoethanol, 5% glycerol,
50 mM NaCl, 1.5 nmol cf>x phage [^H] DNA and 1 yg of Ustilago HI histone
were incubated at 25°C. 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.

-O
X
Relative Activity (%)
rv) ai ' -si
CJl o CJl
100
C-n
'"4

Figure 16. Ability of the Ustilago Hi histone to bind superhelical
DNA.
Reaction mixtures (50 yl) containing 50 mM sodium acetate, pH 6.0,
0.1 M, NaCl, 5 mM 2-mercaptoethanol, 5% glycerol and either f8 RF I
[^H] DNA (•) or f8 RF IV [%] DNA (°), were incubated with Ustilago
Hi histone at 25°C. Reactions were stopped by the addition of 1 ml of
reaction buffer and processed as described in Appendix C. Under these
conditions, 1 Pg of Ustilago HI retained 0.1 nmol of f3 RF I DNA to a
nitrocellulose filter.

Relative DNA Binding (%)
CJ1
o
o
o
cn
<0

60
The Hl-like protein stimulates topoisomerase activity! However,
stimulation is pH-dependent (Fig. 17). At pH 7.5, where the Hl-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
Hl-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 ó - 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 Hl-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 carboxyraethylcellulose 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 yl) containing either 25 mM Tris-HCl, 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 MgCl2, 0.5 mM 2-mercaptoethanol, 5% glycerol, 10 yg/ml BSA and the
following additions were incubated at 25°C. 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 yg Ustilago Hi histone; G, 2ng
topoisomerase, 0.25 yg Ustilago HI histone; H, 2 ng topoisomerase, 1.0 yg
Ustilago HI histone; I and J, 2 ng topoisomerase, 50 ng calf thymus Hi
histone.

) Topo
) pH 75
) Topo
) pH 6.0
) pH 75
I + 30K
) pH 6.0
) + 30K
l pH 75
» +HI
l pH 6.0
» + HI
On
N)

63
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, DBF 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 HI, but unlike the Ustilago Hl-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 HI,
the Ustilago Hl-like protein binds DNA in a pH-dependent fashion (78).
The ability of the Hl-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).

65

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 (3-lactoglobulin. A, Perchloric acid
precipitate; B, Perchloric acid supernatant.

43 K
25.7 K
18.4 K

Figure 20. Stimulation of the topoisomerase by DBP IV.
Reactions (25 yl) containing 25 mM Tris-HCl, 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
MgCl2> 0.4 mM 2-mercaptoethanol, 5% glycerol, 10 yg/ml BSA and the
following additions were incubated at 25°C. 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 DBF IV; G and H, 100 ng DBP
IV; I, no additions.

69
ABC DEFG H I
l + - - + —4 | 4

Figure 21. Effect of pH on the ability of DBP IV to bind
single-stranded DNA.
Reaction (50 Pi) containing either 20 mM potassium phosphate (•) or
sodium acetate ( o) and 1 mM EDTA, 2 mM 2-mercaptoethanol, 5% glycerol , 50
mM NaCl, 1.5 nmol ^yphage [%] DNA (2x 10^ cpm/ymol) and 250 ng DBP
IV were incubated at 25°C. 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.

Relative Activity (%)
no cn ->)
cn o cn
100

72
is also pH dependent and directly correlates with its ability to bind
DNA. This suggests an interaction between the Ustilago Hl-like protein
and DNA is necessary for stimulation of the topoisomerase.
Hl-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 Hl-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 HMG. 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 MgCl2 leads to the recircularization of the DNA (39).
74

75
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 f9
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 f8 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 f9 DNA (Fig.
22 B).
Attempts to recircularize the f 9 DNA which had been broken by the
Ustilago topoisomerase were unsuccessful. Reactions containing circular
single-stranded f 9 DNA previously broken by the topoisomerase were
incubated in the presence of 0.2 M salt or 10 mM MgCl2 for 2 hr at
25 °C. 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 yl) containing 10 mM Tris-HCl, pH 9.0, 1 mM EDTA, 0.2
mM dithiothreitol, 50 yg/ml BSA, 5% glycerol, 2.4 nmol f3 phage DNA, and
either 1 yg 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 yl of 0.3 M NaOH. Reaction B was treated with proteinase K (100
yg/ml) for 30 min at 37°C before adding 4 yl of 0.3 M NaOH. Fifteen yl
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 yg/ml eithidium
bromide before photographing under ultraviolet light.

77
ABC
i
circles
linears

78
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 <{>xl74 DNA which had been
labeled by nick translation with [a-^P] 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 yl reaction containing 6 mM Tris-HCl, pH 9.0, 0.2 mM
dithiothreitol, 50 yg/ml BSA, 150 pmol denatured nick translated [^H]
PBR DNA (3.3 x 10? cpm/nmol) and 1 yg 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 37°C. The reaction was then made 30 mM in
Tris-HCl and the pH titrated to 9.0 with HC1. After adding CaCl2 to a
final concentration of 2 mM, 0.1-0.2 units of Micrococcal nuclease were
added. After incubating at 37°C 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-HCl, 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-0mat AR 5 film with a Dupont lightning-plus intensifying screen at
-70°C.

80
205 K
I 16 K
94 K
68 K
43 K
30 K
tracking dye

81
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 [%] labeled circular single-stranded f9 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 f9 phage [%] DNA (2 x 104
cpm/nmol) and topoisomerase were treated with exonuclease I (exo I) or
exonuclease VII (exo VII). A 75 pi reaction containing 10 mM Tris-HCl,
pH 9.0, 0.2 mM dithiothreitol, 50 yg/ml BSA, 5 nmol f3 phage [^Hj DNA
and 1 yg topoisomerase was incubated at 25°C. After 10 min three 20 yl
aliquots were removed and analyzed as described in Appendix C. One ali¬
quot was diluted into a 200 yl reaction containing 0.8 units of exonucle¬
ase I. Another aliquot was diluted into a 200 yl reaction containing 0.6
units of exonuclease VII. The third aliquot was electrophoresed through a

82
DIGESTION
TABLE V
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

83
1.2% alkaline agarose gel as described in the legend to Figure 22. A
control reaction containing no topoisoraerase 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 f3 DNA, produced by digesting
f3 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] ¿exp 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 topoisómerase complexed with
DNA labeled at the 3' or 5' end.
Form III f9 DNA, prepared by digesting form I f0 DNA with the
restriction endonuclease Sau 96, was labelled with [^p] 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 yl) containing 10 mM Tris-HCl pH 9.0, 0.2 mM dithio-
threitol, 50 yg/ml BSA, and 0.3 nmol of denatured 5' (lanes A-C) or 3'
(lanes D-F) [^P] end labeled DNA were incubated with (lanes B, C, E,
F) or without (lanes A and D) 1 yg of topoisomerase at 25°C for 30 rain.
Reactions were terminated by adding 4 yl of 0.3 M NaOH. Prior to adding
NaOH, samples in lanes C and F were treated with proteinase K (100 yg/ml)
for 30 min at 37°C. Twenty yl of each sample was analyzed by electro¬
phoresis through a 1.6% agarose gel containing 30 mid NaOH, 1 mM EDTA at
10 v/cm for 5 hr at 5°C. Following electrophoresis the gel was soaked
for 10 rain in 500 ml of 0.1 M Tris-HC.l 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.

85
5' 3'
ABC D E F

86
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 <¡>Xl74 RF DNA
which had been labeled by nick translation with [y-^^P] 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 HCl at 110°C 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 [^P] 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 yl 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 HC1 removed in vacuo in the presence of NaOH pellets. The
resulting residue was resuspended in 30 y 1 of H2O 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, P¿. 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

89
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 f3 DNA broken by the
Ustilago topoisomerase does not recircularize in the presence of either
0.2 M salt or 10 mM MgCl2j 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

90
present unknown. The covalent attachment of eukaryotic type I topoisom-
erases to the 3' rather than the 5' end of the DNA suggests they are
probably not involved in the priming of DNA replication. However, this
does not preclude eukaryotic type I topoisoraerases from playing a role in
removing any topological constraints which might arise during the course
of DNA replication.

CHAPTER V
INVOLVEMENT OF THE Ustilago TOPOISOMERASE IN
GENETIC RECOMBINATION
The biological role of topoisomerases in prokaryotic genetic
recombination has recently been established. Mutations in the gene coding
for E. coli DNA topoisomerase I (omega protein) have been found to
dramatically reduce the transposition frequency of the antibiotic-
resistance-carrying transposons Tn 5, Tn 9 and Tn 10 (37). Studies on
another topoisomerase in E. coli, DNA gyrase, have provided evidence for
a role of this enzyme in prokaryotic illegitimate and site-specific
recombination (26,38).
The role of topoisomerases in eukaryotic genetic recombination is
less clear. Although in vitro studies on several eukarytoic type I
topoisomerases suggest a possible involvement of these enzymes in both
homologous and illegitimate forms of recombination, there is no genetic
evidence to support a similar involvement in vivo (3).
Recently, biochemical as well as genetic evidence have been
obtained which suggest an involvement of the Ustilago topoisomerase in
DNA recombination. The evidence and its possible significance are
discussed in this chapter.
The Ustilago Topoisomerase Recombines Complementary
Single-Stranded DNA Rings
In the past it was believed that homologous recombination required
DNA with free ends. However, with the discovery that type I topoisomer¬
ases could catalyze recombination between complementary single-stranded
91

92
DNA circles to form closed circular duplex DNA, a new alternative to
strand transfer was established bypassing the need for free ends in the
DNA (22,23).
The Ustilago topoisomerase also appears to catalyze intertwining of
complementary single-stranded circles of DNA. To illustrate this prop¬
erty, complementary single-stranded circles of <¡>xl74 [^H] DNA were
incubated with the Ustilago topoisomerase under the reaction conditions
described in the legend to Figure 26. The products of the reaction were
then centrifuged through an alkaline sucrose gradient. As is evident
from Figure 26 A, treatment of complementary single-stranded circles of
<¡>Xl74 DNA with the topoisomerase leads to the formation of a DNA species
which sediments at a rate similar to denatured covalently closed duplex
<¡>X174 DNA suggesting that the topoisomerase is able to topologically
intertwine complementary circles of DNA. If the topoisomerase is
incubated with Xl74 phage DNA, which consists of only "plus" strands, no
fast sedimenting DNA species is observed suggesting this reaction
requires that the DNA circles be complementary (data not shown).
The ability of type I topoisomerases to catalyze intertwining of
complementary circular strands of DNA suggests an in vivo role for these
enzymes in removing topological barriers which might arise during DNA
recombination. Such barriers could easily be envisioned to occur during
strand exchange between two DNA molecules.
Relaxing Activity Reduced in the rec 2 Mutant
Mutants of Ustilago defective in DNA replication, repair, and
recombination were screened for the presence of topoisomerase activity
using the agarose gel assay. A mutant defective in recombination, rec 2,

Figure 26. Interlocking of complementary single-stranded rings of
DNA by the topoisomerase.
Reactions (50 yl) containing 40 mM Tris-HCl, pH 7.5, 10 mM MgCÍ2,
25 mM KC1, 5% glycerol, 2 nmol complementary single-stranded (jiyt^H]
DNA rings, and (A) 200 ng of topoisomerase or (B) no topoisomerase were
incubated at 30°C. After 20 min the reactions were terminated by adding
50 yl or 1 M NaCl, 10 yl 0.25 M EDTA and 100 yl 0.5 M NaOH. Samples were
then loaded onto 5-20% sucrose gradients containing 0.25 M NaOH 1 mM
EDTA, and 0.75 M NaCl. The gradients were centrifuged at 40,000 rpm in
the SW 40.1 rotor of a Beckman L5-50 centrifuge at 4°C for 4 hr.
Forty-two fractions (0.25 ml each) were collected from the bottom of each
gradient and each fraction was neutralized with 0.25 ml of 0.25 M HC1
before adding 5 ml of Formula 950 scintillation flúor (New England
Nuclear). The radioactivity of each sample was then determined.
Purification of the [^H] i)>x complementary single-stranded rings of DNA
used in the above reactions is described in Appendix B.

ro
( 01 x wdo) VNQ

95
showed a significantly reduced level of DNA relaxing activity. As shown
in Figure 27, an extract prepared from rec 2 cells contained less than
20% of the relaxing activity of extracts from rec + cells.
The heat stabilities of enzyme partially purified from rec 2 and
rec + cells were also compared and suggests that the topoisomerase from
the mutant may be structurally altered (Dr. M. Brougham, personal
communication). However, these results are preliminary in nature and
additional studies will be needed to determine if the rec 2 mutant
contains a structural defect in the gene coding for the Ustilago
topoisomerase.
Discussion
The Ustilago topoisomerase can carry out several biochemical
reactions which may be of consequence in genetic recombination. One of
these reactions, the topological isomerization of DNA, may determine the
effectiveness of DNA as a substrate in recombination. The role of
superhelicity in bacteriophage lambda as well as transposon yg
recombination points out how the topological state of DNA can be a
critical factor in determining the DNA's effectiveness as a substrate for
recombination (26,31). An ability of the topoisomerase to catalyze
topological linking of complementary single-stranded rings of DNA also
implies that this enzyme can act to resolve topological problems which
might occur during recombination.
Breakage of single-stranded DNA to form a covalent protein-DNA
complex is another reaction carried out by the Ustilago topoisomerase
which may be biologically significant in recombinational processes. Type
I topoisomerases from rat liver and HeLa cells form similar complexes

Figure 27. Topoisomerase activity in the rec 2 mutant.
Cells from haploid strains 58 (rec +) and 221 (rec 2) were grown in
2 liters of medium and processed through the polyethylene glycol step as
described in Appendix D. Reactions (50 yl) containing 75 mM potassium
phosphate, pH 7.5, 5 raM EDTA, 0.4 mM 2-mercaptoethanol, 50 yg/ml BSA, 5%
glycerol, 2 nmol XRF I DNA and the following amounts of enzyme were
incubated at 25°C. AFter 30 min the reactions were terminated an
analyzed by gel electrophoresis as described in Appendix C. Lanes A-C
contained 1, 2, and 5 Vg of protein from strain 58; lanes D-G contained
0.5, 1, 2, and 5 Ug of protein from strain 221.

97
A B C D E F G

98
with single-stranded DNA and in addition they can covalently join the DNA
in this complex to another DNA strand (14,39). Studies are in progress
to determine if the Ustilago enzyme can also covalently join two DNA
molecules.
Topoisomerase activity is significantly reduced in the recombin¬
ation defective rec 2 mutant. Enzyme partially purified from this mutant
is considerably less stable to heating than enzyme from rec + cells
indicating that the mutant enzyme may be structurally altered.
The rec 2 mutant has a reduced frequency of mitotic crossing over.
Although spontaneous gene conversion is normal, UV-induced gene conver¬
sion is substantially reduced. The most unusual feature of the rec 2
mutant is its inability to undergo meiosis. It has been speculated that
the blockage in meiosis may be a result of an impaired ability of rec 2
to carry out rejoining of single strands of DNA at the late steps of
chiasmata formation (92). The notion of topoisomerase playing a part in
this step of meiotic recombination is tantalyzing.

y
APPENDICES

APPENDIX A
PHAGE, BACTERIAL AND FUNGAL STRAINS
Ustilago maydis 58 and 221 were acquired from Dr. Robin Holliday,
National Institute for Medical Research, London. The cells were
cultivated in medium consisting of 2% peptone, 2% sucrose, 1% yeast
extract in a VirTis fermenter with vigorous aeration. Bacteriophage
<¡>Xl74am3 and its host E. coli HF4704 were acquired from Dr. Charles
Radding, Yale University. Bacteriophage f8 and its host E. coli K37
were acquired from Dr. W. Konigsberg. Bacteriophage P22 and its host
Salmonella typhimurim DB25 were acquired from Dr. David Botstein,
Massachusetts Institute of Technology.
100

APPENDIX B
PREPARATION OF DNA
Protocols used for preparing xl74am 3 phage DNA, f3 phage DNA,
174 RFI [^hJ-DNA and f3 RFI [^H]-DNA were described by Cunningham
et al. (93). Preparation of P22 [^H]-DNA was described by Carter and
Radding (94). Relaxed closed circular DNA (RF IV) was obtained by
treating either (j>x RF I or f9 RF I DNA with purified Ustilago
topoisomerase. Following relaxation, the topoisomerase was removed by
phenol extraction. Conversion of f3 RFI DNA to linear DNA(RFIIl) was
achieved by digestion with the restriction endonuclease Sau 96.
Denaturation of duplex DNA was carried out by adding NaOH to 50 mM and
incubating the DNA at 37°C. After 10 -15 min the DNA solution was
reneutralized by adding HC1 to 50 mM and Tris-HCl, pH 7.5 to 25 mM.
Labeling of X RF I DNA by nick translation with [a-^P] dCTP
used a modification of the procedure described by Muzyczka (95).
Briefly, a reaction (55 yl) containing 50 mM Tris-HCl, pH 7.6, 5 mM
MgCl2, 10 mM 2-mercaptoethanol, 25 yM dGTP, 25 yN dATP, 25 pM dTTP,
200 yCi [a-32p] dCTP (410 ci/mmol), 3 nmol x RF I DNA, 1 ng
pancreatic DNase, and 5 units E. coli DNA polymerase I were incubated at
15°C. After 1 hr, the reaction was terminated by adding EDTA to 15 mM.
The reaction was then phenol extracted 1 time and the aqueous phase
loaded onto a Sephacryl S-200 column equilibrated in 10 mM Tris-HCl, pH
7.5, 1 mM EDTA. The DNA eluted in the void volume. The specific
activity of the DNA was between 2.5 - 5.0 x 10^ cpm/nmol DNA.
101

102
Labeling f9 RF III at the 5' end was accomplished by using
[Y-32p] atp an was dephosphorylated using bacterial alkaline phosphatase as described
previously (96). Briefly, a reaction (80 pi) containing 50 mM Tris-HCl,
pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 8 nmol dephosphorylated f3
RF III, 50 PCi [Y-32P] ATP (3000 Ci/mmol), and 5 units of T4
polynucleotide kinase was incubated at 37°C for 90 min. The reaction
was then dialyzed against 3 - 250 ml changes of 10 mM Tris-HCl, pH 7.5,
1 mM EDTA, 1 M NaCl and then 3 - 250 ml changes of 10 mM Tris-HCl, pH
7.5, 1 mM EDTA. The specific activity was 4 x 10^ cpm/pmol.
Labeling f9 RF III at the 3' end was accomplished by using
[a-32P] dGTP and the large Klenow fragment of E. coli DNA polymerase
I (97). A reaction (80 pi) containing 10 mM Tris-HCl, pH 7.5, 10 mM
MgC 12, 10 yH dGTP, 125 pCi [cx~32P] dCTP (410 Ci/mmol) 25 nmol f9
RF III DNA (Sau 96 restriction) and 3 units of the large Klenow fragment
of E. coli DNA polymerase I was incubated at room temperature. After 10
min the reaction was stopped by adding 12.5 pi 0.2 M EDTA and 25 pi 20%
sodium acetate, pH 6.0. The reaction volume was then adjusted to 150 Pi
and dialyzed as described previously for DNA labeled with T4
polynucleotide kinase. The specific activity of the DNA was 1 x 10^
cpm/pmol.
Isolation of complementary single-stranded rings of X DNA was
achieved using a protocol described by Stettler et al. (98). Superheli¬
cal X 174 DNA was nicked in one strand with pancreatic DNase in the
presence of ethidium bromide. The strands were then separated by
centrifugation through an alkaline sucrose gradient. The nicking

103
reaction (1 ml) contained 20 mM Tris-HCl, pH 8.0, 15 mM MgCl2, 0.1 M
NaCl, 300 Pg/ral ethidium bromide, 800 nmoles xRFI [^H] DNA and 240
ng pancreatic DNAse. The reaction was incubated at 30°C for 20 min and
then stopped by adding EDTA to 20 mM. The reaction was then phenol
extracted 3 times to remove both protein and ethidium bromide. The DNA
was precipitated from the aqueous phase by adding 2 volumes of ethanol
and placing in a dry ice-ethanol bath for 10 min. After centrifugation,
the DNA pellet was resuspended in 0.4 ml of 10 mM Tris-HCl, pH 7.5, 1 mM
EDTA. Sodium hydroxide (10 M) was then added to a final concentration
of 0.3 M. The denatured DNA was then divided in half and loaded on two
11-ml 5 - 20% sucrose gradients containing 0.25 M NaOH, 0.75 M NaCl, 1
mM EDTA. Gradients were centrifuged in the TÍ40.1 rotor of a Beckman
L5-50 centrifuge at 34,000 rpm for 18 hr at 4°C. Fractions (0.2 ml)
were collected and the radioactive content of each was determined. The
fractions containing the single-stranded circles were pooled and
neutralized by adding 1 M Tris-HCl, pH 7.5.

APPENDIX C
ASSAYS
Topoisomerase
Topoisomerase activity was monitored during the enzyme purifica¬
tion using the D-loop assay. Reactions (50 yl) containing 75 mM potas¬
sium phosphate, pH 7.5, 1 mM EDTA, 5% glycerol and 3 nmol of xRF
I[3H] DNA (1-2.5 x 10^ cpm/nmol) were incubated with topoisomerase
at 25°C. After 30 min, 10 yl of 3 M KC1 was added to stop the reac¬
tions. The reaction was then split into two 30-yl aliquots. One ali¬
quot was used to assess the amount of nuclease activity using an assay
described by Kuhnlein et al. (45). To the second aliquot was added
single-stranded fragments of x DNA to a final concentration of 0.2 mM.
After incubating the reaction at 75°C for 3 hr the reaction mixture was
diluted into 2 ml of cold 10 x SSC and passed through a nitrocellulose
filter which had been previously washed with 5 ml 10 x SSC. Following
two 5-ml washes with 10 x SSC the filters were dried and then counted in
2 ml of Econofluor. One unit of topoisomerase activity equals the
amount of enzyme that prevents 1 nmol of x RF l[3H] DNA from sticking
to a filter. The efficiency of D-loop formation in the absence of added
topoisomerase was approximately 50%.
The high salt nitrocellulose filter assay has also proven to be a
convenient way to measure topoisomerase activity. After incubating the
topoisomerase with X RF I [3H]-DNA under the reaction conditions
described in the D-loop assay, the reaction was split into two 25 y 1
aliquots. One aliquot was used to assess endonuclease activity as
described before. The second aliquot was combined with 1 ml of 50 mM
104

105
Tris HC1, pH 7.5, 1 mM EDTA, 3.5 M NaCl and passed through a nitrocellu¬
lose filter previously washed with 5 ml of the same high salt buffer.
After two 5-ml washes with the high salt buffer, the filter was dried
and the radioactivity measured as in the D-loop assay.
The conversion of superhelical to relaxed DNA has also been fol¬
lowed using an agarose gel assay. Reactions (25 yl) were carried out
using the same conditions as in the D-loop assay. To stop the reac¬
tions, 3 yl of a solution containing 5% SDS, 0.05% bromophenol blue, 50%
glycerol was added. The sample was then electrophoresed through a 1.2%
agarose gel containing 40 mM Tris-HCl, pH 7.9, 1 mM EDTA, 5 mM sodium
acetate at 3V/cm for 12 - 14 hr at room temperature. Following electro¬
phoresis the gel was stained with a 1 yg/ml solution of ethidium bromide
for 10-30 min before photographing under ultraviolet light.
DNA-Binding Protein
The assay used to detect an interaction between protein and DNA
has been described by Tsai and Green (99) and measures the retention of
[3H] DNA on a nitrocellulose filter in the presence of protein. To
assay for DBP IV during the purification, reactions (50 yl) containing
20 mM Tris, pH 8.0, 1 mM EDTA, 2 mM 2-mercaptoethanol, 5% glycerol, 50
mM NaCl, and 2-3 nmol of denatured P22 [^H] DNA (1.1 x 10^ cpm/
nmol) were incubated with DNA binding protein at 25°C. After 10 min the
reactions were terminated by adding 1 ml of the reaction buffer and then
processed as described by Banks and Spanos (100). One unit is defined
as the amount of protein required to cause the retention of 1 nmol of
DNA on a nitrocellulose filter.

106
Exonuclease
This assay measures the conversion of DNA to an acid-soluble form.
Reactions with exonuclease I were carried out in 200 yl containing 50 mM
glycine, pH 9.5, 10 mM MgCl2 at 37°C. After 30 min the reactions were
terminated by adding 0.3 ml of ice-cold carrier salmon sperm DNA (1
mg/ml) and 0.5 ml 10% trichloroacetic acid. After standing in ice for
10 min the tubes were centrifuged in an Eppendorf Microfuge. After 2
min, 0.5 ml of the supernatant was removed and counted in 5 ml of Triton
scintillation flúor. Reactions with exonuclease VII were carried out in
200 yl containing 50 mM potassium phosphate, pH 7.9 at 37°C. After 30
min the reactions were terminated and processed as described for exo¬
nuclease I.
Protein
Protein content was measured using the method described by
Bradford (101).

APPENDIX D
PROTEIN PURIFICATION
Topoisomerase
All of the steps during the purification were carried out between
0 - 4°C. Activity was determined using the D-Ioop assay. Cells (100 g)
stored at -20°C were thawed in 200 ml of 20 mM potassium phosphate, pH
7.0, 1 raM EDTA, 10 mM 2-mercaptoethanol, 1 M KC1, 5% glycerol (buffer
A). The cells were broken in a French press at 20,000 psi and then cen¬
trifuged at 18,000 rpm for 30 min in the JA 20 rotor of a Beckman J21-C
centrifuge (fraction I). After the lipid was removed from the supernat¬
ant , 24 g of polyethylene glycol was added and the mixture was allowed
to stir for 4 hr before it was centrifuged. The supernatant was saved
and the pellet was reextracted with 70 ml of buffer A and centrifuged
again. The supernatants were combined (fraction II) and loaded onto a
130 ml hydroxylapatite column previously equilibrated in buffer A. The
column was washed with 300 ml of buffer A before eluting the enzyme with
a 1 liter gradient of (0.02 - 1.0 M) potassium phosphate, pH 7.0, in
buffer A. The broad peak of activity (fraction III) which eluted about
midway through the gradient was loaded directly onto a 40 ml octyl-
Sepharose column equilibrated in 20 mM potassium phosphate, pH 7.0, 1 mM
EDTA, 10 mM 2-mercaptoethanol, 10% glycerol, and 0.1 M KC1 (buffer B).
After washing the column with 200 ml of buffer B, a 200 ml 0-2% gradient
of Triton X-100 was initiated. Activity eluted approximately two-
thirds of the way through the gradient. The active fractions were
pooled (fraction IV) and loaded directly onto a 2.5 ml phosphocellulose
107

108
column previously equilibrated in buffer B. After washing the column
with 30 ml of buffer B, a 30 ml gradient from 0.1 - 1.0 M KC1 in buffer
B was applied. Activity eluted as a broad peak covering the latter
third of the gradient. After pooling the active fractions (fraction V) ,
a small aliquot (3 ml) was loaded onto a column of Sephacryl S-200 (1.4
x 56 cm) which had been previously equilibrated in buffer B containing
0.5 M KC1. Fractions (1 ml) were collected at a flow rate of 10 ml/hr.
The peak of activity eluted between ferritin and catalase and was pooled
( fraction Vi).
25,000 Dalton Acid-Soluble Protein
The presence of the 25,000 dalton acid-soluble protein was deter¬
mined at each step of the purification by SDS gel electrophoresis as
described in Appendix E. All steps were carried out at 0 - 4°C. Cells
(100 g) were suspended and broken in 300 ml of 10 mM Tris-HCl, pH 7.5, 1
mM EDTA, 0.35 M NaCl by passage through a French press at 20,000 psi and
then centrifuged in the JA20 rotor of a Beckman J21-C centrifuge at 3500
rpm for 30 min. The supernatant was saved and made 5% in perchloric
acid by adding 23 ml of 70% perchloric acid. After standing for 10 min
the precipitate was removed by centrifugation. Solid trichloroacetic
acid was then added to a final concentration of 25%. This was allowed
to stir for 10 min before centrifuging. The pellet was saved and washed
1 time with 100 ml of acid acetone (1.0 ml concentrated HC1 in 100 ml
acetone) and 2 times with 100 ml of acetone. The residual acetone was
removed under vacuum. The small white pellet was dissolved in 5 ml of
10 mM sodium borate, pH 9.0. Half of this volume was loaded onto a 2 ml
carboxymethylcellulose column equilibrated in 10 mM sodium borate, pH

109
9.0, 0.1 M NaCl (buffer C). The column was washed with 20 ml of buffer
C and then 10 ml of 0.2 M NaCl in buffer C. Finally the column was
eluted with 10 ml of 0.5 M NaCl in buffer C and 1 ml fractions were
collected. The 25,000 dalton protein eluted in fractions 2 and 3.
These fractions were combined and contained approximately 60 pg of
protein as judged from an SDS gel.
DBP IV
All steps of the purification were carried out at 0 - 4°C. The
assay used to detect DBP IV is described in Appendix C. A summary of
the purification is presented in Table VI. Frozen cells (100 g) were
thawed and suspended in 200 ml of 20 mM potassium phosphate pH 7.0, 1 mM
EDTA, 2 mM 2-mercaptoethanol, 0.1 mM PMSF, 5% glycerol (buffer D). The
cells were broken in a Mantin-Gaulin cell crusher at 9000 psi and then
centrifuged in the JA10 rotor of a Beckman J21-C centrifuge at 10,000
rpm for 30 min. The supernatant was discarded and the pellet resuspend¬
ed in 100 ml of buffer D containing 1 M KC1 and centrifuged again, this
time in the JA 20 rotor at 18,000 rpm for 20 min. The pellet was
discarded and to the supernatant was added solid polyethylene glycol to
a final concentration of 7%. This mixture was allowed to stir for 2 hr
and then centrifuged in the JA 20 rotor at 18,000 rpm for 20 rain. The
supernatant was saved and dialyzed against two 2-liter changes of 20 mM
Tris-HCl, pH 8.0, 2 mM 2-mercaptoethanol, 1 mM EDTA, 50 mM NaCl, 5%
glycerol (buffer E). The dialysate (fraction I) was loaded onto a 15 ml
DNA agarose column equilibrated in buffer E. After washing the column
with 150 ml of buffer E containing 0.1 M NaCl, the column was eluted in

TABLE VI
PURIFICATION OF DBF IV
Fraction
Volume
ml
Activity
units
Protein
mg
Specific
Activity
units/mg
I
100
2400
36.5
66
II
15
5100
0.5
10,200
III
10
9200
0.2a
46,000
aProtein concentration was estimated from an SDS polyacrylamide gel.

Ill
a stepwise fashion with 75 ml each of 0.25 M NaCl, 0.5 M NaCl, 1.0 M
NaCl, and 2.0 M NaCl in buffer E. The eluate was collected in 5 ml
fractions. DNA binding activity was present in both the 0.25 M and 0.5
M salt bumps. The active fractions from the 0.5 M salt bump were pooled
and dialyzed against 1 liter of buffer E. The dialysate (fraction II)
was then loaded onto a 5 ml carboxymethylcellulose column equilibrated
in buffer E. After washing the column with 60 ml of buffer E, the
column was washed in a stepwise manner with 35 ml of 0.25 M NaCl and
then 1.0 M NaCl in buffer E. The eluate from the column was collected
in 5 ml fractions. Fractions from the 1.0 M salt elution contained DBP
IV and were pooled (fraction III).

APPENDIX E
ELECTROPHORESIS
Analysis of protein by SDS gel electrophoresis used the method
described by Laemmli (102). Following electrophoresis the gels were
usually fixed in ethanol/acetic acid/water (40/10/50, v/v/v) before
staining with Coomassie blue. Occasionally, gels were stained with
silver using the technique described by Merril et al. (103.)
Ionophoresis of acid hydrolysates on Whatman 3 MM paper was
carried out at pH 3.5 in pyridine/acetic acid/^O (5/50/945, v/v/v) at
2000 V for 75 min (104). Following electrophoresis the paper was dried
in a 50°C oven. Marker amino acids were visualized by spraying the
paper with 0.3 % (wt/vol) ninhydrin in 1-butanol and then heating to
100°C for several minutes (105). Phosphate was detected by spraying the
paper with a solution of 0.42% ammonium molybdate in 1 M sulfuric
acid/10% ascorbic acid (6/1, v/v).
112

APPENDIX F
REAGENTS AND MATERIALS
Chromatographic Media
Affi-Gel blue, a Cibacron blue F3GA agarose was obtained from
Bio-Rad Laboratories. Octyl-Sepharose and Sephacryl S-200 were obtained
from Pharmacia Fine Chemicals. Phosphocellulose Pll and carboxymethy1-
cellulose were obained from Whatman. HA-Ultrogel was from LKB
ins truments.
Enzymes y
Bacterial alkaline phosphatase and pancreatic DNase were obtained
from Worthington Biochemicals Inc. E. coli DNA polymerase I was
obtained from Boehringer Mannheim Biochemicals. Polynucleotide kinase,
large Klenow fragment of E. coli DNA polymerase I, and restriction
endonuclease Sau 96 were obtained from Bethesda Research Laboratories.
Micrococcal nuclease and catalase were obtained from Sigma Chemical Co.
Exonuclease I and Exonuclease VII were generously donated by M. Yarnell
and S. Smallwood.
Miscellaneous
Histones, spermidine, poly-L-lysine, novobiocin, nalidixic acid,
p-chloromercuriphenylsulfonate, 5,51 dithiobis(2-nitrobenzoate), ATP ,
GTP, UTP, TTP, dATP, ADP, AMP, dithiothreitol, N-ethyl maleimide
phosphoserine and phosphothreonine were from Sigma Chemical Co.
Electrophoresis grade agarose was from BioRad Laboratories. Poly(rA),
poly(dG), poly(dC), poly(dA), poly(dT), g,y-methylene adenosine
triphosphate, a,g-methylene adenosine triphosphate and poly(d(G-C))
113

114
were obtained from P-L Biochemicals. Adenosine-5'-0-(3-thiotriphos-
phate), adenylylimidodiphosphate and CTP were from Boehringer Mannheim
Biochemicals. Triton X-100 was from New England Nuclear. Nitrocellu¬
lose filters (BA85) were from Schleicher and Schuell. Phosphothreonine
was kindly provided by Dr. D. Rawlins. Berenil was a generous gift from
the Squibb Pharmaceutical Co. Phosphotyrosine was synthesized according
to Rothberg et al. (105).

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BIOGRAPHICAL SKETCH
Tom was born on December 8, 1951, the fourth child of John Rowe and
Marie Cardón. He has three older brothers, John, Rich and Bob, as well
as a younger sister Beverly. Tom was raised in Boulder, Colorado, and
graduated from Boulder High School in 1970. He then attended Colorado
State University where he received his Bachelor of Science degree in
chemistry. Since then, Tom has been a graduate student working in the
laboratory of Dr. Bill Holloman in the Department of Immunology and
Medical Microbiology at the University of Florida. Upon receiving his
degree of Doctor of Philosophy, Tom will join the laboratory of Dr. Leroy
Liu in the Department of Physiological Chemistry at The Johns Hopkins
University School of Medicine in Baltimore, Maryland.
121

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Professor of Immunology and
Medical Microbiology
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
George E. Gifford
Professor of Immunology and
Medical Microbiology
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Nicholas Muzyczka
Associate Professor of Immunolog
and Medical Microbiology

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
wc'r'HjiC}
/S'
íes B. Flanegan
assistant Professor of Immunology
and Medical Microbiology
I certify that I
conforms to acceptable
adequate, in scope and
of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation forthe degree of Doctor
Molecular Biology
This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
December 1982
iean,College of Medicine
Y/'7/r*
Dean for Graduate Studies
and Research

o Vr'i""1,111,11 "I* III
3 1262 08554 5191


o Vr'i""1,111,11 "I* III
3 1262 08554 5191




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