The Effect of Supercoiling on Small Molecule-DNA Interactions

by

William Albino LaMarr

B.S. in Biochemistry

Hofstra University, 1992

SUBMITTED TO THE DIVISION OF TOXICOLOGY IN PARTIAL

FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

Ph.D. IN TOXICOLOGY

AT THE

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

June, 1998

© 1998 Massachusetts Institute of Technology. All rights reserved.

Signature of Author:

Division of Toxicology

June, 1998

Certified by:

Peter C. Dedon

Associate Professor of ToxicologyThesis Supervisor

Accepted by:

MASSACHUSETTS INSTITUTEOF TECHNOLOGY

SJlUNo8~

U9RARHe~

Peter C. Dedon

Chairman, Committee on Graduate Students

d Division of Toxicology

", I. / I

This doctoral thesis has been examined by a committee of the Division of Toxicology as follows:- A __

Professor John M. EssigmannlL I Chairman

Associate Professor Peter C. Dedon

/ .--- ,Sunervisor

Professor William G. Thilly

Professor Steven R. Tannenbaum

Associate Professor Ram Sasisekharan

\ f~-L

The Effect of Supercoiling on Small Molecule-DNA Interactions

by

William Albino LaMarr

Submitted to the Division of Toxicology

on May 12, 1998 in partial fulfillment of the

requirements for the degree of

Ph.D. in Toxicology

ABSTRACT

DNA supercoiling is an important aspect of genomic architecture and has been implicated

in a variety of biological processes. The effects of supercoiling on DNA structure and dynamics,

in conjunction with its physiologic importance, suggest that superhelical tension may be an

important determinant of genotoxin target selection in vivo.

With the premise that supercoiling will affect DNA-genotoxin interactions, I have

compared DNA damage in plasmid substrates with either negative or positive superhelical

tension. A new technique, utilizing the archaeal histone HMf, was developed to prepare large

quantities of positively supercoiled DNA. The supercoiled substrates were then treated with

oxidants such as y-radiation, peroxynitrite, iron (II)-EDTA/hydrogen peroxide, iron (II)/hydrogen

peroxide, copper (II)/hydrogen peroxide and calicheamicin ylI , and the alkylating agent dimethyl

sulfate.

Four of the oxidizing agents (y-radiation, peroxynitrite, iron (II)-EDTA/hydrogen

peroxide and iron (II)/hydrogen peroxide) induce equal levels of DNA strand breaks in both

substrates. This is consistent with molecules that produce highly reactive species but

presumably have little if any interaction with DNA.

Copper and dimethyl sulfate, however, exhibit a supercoiling bias in which more damage

occurs in the positively supercoiled substrate than the negative. Though these molecules have

different modes of action, they share the N7 position of guanine as their primary site of

interaction with DNA. These results suggest that guanine-N7 is either more accessible or more

reactive in DNA with positive superhelical tension than in negatively supercoiled DNA.

Calicheamicin 711 also shows a sensitivity to superhelical tension by causing -50% more

strand breaks in the negatively supercoiled substrate. This difference is due to a reduced

accessibility of glutathione to drug bound in the minor groove of positively supercoiled DNA.

These results have important implications for any in vivo chemistry involving glutathione which

occurs on or near DNA, such as genotoxin activation, damage chemistry partitioning, protection

and repair.

My work demonstrates that supercoiling can indeed play a role in the interaction of DNA

with small non-intercalative molecules. These results shed light on the complex issue of

genotoxin target selection inside a cell.

Thesis Supervisor: Peter C. Dedon

Title: Associate Professor of Toxicology

Dedicated to my wife Beverly.

Without your unconditional support,

I never would have had the strength

to make it where I am today.

Acknowledgments

I would first and foremost like to express my gratitude to my wife and my family. Your

constant words of encouragement, your faith in my abilities, and your support throughout the

years brought me to this point. I do not feel that I would have ended up where I did without

your help.

Secondly, I would like to thank all the members of the Dedon lab, past and present;

Aaron, Jinghai, Punam, Yu Li, Ken, Choi, Stan, Qi, Jiongru, Daniel and William. It was my

interactions with each and every one of you that kept me sane throughout my time here. Though

it may not have seemed it at times (insert chuckle here), I enjoyed working with you all.

I would also like to thank Teresa Wright for proofreading my thesis. I have always had

problems crossing the I's and dotting the T's (Oops!). I really appreciate your reading this time

and time again, especially knowing how boring it is.

I would particularly like to thank Mr. Aaron A. Salzberg. You played many roles in my

graduate career, and for that, I am grateful. As a colleague, you never let an opportunity to help

me go by. As a mentor, you taught me a lot about science and being a scientist. Most

importantly, as a friend, you were always there for me. I will never forget the concerts, the

fishing trips, the poster making sessions, the pranks and everything. Thanks.

I would also like to express my appreciation to all the people who supplied me with the

items I needed to complete my thesis work. First I thank Ken Moore for providing the

peroxynitrite I used in my early studies. Secondly, I would like to thank Dr. George Ellestad and

Dr. K.C. Nicolaou for providing the calicheamicin 7yl and the calicheamicin 011, respectively.

And last, but certainly not least, I would like to thank Professor John Reeve and all the members

of his laboratory at Ohio State University for providing the HMf proteins. Without this

generous gift, none of this research would have been possible. Special thanks go to Dr. Kathleen

Sandman. Without your input and advice, I would not have been able to develop the technique

for producing positively supercoiled DNA. It was my discussions with you that allowed my

research to progress beyond just the development of a technique.

In addition, I would like to thank the members of my thesis committee; Dr. John

Essigmann, Dr. Steven Tannenbaum, Dr. William Thilly & Dr. Ram Sasisekharan; for their

intellectual support, and the National Institute of Health for it's financial support, throughout the

course of my studies.

Finally I would like to thank Dr. Peter Dedon for his support during my thesis work. In

particular, I thank you for giving a chance to someone who didn't really know what he wanted to

do with his life. Not everyone would have given me this opportunity.

Table of Contents

Abstract --------------------------------------------------------------------------------------------------- 3

Acknowledgments ---------------------------------- -------------------------- 6

Table of Contents -------------------------------------------------------------- 8

List of Abbreviations -------------------------------------------------------------- 10

List of Figures ------------------------------------------------------------------ 12

List of Tables --------------------------------------------------------------- 14

Publications & Presentations -------------------------------------------------------- 15

Biographical Note ------------------------------------------------------------ 16

Chapter 1. Introduction ------------------------------------------------------------- 17

I. DNA Supercoiling --------------------------------------- 18

II. Small Oxidative Agents -------------------------------------------------- 22

III. DNA Alkylators ------------------------------------------------------- 25

IV. Enediynes ------------------------------------------------------------ 26

V. Figures --------------------------------------------------------- 28

Chapter 2. Large scale preparation of positively supercoiled DNA using an archaeal histone -- 34

I. Abstract --------------------------------------------------------- 35

II. Introduction --------------------------------------------------------- 36

III. Materials and Methods -------------------------------------------- 37

IV. Results and Discussion ----------------------------------- ------------------ 38

V. Figures --------------------------------------------------------- 40

Chapter 3. Differential effects of DNA supercoiling on radical-mediated DNA strand breaks--- 41

I. Abstract ------------------------------------------------------------- 42

II. Introduction --------------------------------------------------------- 43

III. Materials and Methods -------------------------------------------- 46

IV. Results and Discussion ------------------------------------ ----------- 48

8

V. Figures --------------------------------------------------------- 54

VI. Tables ---------------------------------------------------------- 56

Chapter 4. Supercoiling affects the accessibility of glutathione to DNA-bound molecules:

Positive supercoiling inhibits calicheamicin-induced DNA damage ---------------- 57

I. Abstract ------------------------------------------------------------- 58

II. Introduction --------------------------------------------------------- 59

III. Materials and Methods -------------------------------------------- 60

IV. Results -------------------------------------------------------------- 63

V. Discussion ----------------------------------------------------------- 66

VI. Figures --------------------------------------------------------- 71

Chapter 5. Conclusion and Future Studies---------------------------------- 76

Bibliography ---------------------------------------------------------------- 83

List of Abbreviations

(in alphabetical order)

8-oxoA

8-oxoG

Ac-4-HAQO

AFB 1

CAL y

CAL 0

CBE

Cu

Cyt Glycol

DMS

DNA

e-aq

E. coli

EDTA

EtBr

Fe

Fpg

y-rad

'H

H20 2

Lk

MNU

NO'

Nth

8-hydroxyadenine

8-hydroxyguanine

4-acetoxy-aminoquinoline- 1-oxide

aflatoxin B

calicheamicin yjl

calicheamicin 011

chicken blood extract

copper

cytosine glycol

dimethyl sulfate

deoxyribonucleic acid

solvated electron

Escherichia coli

ethylenediaminetetraacetic acid

ethidium bromide

iron

formamidopyrimidine-DNA glycosylase

y-radiation

hydrogen atom

hydrogen peroxide

linking number

methylnitrosourea

nitric oxide

endonuclease III

02'- superoxide

'OH hydroxyl radical

ONOO- peroxynitrite

ONOOH peroxynitrous acid

Tw twist

G superhelical density

Wr writhe

List of Figures

Figure 1) The two components of supercoiling: twist and writhe ------------------------- 28

Figure 2) While linking number must remain constant in any closed system, twist and writhe can

interconvert ------------------------------------------------------------------ 29

Figure 3) Superhelical tension is generated in vivo by the act of transcription: negative

supercoiling in the 5' end of the gene and positive supercoiling in the 3' end ------------------ 30

Figure 4) Structures of the enediynes ----------------------------------- ------------- 31

Figure 5) Enediynes: mechanisms of activation and DNA damage ----------------------------- 32

Figure 6) Deoxyribose radical degradation pathways as a result of minor groove hydrogen

abstraction ----------------------------------------------------------------------------- 33

Figure 7) Identification and characterization of positively supercoiled plasmid DNA ---------- 40

Figure 8) Quantitation of y-radiation, peroxynitrite and Fe+2-EDTA/H 20 2 induced strand breaks

in negatively and positively supercoiled DNA substrates ----------------------------------- 54

Figure 9) Quantitation of Fe+2/H 20 2 and Cu+2/H 20 2 induced strand breaks in negatively and

positively supercoiled DNA substrates ------------------------------------------ ---------- 55

Figure 10) Quantitation of calicheamicini lI/glutathione induced strand breaks in negatively and

positively supercoiled DNA substrates ------------------------------------------ ---------- 71

Figure 11) Calicheamicin yll binding causes an increase in the sedimentation rate of negatively

supercoiled plasmid DNA ----------------------------------------------- ---------------------------------- 72

Figure 12) Determination of the relative binding affinity of calicheamicin ylI for negatively and

positively supercoiled plasmid DNA ---------------------------------------- 73

Figure 13) Quantitation of calicheamicin 011 induced strand breaks in negatively and positively

supercoiled DNA substrates -------------------- -------------------- --------------------------- 74

Figure 14) Comparison of calicheamicin 711 induced DNA damage in negatively and positively

supercoiled plasmid DNA when activated by either glutathione or methylthioglycolate -------- 75

List of Tables

Table 1) Quantitation of total methylation induced by dimethyl sulfate in negatively and

positively supercoiled plasmid DNA ----------------------------------- -------------- 56

Publications of William Albino LaMarr

LaMarr, W.A.; Yu, L.; Nicolaou, K.C.; Dedon, P.C. (1998) Supercoiling affects the accessibility

of glutathione to DNA-bound molecules: Positive supercoiling inhibits calicheamicin-induced

DNA damage, Proceedings of the National Academy ofSciences, USA, 95(1), 102-107.

LaMarr, W.A.; Sandman, K.M.; Reeve, J.N.; Dedon, P.C. (1997) Differential effects of DNA

supercoiling on radical-mediated DNA strand breaks, Chemical Research in Toxicology, 10(10),1118-1122.

LaMarr, W.A.; Sandman, K.M.; Reeve, J.N.; Dedon, P.C. (1997) Large scale preparation of

positively supercoiled DNA using the archaeal histone HMf, Nucleic Acids Research, 25(8),1660-1661.

Published Meeting Abstracts

LaMarr, W.A. and Dedon, P.C. (1997) DNA supercoiling alters calicheamicin 711 activation by

glutathione, Journal of Biomolecular Structure and Dynamics, 14(6), 869-871.

LaMarr, W.A. and Dedon, P.C. (1997) Sensitivity of copper, but not iron, to DNA supercoiling,

Proceedings of the American Association of Cancer Research, 38, 127.

LaMarr, W.A.; Sandman, K.M.; Reeve, J.N.; Dedon, P.C. (1996) Supercoiling and radical-

mediated DNA damage, Proceedings of the American Association of Cancer Research, 37, 143.

Presentations

LaMarr, W.A. and Dedon, P.C. (1998) The effect of DNA supercoiling on the damage produced

by calicheamicin and other small oxidative genotoxins, Department of Research and Development,

Wyeth-Ayerst, Pearl River, NY.

LaMarr, W.A. and Dedon, P.C. (1998) Superhelical tension and small molecule-DNA

interactions, Boston Mutagenesis Society.

LaMarr, W.A. and Dedon, P.C. (1997) The effect of supercoiling on oxidative DNA damage, New

England Pharmacologists, 26th annual meeting.

Biographical Note

William A. LaMarr was born on December 23, 1969 in Flushing, New York. In his senior

year at Bloomfield High School, he completed a work-study program with the Department of

Obesity at Hoffman La Roche, in Nutley N.J. Shortly thereafter, he was accepted into the

Department of Biochemistry at Hofstra University in Hempstead, N.Y. He was graduated from

Hofstra University in June of 1992 with a B.S. in Biochemistry. In September of 1992 he joined

the lab of Professor Peter C. Dedon in the Division of Toxicology at the Massachusetts Institute

of Technology, where he investigated the effect of supercoiling on small molecule-DNA

interactions. He defended his Ph.D. thesis on May 12, 1998 and is currently pursuing a career in

the pharmaceutical industry.

William was married on September 26, 1998 and he and his wife, Beverly, currently reside

in Somerville, MA.

CHAPTER 1

INTRODUCTION

SUPERCOILING

Supercoiling is the response of DNA to torsional stress and it involves three aspects of

DNA conformation. First, the DNA can change its helical repeat (twist). Second, the double

helix can rotate around itself (writhe). And thirdly, the DNA molecule can adopt a variety of

unusual secondary structures such as Z-DNA, cruciforms and unpaired regions.

There are many reviews of the mathematical principles of DNA supercoiling.1- 6 The

main concepts of DNA supercoiling can be summed up in the following simple equation:

Lk= Tw + Wr

Lk: linking number; Tw: twist; Wr: writhe

The linking number (Lk) is simply the number of times one of the strands of the double helix

crosses the other strand. There are two variables that contribute to this value, the twist (Tw) and

the writhe (Wr) (Figure 1). Tw is the number of times one of the strands wraps 3600 around the

helical axis (equivalent for both strands). Wr is the number of times the DNA helical axis crosses

itself. These concepts were borrowed from mathematical models of a closed ribbon in space.7

When the ends of a DNA molecule are restrained, as in a circular plasmid or a loop of chromatin

in a eukaryotic cell, the Lk of the DNA molecule must remain constant, unless one of the strand

is broken. However, Tw and Wr can interconvert (Figure 2).

Supercoiling is a biologically important aspect of DNA physiology in both prokaryotic

and eukaryotic cells. Supercoiling is most commonly expressed in terms of superhelical density

(a) which is the number of writhes per helical turn. The E. coli genome has a a of -0.05 while the

genomes of Drosophila melanogaster and human cells have no net superhelical tension.8 This is

presumably the result of several enzymes, topoisomerases,9- 11 which are responsible for

maintaining levels of superhelical tension in vivo by either relieving superhelical stress 12, 13 or

introducing it.14,15 However, there are local "pockets" of both negative and positive superhelical

tension in eukaryotic cells, presumably caused by the act of transcription'6- 2 1 as proposed in the

twin supercoiled domain model of Liu and Wang (Figure 3).22

This model proposes that separation of the DNA strands during transcription, caused by

the helicase activity of the polymerase complex, causes an underwinding of the DNA helix behind

the transcription machinery and an overwinding in front. The resulting torsional tension is

released when the transcription complex dissociates from the DNA. It is proposed however that

in highly transcribed sequences, there are many polymerase complexes, one after the other, on the

active gene sequence, and that this constant transcriptional activity can set up steady state levels

of supercoiling in the ends of the gene. This model is supported by the work of Ljungman and

Hanawalt, in which they showed stable domains of negative and positive supercoiling at the 5'

and 3' ends of the active dihydrofolate reductase gene sequence, respectively. 19

In addition to measurements of superhelical tension, a variety of unusual DNA structures

that are caused by supercoiling have been detected in vivo, including Z-DNA,23 2 5 cruciforms, 26

and regions of single strandedness. 27 A perfect example of one of these supercoiling-induced

changes in secondary structure is the B-DNA to Z-DNA transition that occurs in repeats of

pyrimidine-purine dinucleotide sequences, such as CpG islands, when under negative torsional

tension.28,29 When negative superhelical tension is applied to these sequences, they undergo a

switch from a right-handed double helix (B-DNA) to a left-handed double helix (Z-DNA). This

transition helps to alleviate the torsional tension introduced by the unwinding of the DNA. In

addition, the energy barrier for the transition from B-DNA to Z-DNA can be lowered by the

presence of magnesium ions30 or , in the case of CpG repeats, by methylation at the C5 position

of cytosine. 31

These and other changes in DNA structure and dynamics caused by supercoiling have

been implicated in many cellular processes such as nucleosome assembly, 32-37 replication, 38,39

recombination,40-42 and the initiation and regulation of transcription. 43,44 For example, a

negative linking number change causes an unwinding of the DNA helix, which may eventually

lead to strand separation. The binding of many transcription factors to DNA can induce bending

and looping of the helix leading to a negative writhing of the DNA, and thus a negative linking

number change. 3,4 If this negative superhelical tension occurs in transcription factor binding sites

at the 5' ends of genes, the resulting strand separation may serve as the starting point of

polymerase activity.

In addition to its physiologic roles, supercoiling has also been shown to affect the

interaction of DNA with small genotoxic molecules. A classic example of supercoiling bias

occurs with molecules that intercalate (e.g. ethidium bromide 45). Intercalation is a process by

which a molecule binds to DNA by insertion, in whole or in part, between base pairs. This

insertion is accompanied by both unwinding and lengthening of the DNA helix. Intercalators bind

with highest affinity to negatively supercoiled DNA because of an intercalation-induced net

positive writhing. Local unwinding of the helix to accommodate the intercalating molecule is

compensated by positive changes in the Tw or Wr of the DNA since Lk must be constant in any

closed system.

This affinity bias for negatively supercoiled DNA is demonstrated by the work done with

two strand cleaving antibiotics that bind to DNA by intercalation, bleomycin and

neocarzinostatin. These compounds bind to DNA in part by intercalation of bithiazole or

naphthoate moieties, respectively. 46-48 It was observed that, in both cases, these drugs induced

more DNA strand breaks in negatively supercoiled DNA than in relaxed DNA. The increase in

DNA damage in negatively supercoiled plasmids was attributed to the increased binding of these

molecules to this DNA substrate.

The idea that supercoiling can modulate the damage produced by intercalating genotoxins

is further supported by the work of Menichini et al., in which it was shown that the level of

DNA superhelical tension altered not only the quantity but also the spectrum of DNA adducts

induced by 4-acetoxy-aminoquinoline-1-oxide (Ac-4-HAQO). 49 In these studies, negatively

supercoiled pAT153 plasmid was two times more susceptible to Ac-4-HAQO-induced

adduction than the same plasmid with a single-strand nick introduced. In addition, it was

observed that negative superhelical tension caused a shift in the spectrum of DNA adducts

produced by Ac-4-HAQO. The three main adducts induced by treatment of DNA with Ac-4-

HAQO are 3-(deoxyguanosin-N2-yl)-4-aminoquinoline- 1-oxide, N-(deoxyguanosin-C8-yl)-4-

aminoquinoline- 1-oxide and 3-(deoxyguanosin-N6-yl)-4-aminoquinoline-1-oxide. These three

adducts constituted 50%, 25% and 10% of the total DNA base adducts respectively in the nicked

plasmid substrate. In the negatively supercoiled substrate, these adducts accounted for 80%,

15% and 5% respectively. This case illustrates the influence of DNA supercoiling on the

physical interaction of a genotoxin with DNA bases.

In addition to the general effect of supercoiling observed with intercalators, some of the

secondary structures induced by supercoiling have also been shown to effect DNA damage. For

example, junctions between B-DNA and Z-DNA have been shown to be high affinity damage

sites for dynemicin A and Ac-4-HAQO relative to bulk DNA. Ichikawa et al.50 have shown that

dynemicin A induces more strand breaks in a B-Z junction of DNA when compared to the

identical DNA sequence in the absence of the B-Z transition. Similarly, Rodolfo et al. 51 have

shown that Ac-4-HAQO is not reactive with Z-DNA, but shows a 2.5 fold increase in DNA

adduction at the B-Z junction when compared to relaxed DNA of the same sequence. These

results add another level of complexity to the effect of supercoiling on small molecule-DNA

interactions.

The preceding examples all involve intercalative molecules. However, there are few

examples in the literature that demonstrate an effect of supercoiling on non-intercalative

genotoxins. There has been some debate in the literature as to whether or not supercoiling

modulates radiation-induced DNA damage. Miller et al.52 have shown a direct correlation between

the level of negative superhelicity and the quantity of X-ray-induced single strand breaks in DNA.

They showed that negatively supercoiled DNA sustained more X-ray induced damage than relaxed

DNA at identical doses. However, this work remains controversial because of the contradictory

results presented by Milligan et al.53 in which it was shown that the superhelical state of DNA had

little to no effect on the quantity of X-ray-induced strand breaks. The work presented in this thesis

supports the observation of Milligan et al. in that no effect of supercoiling on radiation induced

strand breaks was observed.

With the exception of the aforementioned work, there is little information regarding the

role of superhelical tension in the interaction of DNA with small molecules that bind to DNA by

other mechanisms. In this work I have investigated the effect of this aspect of DNA

conformation and dynamics on the quantity, chemistry and location of oxidative DNA damage.

SMALL OXIDIZING AGENTS

Oxidative DNA damage has been implicated in a variety of biological processes including

mutagenesis, carcinogenesis and aging. 54-56 Among the most important oxidizing agents in this

respect are y-radiation (y-rad), peroxynitrite (ONOO-), iron (Fe) and copper (Cu).

Fe and Cu are both physiologically important metals that have been implicated in DNA

damage in vivo.57-61 A generally accepted model for Fe-induced and Cu-induced DNA damage

involves Fenton-like chemistry that yields either hydroxyl radical ('OH) or another activated

oxygen species:62-64

Me + n + H202 * Me+(n+l) + OH- + "OH or Me=O + n

Although Fe and Cu presumably damage DNA in the same way, supercoiling may affect the

binding of these metals and thus the quantity, chemistry and location of the DNA damage. For

example, Cu+2 has been shown to bind to the N7 of guanine, 65-67 which could lead to oxidation at

neighboring positions (i.e. C8). Any supercoiling-dependent deformation of DNA secondary or

tertiary structure that would make guanine residues more or less accessible could affect the

binding of Cu+2 and therefore the amount or type of DNA damage formed. A similar argument

could be made for Fe; however, it is less redox active and binds to DNA with lower affinity than

Cu.68

Another agent studied in the following work is ionizing radiation. The radiolysis of water

forms three radical species; 'OH, the solvated electron (e-aq) and the hydrogen atom ('H).6 9

H20 + y-rad * H20' + + e-

H20*+ + H20 c H30+ + 'OH

H20 + y-rad * H20* * Ho + 'OH

These oxygen species can react with DNA in vitro and in vivo to produce a wide range of

deoxyribose and base damage products. 69,70 As mentioned earlier, there has been some

controversy in the literature as to whether superhelical tension plays a role in modulating DNA

damage caused by ionizing radiation. 52,53 One of the goals of my research was to resolve this

discrepancy.

The final oxidizing agent studied here is ONOO-. The ONOO- anion is formed by a

reaction of superoxide (02*-) with nitric oxide (NO').7 1,72 There is evidence that this potent

oxidant is formed in vivo during the simultaneous production of O2' and NO' by activated

macrophages. 73-75 When ONOO- is present under physiologic conditions, ONOO- is in

equilibrium with its conjugate acid peroxynitrous acid (ONOOH). The acid form is believed to

be a precursor to a high energy intermediate that reacts with DNA to produce *OH-like damage

products.

ONOO- - ONOOH (pKa ~ 6.8)

ONOOH c ONOOH* r DNA damage

However, recent studies suggest that 'OH does not arise from ONOOH.76,77 It has also been

shown that ONOO- can cause DNA strand breaks 78 and base damage79-81 but with a different

spectrum of damage products than either Fe+2 /EDTA/H202 or y-rad. These observations are

consistent with differences in reactivity between ONOO- and 'OH.

The four agents described here produce similar DNA damage products in that they all

yield oxidized deoxyribose sugars and nucleobases. The most common sugar damage occurs

when reactive oxygen species abstract deoxyribose hydrogen atoms. The products of minor

groove hydrogen atom abstraction (1', 4' and 5') include strand breaks and abasic sites. These

lesions have been well characterized and are of particular interest to my lab in that they are the

only products of enediyne-induced DNA damage (Figure 6). Although 2' hydrogen atom

abstraction is not observed, 3' hydrogen atom abstraction does occur in the major groove resulting

in DNA strand breaks leaving a 3'-glycoaldehyde-ended fragment and a 5'-phosphate.

3'-Phospho-

B Glycoaldehyde

B

P (H Base1te Q'00Propenoic Acid

reactiveoxygen OOHspecies

The products of base damage are less well characterized. Of the compounds presented

here the best characterized in terms of base damage spectra are Fe and Cu. The Fenton-like

chemistry of these metals produce three major base lesions; 7,8-dihydro-8-oxoguanine (8-oxoG),

7,8-dihydro-8-oxoadenine (8-oxoA) and cytosine glycol (Cyt Glycol). 68

NH 2 0 1'n2

N . H N"O OH

N NN IH2N N

8-oxoA 8-oxoG Cyt Glycol

Though the products of base damage induced by ONOO- are under some

controversy, 80,81 there is evidence for the formation of 8-nitroguanine and 8-oxoG.79,81 There is

also some suggestion that peroxynitrite can go on to further react with 8-oxoG to produce an as

yet uncharacterized base product.80

O O

H HH

NN O NNO 2H2N N H2N N

8-oxoG 8-nitroguanine

The well-studied chemistry of y-rad-induced DNA damage is more complicated than the

other oxidants. 69 However, all the above products, as well as others, are seen in y-rad-induced

DNA damage.

DNA ALKYLATORS

Alkylating agents are chemicals which add an alkyl group to the sugar-phosphate

backbone and/or bases of deoxyribonucleic acid. Although DNA alkylation in the form of C5'-

cytosine methylation appears to be important in gene expression,82,8 3 other types of DNA

alkylation 84,85 have been linked to a variety of genotoxic outcomes including depurination of

DNA substrates, 86-88 chromosomal aberrations, 89,90 effects on the immune response, 9 1

mutagenicity, 92 -94 carcinogenicity 95 and cytotoxicity. 96,97

The specific DNA alkylating agent used in the following studies is the SN2 acting DNA

methylator, 9 1 dimethyl sulfate (DMS).

H3C-O 0

H3 C-O

Upon addition to aqueous solution, DMS rearranges into a directly methylating intermediate. 91

DMS has been shown to methylate DNA in vitro and in vivo, predominantly at the N7 position

of guanine (-75 %), with minor contributions from other methylated products, 98-100 such as

methylation at the N3 position of adenine, which constitutes <20 % of the total alkylation. 86,101

Though the sequence specificity of DMS is low, it does show some selectivity for runs of two or

more guanines. 10 2,10 3 Except for this limited information, little is known about the interaction of

DMS with different DNA structures. The following work sheds light on the effect of the

structural and dynamic properties of supercoiled DNA on DMS-induced alkylation.

ENEDIYNES

The other group of genotoxins on which I have focused is the enediyne family of

antibiotics (Figure 4). The enediynes possess extreme cytotoxicity with an LD 50 dose in the

picomolar to femtomolar range in cultured cells 104-10 6 and as such they are the focus of continued

clinical trials for use as anticancer drugs. Much is already known about the target selection

mechanisms of the enediynes. Their structural diversity and common mechanisms of activation

and DNA damage (Figure 5) make them an excellent "tool box" for understanding the relationship

between drug structure and DNA conformation in the target selection process.

Although the enediynes vary widely in structure, they all bind in the minor groove of

DNA and, upon activation, form a diradical species that abstracts hydrogen atoms from

deoxyribose. 107-109 The oxygen-dependent reactions resulting from minor groove hydrogen atom

abstraction produce an array of deoxyribose degradation products (see Figure 6).

An example of the enediyne family, CAL y, has been shown to produce high levels

(>95%) of double-strand DNA damage. 1'0 7 Its mechanism of action is representative of the

entire enediyne family. The drug binds in the minor groove of DNA and, upon thiol activation

(Figure 5), abstracts the 4' and 5' deoxyribose hydrogens with 75% of the damage consisting of an

abasic site in one strand and a direct strand break in the other strand, respectively.1 10

Historically it has been assumed that CAL y targets tetrapurine sequences in DNA. 111114

However, recent work has suggested that it may not be the tetrapurine sequence itself, but rather

a 3' pyrimidine interruption at the end of the oligopurine tract that is the true target of CAL

.115,116 Work performed in our laboratory has shown that CAL y bends DNA upon binding. 117

This work suggests that the end of the oligopurine tracts acts as a flexible hinge in DNA and that

calicheamicin may target these sites for their intrinsic bendability.

One hypothesis explored in my studies required a thiol-independent CAL y analogue,

CAL 0. This molecule has a thioacetyl rather than the trisulfide moiety in the trigger portion of

the molecule (Figure 4a). Unlike the parent CAL y, CAL 0 does not require thiols to induce

DNA damage but rather can be activated by simple hydrolysis of the thioacetyl group in aqueous

solution. CAL 0 has been shown to cause high levels of double strand damage with the same

sequence selectivity and damage chemistry as CAL y but with an increased cytotoxicity (up to

1000-fold) in some cell lines. 106 A comparison of these two molecules is an important

component in the determination of the structure-function relationships of the enediynes.

One observation serves as the basis for my studies with the enediynes. It was observed

that calicheamicin 711 (CAL y)-binding increases the sedimentation rate of negatively supercoiled

plasmid molecules, which is indicative of drug-induced negative writhing. This led me to

hypothesize that superhelical tension may play an important role in the interaction of CAL y

with DNA, and as such CAL y became the focus of my experimental investigation.

TWIST (Tw)B-DNA

WRITHE (Wr)

-10.5 basepairs

Twist the number ofhelical turns per

molecule

Writhe: the numberof times the doublehelix crosses itself

Figure 1: The energy imparted to DNA by superhelical tension can be relieved in two different ways. Twist is a change inthe helical repeat of DNA. Writhe is a crossing of the double helix over itself. Negatively supercoiled DNA is underwoundand positively supercoiled DNA is overwound. The third way that DNA can releive superhelical tension is by theformation of unusual secondary structures ( i.e. cruciforms, hairpins, Z-DNA, unpaired regions, etc.). To date, thesestructures have only been observed in negatively supercoiled DNA.

Tw = -3 Tw = -2 Tw = -1 Tw = 0

Wr = 0 Wr = -1 Wr = -2 Wr = -3

A Lk -3Figure 2: In any closed system, twist and writhe can interconvert as long as the linking number change (ALk) remainsconstant. The energy of superhelical tension can be expressed as all twist, all writhe, or a mixture of the two. Severalstudies suggest that up to 30% of superhelical energy can be expressed as changes in twist. This is important sincechanges in DNA twist will have a greater effect on local DNA conformation, such as the width and depth of the grooves,than writhing.

Negative underwoundA Lk<O

Positive overwoundA Lk>O

POLYMERASE

Figure 3: As first proposed in the twin supercoiling domain model of Liu and Wang, 15 superhelical tension isgenerated in vivo by the act of transcription. The polymerase machinery tracking along the DNA helix generatesnegative supercoiling at the 5' end of the gene and positive supercoiling at the 3' end of the gene. A rigorousmodel system for the study of supercoiling should therefore include both negatively and positively supercoiledsubstrates.

Figure 4: The enediyne family of antitumor antibiotics. Though these molecules havea variety of structures, they all have a common mode of activation and DNA damageinduction.

R Q SSCH3 DihydrothiopheneR~j,

R

/ OR H

Figure 5: The enediynes all have a common mechanism for damaging DNA. Upon activation,presumably by glutathione in vivo, the molecule undergoes a rearrangement to form a diradicalspecies. When this diradical is positioned in the minor groove of DNA, it abstracts deoxyribosehydrogen atoms. The sugars then undergo oxygen-dependent breakdown reactions that result inDNA strand breaks or abasic sites. Calicheamicin forms >95% double strand breaks by exclusiveabstraction of the 4' hydrogen on one strand and the 5' hydrogen on the other.

0 -O + B

R-

\ , BP

O

,O0 B

P-1

B P-

0=_'P-O

P +

BP P

R

Figure 6: Deoxyribose radical breakdown pathways. The enediynes abstract minor groove hydrogenatoms from deoyxribose (1', 4' & 5') . The resulting sugar radicals then undergo a series of oxygen-dependent reactions that yield a variety of lesions, including strand breaks and abasic sites.

4'rCH

R

B

-0 O

CCR

5,

J H

_10-

CHAPTER 2

LARGE SCALE PRODUCTION OF

POSITIVELY SUPERCOILED DNA

USING THE ARCHAEAL HISTONE HMf

ABSTRACT

A technique to prepare relatively large quantities (2100 4g) of highly positively

supercoiled DNA is reported. This uses a recombinant archaeal histone, rHMfB, to introduce

toroidal supercoils, and an inexpensive chicken blood extract (CBE) to relax unrestrained

superhelical tension. Preparation of positively supercoiled pUC19 DNA molecules, >50% of

which have linking number changes ranging from +8 to +17, is demonstrated. Advantages include

the high degree of positive supercoiling that can be achieved, control over the extent of

supercoiling, easy production of relatively large quantities of supercoiled DNA, and low cost.

INTRODUCTION

There is growing interest in the biological roles of superhelical tension, given the evidence

for its presence in both prokaryotic and eukaryotic cells,8, 19,118 and its apparent involvement in

diverse cellular processes.32,34,38,40,41,44,119 Superhelical tension also alters the structure and

dynamics of DNA in ways that may affect the selection of targets by genotoxic chemicals such as

DNA intercalators. 19,49,120,12 1 The full physiological range of superhelical tension has not been

experimentally accessible as methods to prepare positively supercoiled DNA are limited and

therefore, to help solve this problem, I have developed a straightforward and inexpensive method

that generates preparations of circular DNAs with high levels of positive supercoiling.

The method exploits the observation of Musgrave et al.122 that the archaeal histones from

Methanothermusfervidus (designated HMf) form nucleosome-like structures in which the DNA

molecule is wrapped around the protein core in a right-handed superhelix. HMf binding to a

circular DNA molecule, followed by relaxation of unrestrained supercoils and removal of all

proteins, results in a DNA molecule that is positively supercoiled. In contrast, exposing circular

DNAs that contain eukaryal nucleosomes 43 to this procedure results in negatively supercoiled

DNA molecules.

Critical reagents are a clean preparation of circular DNA, purified recombinant HMfB

(rHMfB), 123 and a topoisomerase-containing extract obtained from chicken erythrocytes. The

technique works equally well with commercially prepared topoisomerase I and the empirically

determined quantities of wheat germ topoisomerase I from Promega (Madison, WI) are noted

throughout the following procedure as an example. Furthermore, the results shown here to

illustrate the procedure were obtained using plasmid pUC19 DNA, isolated from lysates of E.

coli DH5ca, by using Qiagen Gigaprep kits (Qiagen, Santa Clarita, CA). Another commercial

plasmid isolation kit (Wizard Megaprep, Promega; Madison, WI) was evaluated but the plasmid

preparations obtained apparently contained materials that interfered with the topoisomerase I

activity.

MATERIALS AND METHODS

Preparation ofrHMfB. rHMfB preparations were purified, exactly as described by

Starich et al.,124 from E. coli JM105 cells that contained pKS323, an expression plasmid that

carries the HMJfB gene. The pKS323 plasmid can be obtained from Professor John Reeve's lab at

Ohio State University; Dr. Kathleen Sandman (ksandman@postbox.acs.ohio-state.edu).

Preparation of chicken blood extract (CBE) that relaxes superhelical tension. As a less

expensive alternative to purchasing purified topoisomerase I, the procedure of Camerini-Otero

and Felsenfeld 125 was used to prepare a topoisomerase-containing extract from chicken

erythrocytes, here referred to as CBE. These preparations contained no detectable endonuclease

or protease activities, and retained activity for at least 6 months when stored at -800 C.

Production ofpositively supercoiled pUC19 DNA. Negatively supercoiled pUC 19

molecules, isolated from E. coli, were relaxed by incubation at 25 gg/ml with CBE in 200 mM

NaCI, 20 mM Tris-HCl (pH 8), 0.25 mM EDTA-Na2 and 5% glycerol for 1 h at 370 C. The

CBE:DNA ratio must be determined empirically for each batch of CBE; the present studies were

performed with 0.5 pl CBE/ig pUC19, or 4 U wheat germ topoisomerase I/gg pUC19. The

relaxed molecules were purified by exposure to proteinase K (100 tg/ml) for 2 h at 370 C in the

presence of 1% SDS, phenol/chloroform extraction, ethanol precipitation, and passage through a

Sephadex G-50 spin column. The purified plasmid (25 jgg/ml) was then complexed with 1.2-1.4

mass equivalents of rHMfB by incubation in 10 mM Tris-HCl (pH 8), 2 mM K3PO4, 1 mM

EDTA-Na2 and 50 mM NaCl for 15 min at 370 C. To remove unrestrained negative supercoils,

the reaction conditions were adjusted by the addition of 1/3 volume of 186 mM Tris-HCl (pH 8),

3 mM K3PO4, 8 mM EDTA-Na2 and 72 mM NaCl, followed by addition of 1/10 volume

(adjusted) of CBE and incubation for 45 min at 370 C. Again, the CBE:DNA ratio must be

determined empirically; the present studies were performed with 6 gl CBE/gg pUC19, or 12 U

wheat germ topoisomerase I per gLg pUC19. As a control, a population of negatively supercoiled

pUC19 molecules was subjected to the same treatments except that CBE dilution and storage

buffers were added instead of CBE. Plasmid DNAs were quantified using the Hoechst dye

33258 (Sigma, St. Louis, MO) fluorescence assay. 126

2D gel electrophoresis: After standard one dimensional electrophoresis, agarose gels were

soaked in 1 X TBE buffer containing 60 jgM chloroquine for 2 hrs. in the dark. The gels were

then submitted to a second electrophoresis step 900 perpendicular to their original orientation, in

the dark, using 1 X TBE containing 60 M chloroquine as the running buffer.

RESULTS AND DISCUSSION

An example of the positive supercoiling achieved with pUC19 is shown in Figure 7.

Maximal supercoiling was obtained with a rHMfB: DNA mass ratio of 1 to 1.4 and, although the

highest resolvable topoisomer had a change in linking number (ALk) of +11 (Figure 7b), >30% of

the DNA molecules had electrophoretic mobilities consistent with even higher ALks, with the

maximum ALk estimated to be +17.

This technique is not limited by the size of the DNA substrate, allows the extent of

positive supercoiling to be controlled, and generates substantial quantities of positively

supercoiled product. I routinely prepare >100 jgg of positively supercoiled pUC 19 as a single

batch, and multiple batches can be prepared concurrently. Positive supercoiling has been

introduced into plasmids and cosmids with a wide range of different sizes, including large

plasmids such as the 94.5 Kbp F-factor from E. coli (John Reeve lab, unpublished results). The

extent of positive supercoiling is determined by the ratio of rHMfB to DNA1 22 and, by

optimizing this ratio, substantial quantities of a plasmid DNA with a specific superhelical

configuration can be obtained by band excision and extraction from a one-dimensional agarose gel.

The use of rHMfB offers several advantages for the preparation of positively supercoiled

DNA. Intercalators, such as ethidium bromide,49 do not generate the high levels of positive

supercoiling achieved with rHMfB and, although high levels of superhelical tension can be

obtained by using reverse gyrase, 127,128 the purification of this enzyme is complicated and

lengthy in comparison with the simple rHMfB purification procedure. 124 Approximately 50 mg

of rHMfB can be purified from a 10 L culture of E. coli, and this is sufficient protein to prepare

-30 mg of positively supercoiled pUC19. Using the CBE rather than purchasing purified

topoisomerase I results in a very substantial reduction in cost; -120 ml of CBE can be obtained

from 250 ml of chicken blood ($100-200) in less than one day that, when used in this procedure,

is functionally equivalent to -240,000 units of commercial topoisomerase I. It should be noted,

however, that I have obtained identical results using commercially available topoisomerase I from

TopoGEN Inc. (human; Columbus, OH), Promega (wheat germ; Madison, WI), and Gibco-BRL

(calf thymus; Grand Island, NY); other sources are likely to give similar results. As mentioned

earlier, there is potential for inhibition of topoisomerase I by DNA contaminants introduced

during DNA isolation.

Circular DNAs, with the high and defined levels of positive supercoiling generated by this

procedure, should now be very valuable in evaluating the role of this superhelical tension in DNA

interactions with small chemical ligands, and also with both sequence specific and non-specific

DNA-binding proteins.

A)

CO

a)CE.5

2nd Dimension B)

Relaxed

(000P00O

S.C ~

ALk0

+2

+4

+6

-+10;> +12

Figure 7: Electrophoretic separations of positively supercoiled topoisomers of plasmid pUC19. (A)

Topoisomers of positively supercoiled pUC19, prepared as described in the text, resolved by two-

dimensional agarose gel electrophoresis. (B) One-dimensional agarose gel separation showing higher

resolution of positively supercoiled pUC19 topoisomers with ALk values indicated to the right of the figure.

CHAPTER 3

DIFFERENTIAL EFFECTS OF

DNA SUPERCOILING ON

RADICAL-MEDIATED STRAND BREAKS

AND DNA ALKYLATION

ABSTRACT

Supercoiling is an important feature of DNA physiology in vivo. Given the possibility

that the reaction of genotoxic molecules with DNA is affected by the alterations in DNA

structure and dynamics that accompany superhelical tension, I have investigated the effect of

torsional tension on DNA damage produced by six small DNA-damaging agents: y-rad, ONOO',

Fe+ 2 /EDTA/H202, Fe+2 /H202, Cu+2 /H202 and dimethyl sulfate (DMS). With positively

supercoiled plasmid DNA prepared by a recently developed technique, I compared the quantity

of strand breaks produced by the five agents in negatively and positively supercoiled pUC 19. It

was observed that strand breaks produced by y-rad, ONOO- and Fe+ 2 /EDTA/H202 are

insensitive to DNA superhelical tension. These results are consistent with a model in which

chemicals that generate highly reactive intermediates (e.g., 'OH), but do not interact directly with

DNA, will be relatively insensitive to the changes in DNA structure and dynamics caused by

superhelical tension. In the case of Fe+ 2 and Cu+2 , metals that bind to DNA, only Cu+2 /H202

proved to be sensitive to DNA superhelical tension. Strand breaks produced by Cu+2 /H202 in

the positively supercoiled substrate occurred at lower Cu concentrations than in negatively

supercoiled DNA. Furthermore, a sigmoidal Cu+2/H202 damage response was observed in the

negatively supercoiled substrate but not in positively supercoiled DNA. The results with Cu+2

suggest that the redox activity, DNA binding orientation, or DNA binding affinity of Cu+1 or

Cu+2 is sensitive to superhelical tension. In addition to the oxidative agents, I also investigated

the effects of supercoiling on the reactivity of the DNA alkylating agent DMS. I observed that

DMS induces -25% more total alkylation in the positively supercoiled substrate. This result, in

conjunction with the Cu+2 studies, implies a difference in either the reactivity or accessibility of

the N7 position of guanine in the two supercoiled substrates.

INTRODUCTION

DNA damage resulting from exposure to endogenous reactive oxygen species and other

radicals plays a role in a variety of biological processes such as mutagenesis, aging and

carcinogenesis. 129,130 Even though there is considerable understanding of radical-mediated DNA

lesions in vitro, the role of genomic organization in defining the location, quantity and chemistry

of DNA damage remains elusive. To better understand how DNA physiology affects DNA

damage, I have examined one feature of genomic organization, DNA superhelical tension, and the

role it plays in determining strand breaks produced by y-rad, ONOO-, Fe+2 /EDTA/H202,

Fe+2 /H202 and Cu+2 /H202. As a control for the results with Cu+2, which binds with relatively

high affinity to guanine-N7, 65, 13 1 I have also investigated the damage produced by the DNA

alkylating agent, DMS. DMS induces cell death96,132 by methylating DNA, predominantly at

the N7 position of guanine.98-100

Supercoiling is an important aspect of DNA physiology in both prokaryotic and

eukaryotic cells and appears to be involved in gene expression,5 DNA replication,5 and DNA

repair. 133 While the Escherichia coli genome has a a = - -0.05, the genomes of Drosophila

melanogaster and human cells have no net superhelical tension.8 However, as first proposed in

the twin supercoiled domain model of Liu and Wang,22 the act of transcription generates localized

torsional tension in genes: negative superhelical tension in the 5'-ends and positive supercoiling in

the 3'-ends. 19,2 1,22,134

The possibility that supercoiling-induced alterations in DNA structure and dynamics

could affect the interaction of small DNA-damaging agents with DNA served as the premise for

the present studies. Torsional tension can affect DNA structure in several ways, including

changes in helical repeat (twist) and writhing of the DNA helix 6 and, in the case of negative

supercoiling, alteration of DNA secondary structure to form Z-DNA, cruciforms and unpaired

regions. 135, 136 Although the interaction of intercalators with negatively supercoiled DNA is well

defined, 19,137 the effect of superhelical tension on the activity of other DNA-binding and DNA-

damaging chemicals has not been thoroughly explored.

To study the role of supercoiling in DNA damage, I have developed a technique to

prepare highly positively supercoiled plasmid DNA.'38 The DNA has a ( = -+0.04, while

negatively supercoiled plasmid DNA, isolated from E. coli, has an average a = -- 0.05. These

levels of supercoiling are similar to those estimated to exist in vivo.5,119

Using positively and negatively supercoiled DNA substrates, I have investigated the

effect of superhelical tension on the quantity of DNA strand breaks produced by five oxidizing

agents; y-rad, ONOO-, Fe+ 2 -EDTA/H202, Fe+ 2/H202 and Cu+2 /H202; and also the extent of

DNA methylation induced by the DNA alkylator, DMS.

Fe and Cu are physiologically important metals that have been implicated in DNA

damage in vivo.58'6 1 In the presence of H202, both metals generate 'OH and/or *OH-like species

through Fenton chemistry, which for Cu+ 2 appears to involve an initial reduction to Cu+ 1:139-

141

2Cu + 2 + H202 > 2Cu+1 + 02 + 2H+ or 2 Cu + 2 > Cu+1 + Cu+3

Cu+l/Fe+2 + H202 r Cu+2/Fe + 3 + OH" + 'OH

Supercoiling-dependent deformation of DNA structure may affect the binding of these metals and

thus the DNA damage they produce. For example, the Cu+l1 and Cu+2 bind preferentially to the

N7 of guanine, and at least Cu+2 is capable of forming chelation complexes with the 06 in the

same guanine l31 and with other nearby bases.65, 131 This may account for the formation of 8-

oxoG 142'143 and strand breaks59,144 at runs of guanines observed with Cu+2 /H202. 145 The

effects may be different for Fe since it is less redox active and binds to DNA with lower affinity

than Cu.68

The EDTA complex of Fe+2 presents a unique opportunity to compare Fe bound to

DNA to Fe free in solution. As with free Fe+ 2 , Fe+ 2/EDTA has been shown to generate a 'OH-

like species in the presence of hydrogen peroxide. 146,147 However, unlike free Cu and Fe, the

negative charge of the Fe+ 2/EDTA complex likely prevents it from binding to DNA phosphates

or bases147,148 and the resulting 'OH-like species may exist in the fluid phase. This could

account for the lack of sequence-selectivity of strand breaks generated by the complex. 147,148 In

this respect, Fe+ 2 /EDTA may be similar to ionizing radiation in its sensitivity to DNA

supercoiling.

In addition to a minor contribution from direct interaction with DNA, ionizing radiation

reacts with water to produce *OH and *H by homolytic fission of oxygen-hydrogen bonds and to

produce hydrated electrons. 69, 146 Radiation-induced 'OH, like that produced by

Fe+ 2 /EDTA/H202, causes both strand breaks 149,150 and base damage.' 51 Both y-rad and

Fe+ 2 /EDTA/H202 are sensitive to local DNA conformation, 146,152 but the effect of superhelical

tension on radiation-induced DNA damage has been controversial. 52,53,153

Nitric oxide, an important mediator of many biological processes, 154 reacts with 02*- to

form ONOO- with a rate constant near the diffusion-controlled limit.73 Peroxynitrite and its

conjugate acid, ONOOH, are highly reactive at physiological pH and are capable of producing

base damage 79,8 1 and strand breaks in DNA.78,81 However, ONOO- appears to have a different

reactivity than 'OH. 142,155

DMS, an SN2 acting DNA alkylator, has been shown to selectively interact with and

methylate DNA predominantly at the N7 position of guanine. 91, 132 DMS alkylation of DNA

has been linked to mutagenicity, carcinogenicity and cytotoxicity. 96,132 Though DMS shows a

slight sequence specificity for runs of guanines, 10 2,103 little is known of the effect of DNA

structure on DMS-induced methylation. The study of this compound provides and interesting

opportunity to explore the effect of supercoiling on the reactivity and/or accessibility of

nucleophilic sites on the DNA bases. In addition, these results will provide for an interesting

comparison with Cu, another agent known to selectively interact with the N7 position of

guanine.

I have found that only the Cu+2 /H202 and DMS systems are sensitive to DNA

supercoiling. A lack of sensitivity to supercoiling for y-rad, ONOO-, and Fe+ 2 -EDTA is

consistent with damage caused by a species that does not bind to DNA prior to inducing damage,

while the lack of effect with Fe+2 /H202 suggests that Fe+ 2 interacts with DNA through a

different mechanism than either Cu ions or DMS.

MATERIALS AND METHODS

Chemicals. All chemicals were reagent grade. 3H-DMS (5 Ci/mmol) was obtained from

American Radiolabeled Chemicals Inc. ONOO- solution was generated from a reaction of ozone

with sodium azide as described by Pryor et al.;156 the product was characterized

spectrophotometrically in 0.1 N NaOH (E30 2 = 1670 M-1 cm-1)156 and by its ability to cause the

nitration of L-tyrosine. 156 Caution should be exercised in the handling of ONOO- as it is a

strongly oxidizing species.

Preparation ofsupercoiledpUC19 substrates. Positive supercoiling of plasmid pUC19

was achieved using a recombinant archaeal histone HMf (rHMf) synthesized in E. coli from the

cloned HMfB gene from Methanothermusfervidus as described previously. 138 The DNA used

had an average a = -+0.04, as determined by two-dimensional gel electrophoresis. 138

Negatively supercoiled pUC 19 DNA was isolated from DH5a E. coli with an average

density of -- 0.05. This DNA was subjected to the same treatment as the positively supercoiled

DNA except for the addition of topoisomerase-containing CBE; sham reactions were instead

performed with the dilution and storage buffer used with the CBE. Both plasmid substrates were

quantitated using the Hoechst dye 33258 (Sigma) fluorescence quantitation assay.126

In both DNA substrates, care was taken to remove trace quantities of metals and other

contaminants that could interfere with the radical-mediated DNA damage. To remove trace

quantities of non-specifically bound positively charged peptides, the DNA was adjusted to 2M

NaCl and incubated at room temperature for 2 h. The DNA was then dialyzed at 40 C against

Chelex-treated phosphate buffer (50 mM, pH 7.4) containing the metal chelating agent,

diethylenetriaminepentaacetic acid, and finally dialyzed against phosphate buffer alone to remove

the chelator.

Treatment ofsupercoiledpUC19 with oxidative agents. Damage reactions were performed

in 25 gL reaction volumes with 30 jgg/mL pUC19 plasmid DNA at ambient temperature for 30

min. y-rad and ONOO- damage reactions were carried out in 50 mM potassium phosphate buffer

(pH 7.4). DNA was irradiated with 0-17.6 Gy in a 6 0 Co y-source at 4 Gy/min and the irradiated

DNA was left at ambient temperature for approximately 30 min before further processing.

ONOO- in 100 mM NaOH was diluted with 10 mM NaOH immediately before addition of 1 pl

aliquots to the DNA solution. Fe+2 /EDTA/H202, Fe+ 2 /H202 and Cu+2/H202 damage

reactions are carried out in 50 mM phosphate buffer (pH 7.4) with 1 mM H202. Fe+ 2 and

Cu + 2 were added to a solution of DNA in phosphate buffer followed by addition of H202 to 1

mM; the Fe+ 2 -EDTA reaction was performed as described elsewhere. 157 Since oxidation of

deoxyribose causes abasic sites as well as direct strand breaks, the damaged DNA was treated

with putrescine (100 mM, pH 7, 1 h, 370C) to convert abasic sites of all types to strand

breaks. 107,108,158,159 As shown in previous studies, putrescine treatment does not appear to

react with oxidized bases to cause formation of abasic sites and strand breaks. 142

Following treatment, DNA samples were purified by passage over G-50 Sephadex spin

columns. The DNA was then relaxed with CBE, to remove any superhelical dependent biases in

subsequent electrophoresis or probing; this treatment did not affect the number of strand breaks

in the DNA substrates. Finally, the DNA was purified by proteinase K digestion (100 gg/mL;

1% SDS; 2 h at 370 C) followed by phenol/chloroform and chloroform/isoamyl alcohol extractions

and ethanol precipitation.

Treatment ofsupercoiledpUC19 with DMS. DMS exposure was performed in 100 gL

reaction volumes with 7.5 gg/mL pUC19 and 50 mM potassium phosphate (pH 7.4). 3H-DMS

was added to a final concentration of 100 gCi/mL (20 jtM) or 500 gCi/mL (100 jtM), the

reaction mixture was then vortexed and allowed to incubate at 370 C for 2 hrs. The products were

dialyzed twice against 3 liters of 10 mM Tris, 1 mM EDTA buffer (pH 7.0) at ambient

temperature for 6 hrs to remove unreacted and/or unadducted DMS. The 3H quantitated by

scintillation counting.

Gel Electrophoresis. Each DNA sample was resolved on a 1.5% agarose gel (Tris-borate-

EDTA) containing 60 ktM chloroquine (Sigma) to resolve plasmid topoisomers. The gels were

washed extensively in water to remove the chloroquine and then stained with 0.5 gtg/mL EtBr for

UV photography. The gels were exposed to short wave UV light for an additional 2 minutes to

nick the DNA and rule out superhelical-dependent effects on hybridization efficiency. The gels

were subsequently dried on a conventional gel dryer for 45 minutes at ambient temperature and

45 minutes at 600 C.

Quantitation ofPlasmid Topoisomers. Gels were probed with a pUC 19 random primer

probe according to the protocol of Lueders and Fewelll160 and the radioactivity associated with

each plasmid form was determined on a phosphorimager (Molecular Dynamics). The quantity of

strand breaks was calculated from the proportion of supercoiled (form I) DNA converted to

nicked (form II) DNA and linear (form III).

RESULTS AND DISCUSSION

Given the biological importance of DNA supercoiling and the changes in DNA

conformation that accompany superhelical tension, I have investigated the effect of DNA

supercoiling on the damage produced by y-rad, ONOO-, Fe+2 /EDTA/H202, Fe+ 2 /H202,

Cu+2 /H202 and DMS. The first five agents produce DNA damage by free radical processes but

differ in the mechanisms by which they associate with and damage DNA, while DMS induces its

damage by methylating DNA predominantly at the N7 position of guanine.

The DNA substrates for these studies consisted of a 2686 base pair plasmid that

possessed a high degree of either negative or positive supercoiling. The negatively supercoiled

plasmid is estimated to have a - -0.05; this represents approximately 13-15 supercoils per

plasmid (ALk - 13-15). The positively supercoiled plasmid has an average a - +0.04, with a

maximal ALk of +17.138

To ensure the removal of contaminants that could interfere with the radical-induced DNA

damage, the DNA was subjected to extensive dialysis against Chelex-treated phosphate buffer

containing a metal chelator. The purity of the DNA is indicated by the absence of detectable

nicking of the plasmid DNA in the presence of 1 mM H202 alone. Furthermore, the negatively

supercoiled DNA was subjected to the same conditions used to prepare the positively

supercoiled DNA. Finally, while I show all of the data, I was careful to draw conclusions only

from the strand break data that fell within "single-hit conditions," which corresponds to -30-40

strand breaks in 106 bases of pUC19 DNA according to a Poisson distribution. 10 7 Above this

level of damage, the number of strand breaks is underestimated because additional nicks in an

already nicked plasmid cannot be detected by topoisomer analysis. The DNA alkylation studies

did not involve the plasmid topoisomer assay; instead they involved the measurement of transfer

of a tritiated methyl group to the DNA.

The results of the supercoiling studies for the oxidative DNA-damaging agents are shown

in Figures 8 and 9. As expected for genotoxins that have little affinity for DNA, strand breaks

produced by ONOO-, Fe+ 2/EDTA/H202, and y-rad were found to be insensitive to DNA

superhelical tension (Figure 8). This insensitivity is likely due to the high reactivity of the 'OH-

like species thought to be responsible for the DNA damage. In any case, the results indicate that

negative and positive supercoiling do not create sites of high reactivity for these agents.

The present results with radiation are consistent with the findings of Milligan et al. 53 who

observed no effect of supercoiling on radiation-induced strand breaks. However, their results and

the results of the present study differ from the contradictory results of Miller et al. 52 and Root et

al. 153 Root et al. concluded that there was 10- to 15-fold more damage in isolated salmon sperm

DNA compared to negatively supercoiled SV40 DNA, 153 while Miller et al. observed a direct

relationship between radiation-induced strand breaks and negative superhelicity in plasmid

pIBI30. 52 In both studies, the purity of the DNA was not addressed and, in the case of

Miller et al., residual ethidium bromide from preparation of supercoiled DNA could interfere

with radiation-induced DNA damage. I have taken great care to ensure that both supercoiled

DNA substrates were free from contaminants that could either enhance or inhibit the DNA

damage. Furthermore, the results with the other oxidizing agents (Figures 8 and 9) lend support

to a model in which chemicals that do not bind to DNA but generate highly reactive oxidizing

species (e.g., 'OH) will be relatively insensitive to the changes in DNA structure and dynamics

caused by superhelical tension.

The observed lack of supercoiling effects on strand breaks does not rule out small, local

changes in the reactivity of DNA due to superhelical tension, such as the variation in 'OH-

mediated strand break formation as a function of minor groove width. 146 ,152 My results do

suggest, however, that there are no supercoiling-dependent "hotspots" for pUC 19 DNA damage

produced by Fe+ 2 /EDTA/H202, y-rad, and ONOO-.

The two physiologic metals which do bind to DNA, Cu and Fe, show different

sensitivities to DNA supercoiling. Fe+ 2 /H202 produced an equal number of strand breaks in

both substrates (Figure 9), which suggests that supercoiling-dependent changes in DNA structure

and dynamics do not greatly affect the binding of Fe to DNA. With Cu+2 /H202, however,

strand breaks in the positively supercoiled substrate occurred at lower Cu concentrations than in

negatively supercoiled DNA. Furthermore, the sigmoidal damage response of the negatively

supercoiled substrate, which has been observed in other studies, 142,161 was absent in positively

supercoiled DNA (Figure 9). The similarity of the damage profiles at high Cu concentrations (>3

gM) is likely due to the fact that the level of strand breaks exceeds single-hit conditions.

There are several explanations for the supercoiling-dependent changes in DNA damage

produced by Cu+2 /H202. First, supercoiling may simply alter the affinity of Cu + 2 or Cu + 1 for

DNA, with higher binding affinity correlated with higher levels of DNA damage. High

concentrations of Cu+ 2 (equivalent to -50 gM under the present conditions; 46 gM base pairs)

have been shown to lower the melting temperature of linear T7-bacteriophage DNA by a

mechanism that may involve unwinding of the helix among other changes in DNA structure. 162

This may explain the observed effects of supercoiling on Cu-induced DNA damage if the

formation of DNA-unwinding Cu complexes is inhibited in positively supercoiled DNA, as

occurs with intercalators. However, even at extremely high concentrations (> 1 mM), Levy and

Hecht observed that Cu+ 2 did not affect the topology of negatively supercoiled plasmid

DNA, 163 so the question of Cu-induced unwinding of DNA remains controversial. Future

studies utilizing a damage competition assay may address the issue of relative binding affinity of

Cu (II) for negatively and positively supercoiled DNA. A similar assay is described for CAL y in

the following chapter (p. 60).

Alternatively, if the reduction of Cu+ 2 to Cu + 1 occurs only with unbound Cu + 2 , then an

inverse relationship may exist between Cu +2 DNA binding affinity and DNA damage. While the

present studies do not address base damage, it has been proposed that Cu bound to high affinity

sites on bases, probably the N7 of guanine, 66 causes base lesions while unbound Cu or Cu bound

to lower affinity sites (e.g., backbone phosphate) produces strand breaks. 161 In light of this

model, the observed increase in strand breaks in positively supercoiled DNA may be

accompanied by reduced base damage if positive superhelical tension limits access to high affinity

binding sites on bases. The converse would also be true, in that the apparent lack of strand

breaks in the negatively supercoiled substrate may be accompanied by a comparatively high

amount of base damage. It is possible, however, that strand breaks and base damage increase

simultaneously due to improved binding site access for both lesions. However, preliminary

studies in our lab have shown that negatively supercoiled DNA sustains even lower levels of base

damage than strand breaks when treated with Cu+2 /H20 2. This casts doubt on the hypothesis

that strand breaks and base damage may be arising as the result of two different binding modes.

The above hypotheses present a situation in which the observed difference in strand

break quantities can be explained by supercoiling-dependent changes in binding affinity or

chemical reactivity of Cu in either substrate. Regardless of which substrate serves as a better

target for Cu, it can be assumed that this "high affinity" or "highly reactive" site is the N7

position of guanine, the only known specific binding site of Cu to DNA. The guanine-N7 may

be either more accessible due to supercoiling dependent changes in DNA structure, or simply a

more attractive binding site for Cu due to changes in the electronic distribution within the DNA

bases.

To address the issue of guanine-N7 accessibility/reactivity in supercoiled DNA

substrates, I have looked at the reactivity of a guanine-N7-selective DNA alkylator, DMS. Upon

addition to aqueous solution, DMS rearranges to a directly acting intermediate 91 that methylates

DNA predominantly at the N7 position of guanine. 98-100 The use of a directly damaging agent

removes the complications introduced by the redox reactions and possible bidentate DNA

binding of Cu.

The DMS results are summarized in Table 1. It can be seen that at two different

concentrations of DMS, there is a higher level of alkylation observed in the positively

supercoiled substrate. Since DMS is such a highly reactive agent, the observed difference implies

that the N7 position of guanine is either more accessible and/or more reactive in DNA with

positive superhelical tension. The results seen with DMS and Cu, two chemicals which have

different mechanisms of action and yet exhibit the same behavior (overall higher reactivity in

positively supercoiled plasmids), suggest that superhelical tension may play a role in the damage

caused by any DNA reactive agent that shows a selectivity for the N7 position of guanine.

In addition, preliminary HPLC results have shown that DMS exhibits an increased

methylation at the N3 position of adenine as well. Though a minor product of DMS-induced

DNA alkylation, the adenine-N3 seems to get the same level of increase in methylation in the

positively supercoiled substrate when compared to DNA with negative superhelical tension.

This may indicate a positive supercoiling-dependent increase in the nucleophilicity of both

purine positions, or perhaps a more general base accessibility in this substrate.

In conclusion, I have observed differential effects of superhelical tension on the reactivity

of DNA with several oxidizing agents and an alkylating agent. Strand breaks produced by

Cu+2 /H202 were found to occur at lower Cu concentrations in positively supercoiled DNA,

while y-rad, ONOO-, Fe+2 /EDTA/H202, and Fe+ 2/H202 did not show significant changes in

reactivity with DNA of different supercoiling states. DMS was also shown to be affected by

DNA supercoiling, causing more total alkylation in the positively supercoiled substrate. The Cu

and DMS results together suggest a reactivity issue of the N7 position of guanine in DNA with

different superhelical densities. These results confirm the hypothesis that superhelical tension

can play a role in the DNA damage induced by small non-intercalative genotoxins.

O - supercoiled

5 * + supercoiled

0 0

5

0 ,

o 5 10 15y-radiation, Gray

O - supercoiled* + supercoiled

0-

0

0

0-

o

0

0 1 2Fe(II)/EDTA, gM

)

Eo-0

0R

5 0 - supercoiled+ supercoiled

00

5 0

0

0 25 50 75ONOO, IM

Figure 8: Strand breaks produced by y-radiation, peroxynitrite and Fe+2/EDTA/H 20 2 are notsensitive to DNA supercoiling. Samples of negatively and positively supercoiled pUC19 were treatedwith y-radiation, peroxynitrite, Fe+2/EDTA/H 20 2, as described in Materials and Methods, and gel-resolved plasmid topoisomers were quantified by phosphorimager analysis of probed gels (n = 3-5).

1

0)

E

o(

40 60Fe(ll), jIM

80

60

50

040

E 30

20

10

100 5 10Cu(ll), IM

Figure 9: Strand breaks produced by Cu+2/H202, but not Fe+2/H20 2 , are sensitive to DNAsupercoiling. Samples of negatively and positively supercoiled pUC19 were treated with Cu+2/H202and Fe+2/H20 2 , as described in Experimental Procedures, and gel-resolved plasmid topoisomers werequantified by phosphorimager analysis of probed gels (n = 3-5).

40

30-O)

E 20

10

O - supercoiled 0* + supercoiled O

0

20 15

Table 1: Supercoiling-dependent differences in methylationof DNA by [3H]-dimethyl sulfate

Radioactivity (cpm) bound to DNA

NegativelySupercoiled DNA

PositivelySupercoiled DNA

Ratio -/+

Low [DMS](20 uM)

High [DMS](100 IrM)

DMS shows a supercoiling bias, methylating the positively supercoiled DNA morethan DNA with negative superhelical tension. Supercoiled substrates were treatedwith [3 H]-DMS and then the DNA was purified. The negatively and positivelysupercoiled substrates are indicated by - and +, respectively. The amount of methyltransfer was quantitated by scintillation counting. DMS causes -25% moremethylation in the positive substrate at two different DMS concentrations.

586 +145 1607 + 134

733 + 67 2100 + 204

0.799 0.765

CHAPTER 4

SUPERCOILING AFFECTS THE ACCESSIBILITY

OF GLUTATHIONE TO DNA-BOUND MOLECULES:

POSITIVE SUPERCOILING INHIBITS

CALICHEAMICIN-INDUCED DNA DAMAGE

ABSTRACT

DNA superhelical tension, an important feature of genomic organization, is known to

affect the interactions of intercalating molecules with DNA. However, the effect of torsional

tension on non-intercalative DNA-binding chemicals has received less attention. I demonstrate

here that the enediyne CAL y, a strand-breaking agent specific to the minor groove, causes -50%

more damage in negatively supercoiled plasmid DNA than in DNA with positive superhelicity.

Furthermore, I show that the decrease in damage in positively supercoiled DNA is controlled at

the level of thiol activation of the drug. My results suggest that supercoiling may affect both the

activity of non-intercalating genotoxins in vivo and the accessibility of glutathione and other small

physiologic molecules to DNA-bound chemicals or reactions occurring in the grooves of DNA.

INTRODUCTION

Superhelical tension plays a role in many aspects of DNA physiology including

transcription, 5 replication, 5 and repair. 133 The genome of E. coli has a net a = -0.05, while

there appears to be no net superhelical tension in the genomes of Drosophila melanogaster and

humans.8 However, as a consequence of the loop organization of the genome in higher

eukaryotes, 164 transcriptionally active DNA contains high levels of localized torsional tension--

negative in the 5'-ends and positive in the 3'-ends17-22 ,134--which is consistent with the twin-

domain model of Liu and Wang for transcriptionally-induced DNA supercoiling. 22 Though

superhelical tension has well-defined effects on the interactions of proteins and small DNA

intercalators, its effects on other molecules that interact with DNA have not been fully explored.

The effects of torsional tension on small molecule-DNA interactions are a direct

consequence of changes in DNA structure and dynamics. Negative and positive superhelical

tension occur when the helix is unwound or over wound, respectively, 136 and it has been

estimated that as much as 30% of torsional tension is expressed in alterations of twist (helical

repeat) with the remaining 70% expressed as writhing of the helix. 165-167 Torsional tension has

several consequences for DNA secondary structure, including formation of Z-DNA, cruciforms

and single-stranded regions. 11,135,136,168 ,169 In the case of negatively supercoiled DNA, the

under wound state of the helix favors binding of intercalating molecules since they induce local

unwinding that is dissipated as global overwinding of the helix. 19,137

By analogy to intercalators, the DNA interactions of other molecules that are sensitive to

twisting and writhing of the helix should also be affected by superhelical tension. I have tested

this hypothesis with the enediyne antibiotic CAL y (Figure 4a). The enediyne family is a

structurally diverse group of DNA-damaging molecules that are cytotoxic at picomolar to

femtomolar concentrations. 104-106 This lethality is likely due to the production of high levels

(>95%) of double strand DNA damage 10 7 following reductive activation, presumably by

glutathione in vivo, to form a diradical intermediate that binds in the minor groove and abstracts

hydrogen atoms from deoxyribose (Figure 5).10 5,112,170,171 In the present studies, I have

employed an analog of CAL 7, CAL 0, in which the methyl trisulfide trigger has been replaced by

a thioacetate group that undergoes hydrolysis in the absence of thiols (Figure 4a). The resulting

diradical intermediate is identical in structure to that arising from CAL 7,106 as are the sequence-

selectivity, 106 the chemistry, and the bistranded nature of the DNA lesions produced by the two

drugs (P. Dedon et al., in preparation).

Using highly positively supercoiled DNA produced by a recently developed technique I

have investigated the effect of superhelical tension on the interaction between DNA and the

calicheamicins. I found that CAL y preferentially damages negatively supercoiled DNA and that

this difference is due to accessibility of glutathione to DNA-bound drug. My results suggest that

DNA supercoiling may have significant effects on small molecule-DNA interactions and on

chemical reactions that occur in proximity to DNA.

MATERIALS AND METHODS

Reagents. CAL y was provided by Dr. George Ellestad (Wyeth-Ayerst Research). CAL

0 was synthesized as described elsewhere. 10 6

Production ofpositively supercoiled substrate. Positive supercoiling of plasmid pUC 19

was achieved using a recombinant archaeal histone HMf (rHMf) synthesized in E. coli from the

cloned HMfB gene from Methanothermusfervidus as described previously 138. Negatively

supercoiled pUC19, isolated from DH5a E. coli by standard techniques, was subjected to the

same treatment as the positively supercoiled DNA except for the addition of topoisomerase-

containing CBE. Sham reactions were instead performed with the dilution and storage buffer

used with the CBE. Both plasmid substrates were quantitated using the Hoechst dye 33258

(Sigma) fluorescence quantitation assay.126

In both DNA substrates, care was taken to remove contaminants that could interfere with

the drug-induced DNA damage. To remove trace quantities of non-specifically bound positively

charged peptides, the DNA was adjusted to 2M NaCl and incubated at room temperature for 2

hr. The DNA was then dialyzed at 40 C against Chelex-treated phosphate buffer (50 mM,

pH 7.4) containing the metal chelating agent, diethylenetriaminepentaacetic acid, and finally

dialyzed against phosphate buffer alone to remove the chelator.

CAL y induced changes in DNA supercoiling. The effect of CAL y on the topology of

negatively supercoiled pUC19 was determined by sucrose density gradient centrifugation as

described elsewhere. 10 8 Briefly, a mixture of [3 H]-labeled, nicked plasmid and [14 C]-labeled

negatively supercoiled plasmid was layered on a 5-20% sucrose density gradient (10 mM

HEPES, 1 mM EDTA, 10 % DMF, pH 7) containing various concentrations of CAL y. The

gradients were centrifuged at 248,000xg for 1.5 hr at 40 C. Fractions were collected from the top

of the tube and [3 H] and [14 C] were quantitated by scintillation counting. The peak radioactive

fraction for each isotope was used to calculate the sedimentation of the form I DNA relative to

the form II DNA. CAL y was found to be soluble and chemically stable for up to 10 hr in the

sucrose buffer, and there was no detectable nicking of the plasmid DNA.

Treatment ofsupercoiledpUC19 with DNA-damaging agents. Damage reactions were

performed in 25 gL reaction volumes (10 mM HEPES, 1 mM EDTA, 8% MeOH, pH 7.0)

containing 30 gg/mL of either positively or negatively supercoiled pUC 19 DNA. CAL y was

added to each sample and allowed to incubate at ambient temperature for 5 minutes. The drug

was then activated by the addition of either glutathione to 10 mM or methyl thioglycolate to 1

mM and the reaction carried out for 30 minutes at ambient temperature; in one experiment, the

reaction was allowed to proceed for up to 24 hr. CAL 0 reactions were performed in a similar

manner except that thiols were omitted and the damage reaction was allowed to proceed for 16 hr

at 370C. In both cases, putrescine was added to each sample to 100 mM and the samples were

incubated at 370 C for 30 minutes to express abasic sites as strand breaks. 10 7 Following

treatment, DNA samples were purified by passage over G-50 Sephadex spin columns. The

DNA was then relaxed with CBE, 138 to remove any superhelical dependent biases in subsequent

electrophoresis or probing; this treatment did not affect the number of strand breaks in the DNA

substrates. Finally, the DNA was purified by proteinase K digestion (100 gg/mL; 1% SDS; 2 hr

at 370 C) followed by phenol/chloroform and chloroform/isoamyl alcohol extractions and ethanol

precipitation.

DNA damage analysis. DNA from each sample was resolved on a 1.5% agarose gel

(Tris-borate-EDTA) containing 60 mM chloroquine (Sigma Chemical Co. St. Louis, MO). The

gels were washed extensively in water to remove the chloroquine and then stained with 0.5

mg/mL ethidium bromide for UV photography. Following exposure to 254 nm light for 5

minutes to ensure nicking of the plasmid DNA, the gels were dried on a conventional gel dryer for

45 minutes at ambient temperature and 45 minutes at 600 C. DNA topoisomers were then

quantified by in situ hybridization in the dried agarose gel with a pUC19 random primer probe

according to the protocol of Lueders and Fewell. 160 The relative intensities of the plasmid forms

were determined on a phosphorimager (Molecular Dynamics) and the quantity of strand breaks

was calculated from the proportion of supercoiled (form I) DNA converted to nicked (form II)

DNA and linear (form III). Drug concentrations were selected so that the number of strand

breaks would not exceed "single-hit" conditions according to a Poisson distribution, which

corresponds to no more than -30% of the supercoiled DNA molecules sustaining damage. 10 7

In all experiments, I have compared the ratios of damage in the two supercoiled forms

rather than comparing the absolute levels of damage. This was necessitated by moderate

experiment-to-experiment variability in the absolute quantity of damage. However, within a

single experiment, the absolute levels of DNA damage are comparable between samples.

Determination of relative binding affinities of CAL y for positively and negatively

supercoiledpUC19. The relative affinity of CAL y for positively and negatively supercoiled

pUC 19 was determined by a competitive DNA damage assay. CAL y-induced DNA damage in a

[3 2 p]-labeled DNA fragment was measured in the presence of varying concentrations of

unlabeled, supercoiled pUC 19, which competed with the labeled DNA for drug binding. The

relative reduction in damage to the labeled strand is directly correlated with the overall binding

constant for CAL y with the supercoiled DNA competitor. While this assay cannot be used to

determine absolute binding constants, the relative binding affinity can be defined for the two

supercoiled DNA substrates. The 215 bp HindIII/PvuII fragment of pUC19 was 5'-[ 3 2 P] end-

labeled at the HindIII site as described elsewhere. 107 The labeled fragment was mixed with calf

thymus DNA (final 0.3 gg/ml) and 0.3 nM CAL y in 10 mM HEPES, 1 mM EDTA, pH 7. This

mixture was then divided into 8 gl aliquots and 1 gl of supercoiled DNA was added to each

aliquot. After 5 min of equilibration, 1 gl of 100 mM glutathione (pH 7) or 10 mM methyl

thioglycolate was added and the damage reaction was allowed to proceed for 30 min at ambient

temperature; the drug reaction was complete within this time as evidenced by a lack of increase

in damage quantity with extended incubations. Abasic sites were converted to strand breaks by

treatment with putrescine (100 mM) for 30 min at 370 C. 107 Following addition of glycerol-

based loading buffer, the entire sample was resolved on a 10% nondenaturing polyacrylamide gel.

Radioactivity in a DNA fragment produced by cleavage at an AGGA site in the labeled DNA

was quantified relative to total radioactivity in the lane by phosphorimager analysis. The ratio of

binding constants for the supercoiled DNA substrates is proportional to the concentration of

supercoiled DNA necessary to produce equivalent amounts of damage in the radiolabeled marker

DNA.

RESULTS

In the presence of glutathione, CAL y produces more damage in negatively supercoiled

DNA than in positively supercoiled DNA. I investigated the effect of superhelical tension on the

damage produced by CAL y in highly positively and negatively supercoiled pUC 19. The

positively supercoiled pUC 19 was prepared using a recently developed technique that employs

HMf proteins 138 and the DNA had an average a- +0.04, with a maximal ALk of +17. The

negatively supercoiled plasmid was estimated to have a - -0.05 (DLk - -14) by two-dimensional

gel electrophoresis (Figure 7).

As shown in Figure 10, CAL y activated by glutathione produced 50% more damage in

the negatively supercoiled substrate than in positively supercoiled DNA. The reaction was

assumed to be complete after 30 minutes since no further changes were noted with incubations

up to 24 hours. As observed in previous studies, 10 7 CAL y caused only bistranded DNA

lesions, as indicated by the fact that only linear (form III) DNA was produced in the damage

reactions.

CAL y induces negative writhing ofplasmid DNA. To define the basis for the

supercoiling-dependent difference in CAL y damage, I studied the effect of CAL y binding on

DNA topology. As shown in Figure 11, binding of CAL y increased the sedimentation of

negatively-supercoiled plasmid DNA relative to nicked-open circular DNA. This is consistent

with drug-induced negative writhing of the plasmid DNA. As noted previously, 10 8 no change in

supercoiling is observed for esperamicin C, an analog of CAL y missing the terminal sugar-

aromatic ring (Figure 4c). This serves as a negative control for the effects of CAL y and

demonstrates the importance of the carbohydrate side chain in CAL y-DNA interactions.

The CAL y-dependent increase in negative writhing could be a response to changes in

DNA twist, a direct response to drug-induced changes in helical writhing, or a combination of

these two extremes. It has been previously demonstrated that calicheamicin e, the aromatized

form of CAL y, binds to flexible DNA sequences and bends the DNA upon binding. 172,173 Such

DNA bending can, in theory, alter the net writhing of the closed-circular plasmid DNA. On the

other hand, the drug-induced increase in negative writhing is consistent with circular dichroism

evidence for helical overwinding caused by binding of CAL y.174 This contrasts with the

behavior of intercalating agents, such as the enediyne esperamicin Al,10 8 that characteristically

unwind the DNA helix upon binding121,175 and cause positive writhing. However, the degree to

which helical winding contributes to the changes in DNA topology cannot be determined from

plasmid sedimentation studies. Furthermore, a predominance of drug-induced overwinding of the

helix would require that the calicheamicins bind to the over wound helix of positively supercoiled

DNA with higher affinity than to negatively supercoiled DNA. The following experiment

demonstrates that this is not the case.

CAL y has equal binding affinities for negative and positive superhelical DNA. To

test the hypothesis that CAL y binds preferentially to positively supercoiled DNA, I determined

relative binding affinities of the drug in the two supercoiled DNA substrates. This experiment

was performed by comparing the level of DNA damage in a [3 2 p]-labeled DNA fragment in the

presence of either positively or negatively supercoiled pUC19; preferential binding of CAL y to

one of the supercoiled substrates would cause a decrease in the damage in the labeled fragment.

As shown in Figure 12, both negatively and positively supercoiled plasmid DNA

compete to the same extent in the damage assay. This is illustrated by the expected 50%

reduction in damage in the labeled DNA fragment when 0.3 gtg/ml of either supercoiled substrate

is added to 0.3 gg/ml of the carrier DNA (average from two experiments; the labeled DNA

fragment is present in trace quantities). Thus, there is no major difference in the affinity of CAL

y for these two supercoiled plasmid molecules. It should be noted that similar results were

obtained with both methyl thioglycolate and glutathione as drug activators.

CAL O produces equivalent amounts of damage in both supercoiled substrates. The

observed supercoiling-dependent bias in drug-induced damage in spite of similar binding affinities

raised the possibility that supercoiling affected the drug activation step rather than the actual

damage events. To test this hypothesis, I examined the effect of supercoiling on damage

produced by CAL 0, an analog of CAL y with a thioacetate trigger that undergoes hydrolysis in

the absence of thiols (Figure 4a).

The results of these studies are shown in Figure 13. CAL 0 alone produced equal

numbers of double-strand breaks in both plasmid substrates. This is consistent with a model in

which glutathione has a reduced accessibility to drug that is bound to positively supercoiled

DNA.

In the presence of methyl thioglycolate, CAL ' shows no supercoiling bias. To test the

hypothesis that the supercoiling-dependence of glutathione activation is due to the size and/or

negative charge of the thiol, I studied the damage produced in supercoiled DNA when CAL y was

activated by methyl thioglycolate, a small, uncharged thiol. The results shown in Figure 14 reveal

O SH

O +H3 N

HSJ o,CH3 NH O

O OO OMethyl thioglycolate Glutathione

that activation of CAL y by methyl thioglycolate causes the enediyne to produce equal amounts

of DNA damage in both supercoiled substrates. Methyl thioglycolate does not affect the binding

of the activated drug since both methyl thioglycolate and glutathione result in the same sequence-

selectivity and double-strand character of CAL y-induced DNA damage. It is also apparent in

Figure 14 that methyl thioglycolate and glutathione cause CAL y to produce equal amounts of

damage in the negatively supercoiled substrate. This indicates that the supercoiling bias observed

with glutathione is due to a reduction in damage in positively supercoiled DNA.

DISCUSSION

Using DNA substrates with high levels of positive and negative supercoiling, I have

demonstrated that the damage produced by CAL y, an equilibrium-binding, minor groove-specific

DNA-damaging agent, is sensitive to DNA supercoiling. The mechanistic model most consistent

with my data is differential accessibility of glutathione to CAL y bound to DNA in different

states of superhelical tension. Given the presence of high levels of both positive and negative

supercoiling in transcriptionally active genes, these results have significant implications for other

physiologically relevant small-molecule DNA interactions.

There are several models that account for the effect of supercoiling on the damage

produced by CAL y. First, the preference for damaging negatively supercoiled DNA could reflect

a higher affinity of the drug for DNA with negative torsion. Alternatively, CAL y may bind with

higher affinity to positively supercoiled DNA and paradoxically experience less activation to

produce DNA damage. This inverse relationship between binding affinity and activation has

been observed by Myers and coworkers with dynemicin analogs. 176 A third model involves

CAL y binding to the supercoiled DNA substrates with similar affinities but in different

orientations. This model requires a nonproductive binding orientation in positively supercoiled

DNA. The recent studies of Epstein et al. 177 suggest a fourth model in which supercoiling

affects the ability of glutathione to repair drug-induced DNA lesions. Finally, the drug may bind

in similar affinity to both DNA substrates with the supercoiling effects due to differences in the

ability of glutathione to activate the drug. Considering the present experimental results, only the

last model is acceptable.

The lack of effect of supercoiling on the results of the competitive drug binding assay

appears to rule out the first two models, which depend on supercoiling-dependent differences in

drug binding. This result also suggests that the main determinant of drug-induced alterations in

plasmid topology is not helical over winding. If drug-induced helical over winding were solely

responsible for the observed negative plasmid writhing (Figure 11), then CAL y should have

bound with higher affinity to positively supercoiled DNA. However, I did not detect any

significant difference in the affinity of CAL y for either state of DNA supercoiling. Assuming

that damage occurs in proportion to the level of bound drug and that CAL y binds to pUC 19 with

a micromolar binding constant, 178,179 a 50% difference in the quantity of damage would require

that binding constants differ by -100-fold. The competitive binding assay would have detected

this difference in binding affinity.

Though supercoiling may not affect the DNA binding affinity of the parent form of the

drug, a parallel argument could be made for differences in the binding of the dihydrothiophene

intermediate of reduced CAL y (Figure 5). However, the lack of supercoiling effect observed with

CAL 0 and with CAL y activated by methyl thioglycolate rules out this scheme.

The third model, similar binding affinities but different binding orientations, is also

inadequate to explain the results. CAL y produces only bistranded DNA lesions and has the

same sequence selectivity in both positively and negatively supercoiled DNA. Grossly different

orientations of the bound drug, or reaction intermediates, would be expected to affect these

features of drug-induced damage. Furthermore, the reactive intermediates for CAL y and CAL 0

are identical, which rules out different binding orientations as the cause of the supercoiling-

dependent differences.

The model that best explains the preference of CAL y for damaging negatively supercoiled

DNA involves thiol accessibility. Two pieces of evidence support this conclusion. First, the

thiol-independent CAL 0 produced equal amounts of DNA damage in both supercoiled

substrates. Second, activation of CAL y by the neutral methyl thioglycolate also produced

equivalent results in both forms of supercoiled DNA. This latter result suggests that glutathione

is limited in its accessibility to DNA-bound CAL y by either its negative charge or its steric bulk.

This argument is similar in nature to a model in which supercoiling differentially affects

the ability of glutathione to "repair" the CAL y-induced damage by hydrogen transfer from the

thiol to the carbon-centered radicals in deoxyribose. This phenomenon has been proposed by

Epstein et al. to occur with DNA damage produced by the enediynes esperamicins Al and C. 177

According to their model, the negative charge of glutathione reduces its accessibility to drug-

induced deoxyribose radicals compared to the smaller, neutrally-charged methyl thioglycolate.

While such repair may have occurred to some extent in the experiments, my observation of biased

DNA damage cannot be explained by supercoiling-dependent differences in thiol-mediated DNA

repair. If the repair phenomenon were the primary determinant of the observed effects, then I

would not expect methyl thioglycolate to cause an increase--relative to glutathione--in

CAL y-induced damage in positively supercoiled DNA (Figure 14). Methyl thioglycolate is

proposed to be more efficient at quenching drug-induced deoxyribose radicals than glutathione, 177

and I would expect methyl thioglycolate to further reduce the damage in positively supercoiled

DNA relative to glutathione.

All of the evidence thus points to a model in which supercoiling influences the

accessibility of glutathione to DNA-bound CAL y. Any mechanistic proposal to explain this

phenomenon must take into account two observations: (1) within 30 minutes, all of the drug

appears to be rendered inactive toward production of DNA damage; and (2) there appears to be

no major difference in the affinity of CAL y for binding to supercoiled DNA of either sign. A

general scheme consistent with these observations centers on a side reaction--predominantly in

positively supercoiled DNA--that results in non-damaging activation of the drug by thiol or

inactivation of some intermediate form of the drug. While the exact spatial relationships of drug,

DNA and thiol that lead to productive activation are unknown, it is possible that positive

supercoiling causes a portion of bound drug, but not "unbound" or freely solvated drug, to be

inaccessible to glutathione, thus shifting the balance to nonproductive activation of CAL y. For

example, positive supercoiling could alter the charge density around a narrowed minor groove,

which could limit the accessibility of the negatively charged glutathione to drug positioned in the

groove. Such changes in DNA conformation are likely given the significant portion of

superhelical tension that is translated into changes in DNA twist. 165-167 Another possibility lies

in the handedness of the writhing of DNA helices in plectonemic supercoiling. 165 Positive

supercoiling causes the helices to cross one another in a left-handed superhelix with the walls of

the major and minor grooves running approximately parallel to each other. In negatively

supercoiled DNA, however, the walls of the grooves adopt an orthogonal orientation. Parallel

placement of the grooves would create a hydrophobic "tunnel" that might protect CAL y from

glutathione activation.

Glutathione is an important physiologic molecule 18 0 and has been implicated in drug

activation, 10 7,10 8,18 1, 182 modulation of DNA damage chemistry partitioning, 183-187 and DNA

protection and repair.187-193 It can be seen that any inhibition of the interaction of glutathione

with DNA may have far reaching consequences. The reduced accessibility of glutathione to the

minor groove may have a protective effect if glutathione is necessary for the activation of a drug

bound to DNA, as is seen with CAL y. This reduced accessibility may also increase the damage

caused by these agents however, by limiting the ability of glutathione to donate a hydrogen to the

sugar and/or base radical products. This is also a complex situation in which supercoiling may

have many roles in the damage process. In the case of CAL y, glutathione is limited in its ability

to activate the drug when bound to positively supercoiled DNA reducing the drug's overall

potency; but once the sugar radical has been formed, glutathione is limited in its ability to act as a

hydrogen donor to the positively supercoiled substrate, thereby also potentially increasing its

overall potency. A supercoiling-dependent exclusion of glutathione from the major or minor

grooves of DNA may affect the chemistry of oxidative DNA damage in transcriptionally active

genes that are subjected to high levels of both positive and negative supercoiling. 19,22

Furthermore, the effect of supercoiling on glutathione-DNA interactions may extend to

other small genotoxic molecules such as the negatively charged 02*-. I have observed that DNA

damage produced by Cu+2/H202 is sensitive to DNA supercoiling. 194 Since glutathione appears

to be capable of mediating Cu+2-induced Fenton chemistry, 181 the location, quantity and

chemistry of Cu-induced DNA damage could thus be further complicated by differential

accessibility of glutathione to sites of bound Cu or DNA damage produced by Cu.

To summarize, I have shown that CAL y, a non-intercalating DNA-damaging agent,

exhibits a supercoiling bias, causing more damage in negatively supercoiled plasmid DNA than in

DNA with positive superhelical tension. Furthermore I have shown that this supercoiling-

induced difference is controlled at the level of glutathione activation of the drug. My findings

suggest a general effect of supercoiling on the accessibility of glutathione and other small

molecules to DNA.

O - supercoiled

15 * + supercoiled

10 o

E0

% damage in - supercoiled DNA 1.5 0.1% damage in + supercoiled DNA

0.0 0.2 0.4 0.6 0.8 1.0

CAL y, nMFigure 10: Calicheamicin y produces 50% more damage in negatively supercoiled DNA than inpositively supercoiled DNA when activated by glutathione. Samples of negatively and positivelysupercoiled pUC19 were treated with calicheamicin y and 10 mM glutathione as described inMaterials and Methods and gel-resolved plasmid topoisomers were quantified by phosphorimageranalysis of probed gels; representative experimental results are shown in the graph. The averageratio of double-strand breaks in negatively supercoiled DNA to double-strand breaks in positivelysupercoiled DNA is 1.5 + 0.1 (n = 5).

I HighlySupercoiled

.2- CAL y i(- or +)

S250,000g

Plasmi Form II Srel ESP C Supercoiled+ drug (- or +)

Form I

ESP Al Relaxed

1.0- . * *0 5 10 15 20

drug/base pairs x 102

Figure 11: Calicheamicin y causes an increase in the density of plasmid DNA. The effectof binding of calicheamicin y (squares), esperamicin C (triangles) and esperamicin Al(circles) on the topology of negatively supercoiled pUC19 was determined by sucrosegradient centrifugation as described in Materials and Methods. Esperamicin Al isincluded to show the characteristic behavior of an intercalating molecule which isconsistent with net positive writhing of the plasmid molecule. The sedimentation ofsupercoiled pUC19 DNA was determined relative to nicked, open-circular pUC19.

0.5

0.4

Q O0

O0.2 00.0 1

0.0 0.5 1.0 1.5 2.0

Supercoiled DNA, mg/ml

Figure 12: Calicheamicin y binds with equal affinity to negatively and positively supercoiled pUC19. Therelative affinity of calicheamicin yfor negatively (open circles) and positively (closed circles) supercoiledpUC19 was determined by a competitive DNA damage assay, as described in Materials and Methods.Calicheamicin y-induced DNA damage in a [32P]-labeled DNA fragment was measured in the presenceof varying concentrations of unlabeled, supercoiled pUC19. The reduction of damage in the labeledDNA correlates directly with the overall binding constant for calicheamicin with the supercoiled DNAcompetitor. Representative experimental results are shown in the graph; the average ratio of damagein the presence of negatively supercoiled DNA to that with positively supercoiled DNA is 1.0 ± 0.1 (n=8).

)

E

00

15-

10-

5-

O - supercoiled* + supercoiled I

20 40 60 80

CAL 0, nMFigure 13: Calicheamicin 0 is not affected by DNA supercoiling. Samples ofnegatively (open circles) and positively (closed circles) supercoiled pUC19 weretreated with calicheamicin 0 as described in Materials and Methods and gel-resolvedplasmid topoisomers were quantified by phosphorimager analysis of probed gels;representative experimental results are shown in the graph. The average ratio ofdouble-strand breaks in negatively supercoiled DNA to double-strand breaks inpositively supercoiled DNA is 0.95 ± 0.08 (n = 5).

Glutathione Activation Methyl Thioglycolate Activation

0.2 0.4

10]

0.6 0.8

O -supercoiled O

* +supercoiled

O

O *0

0

0.6 0.8

CAL y, nM CAL y, nM

Figure 14: Activation of calicheamicin yby methyl thioglycolate abolishes supercoiling-dependent biases in DNA damage caused by glutathione. Samples of negatively (open circles)and positively (closed circles) supercoiled pUC19 were treated with calicheamicin y and 1 mMmethyl thioglycolate or 10 mM glutathione as described in Materials and Methods and gel-resolved plasmid topoisomers were quantified by phosphorimager analysis of probed gels;representative experimental results are shown in the graph. The average ratio of double-strandbreaks in negatively supercoiled DNA to double-strand breaks in positively supercoiled DNA formethyl thioglycolate is 0.97 ± 0.08 (n = 5); the average ratio for glutathione is 1.5 ± 0.1 (n=5).

10

5-

0.2 0.4

O - supercoiled

* + supercoiled

CHAPTER 5

CONCLUSION AND FUTURE STUDIES

Superhelical tension is an important aspect of genomic organization, 3,5,195 and is involved

in a variety of biological processes. 33,36 3 8,42,44 Supercoiling has been linked to the act of

transcription, 22 and has been measured in active genes in vivo.19-21 Thus, there is the potential

for supercoiling effects on the location and quantity of damage produced by any DNA reactive

agent entering the cell. For example, the DNA intercalator psoralen produces more damage in the

negatively supercoiled 5' end of the actively transcribing dihydrofolate reductase gene than in the

positively supercoiled 3' end of the gene, 19 as might be expected for an intercalating molecule.

However, with the exception of some controversial work with radiation, 52,53 the role of

superhelical tension in the interaction of non-intercalative molecules with DNA has remained

largely unexplored.

The results presented in this thesis represent some of the first evidence that supercoiling-

induced alterations in DNA structure and dynamics can effect the interaction of non-intercalative

molecules with DNA. While y-rad, ONOO', Fe+2/EDTA/H 20 2 and Fe+2/H20 2 are not sensitive

to DNA superhelical tension, I have shown that a physiologic metal (Cu), a DNA alkylating

agent (DMS) and an equilibrium groove-binding antibiotic (CAL y) are all sensitive to DNA

supercoiling. The two guanine-N7 selective agents, DMS and Cu, both exhibit a higher overall

reactivity in the DNA with positive superhelical tension, while the antitumor antibiotic shows a

higher reactivity in the negatively supercoiled substrate.

I have shown that the Cu+2/H20 2 system produces more strand breaks in a DNA

substrate with positively supercoiling. This increase in strand break activity could be due to

many factors including an increased access to or binding affinity for the N7 position of guanine.

Another possibility, however, is an alteration in the redox potential of Cu +2 when bound to

negatively or positively supercoiled DNA, perhaps the result of differing binding orientations.

Though I have mentioned that Cu seems to produce less base damage than strand breaks

in the negatively supercoiled substrate, the issue of Cu-induced base damage warrants further

study. By combining glycosylase treatment (formamidopyrimidine-DNA glycosylase (Fpg) and

endonuclease III (Nth)) to express base damage as strand breaks and the plasmid topoisomer

assay, one can quantitate the Cu-induced base damage in both substrates. Fpg and Nth convert a

broad spectrum of purine and pyrimidine damage products, including the major forms of Cu-

induced base damage,68 into single-strand DNA breaks. 196,197 In this way one can directly

compare the quantity of base damage formed in the opposing supercoiled substrates. These

types of studies can begin to answer the question posed in Chapter 3, "Do Cu-induced strand

breaks and base damage arise from different binding modes or binding orientations of Cu to

DNA?"

I have also raised the issue of the accessibility of the N7 of guanine vs. the binding

affinity for the guanine-N7 in substrates of equal but opposite DNA superhelical tension. As

mentioned earlier, the competitive damage assay described for CAL y in Chapter 4 may also be

used to determine the relative binding affinities of Cu for negatively and positively supercoiled

DNA. Other studies in our laboratory have identified a damage "hotspot" (a run of four

guanines) in a 215 base pair fragment of the 5s rRNA gene system. This DNA fragment can be

used as the "indicator" DNA for Cu+2/H20 2 just as the AGGA site in the 215 bp HindIII/PvuII

fragment of pUC19 was used for the CAL y studies. I suggest that Cu will indeed exhibit a higher

binding affinity for positively supercoiled DNA, an hypothesis based mainly on the results

presented here for DMS.

My research has demonstrated that DMS induces -25% more total methylation in

positively supercoiled DNA than DNA with negative superhelical tension. This can be due to

one of two factors: (1) an increased accessibility to bases in the positively supercoiled substrate;

or (2) a positive supercoiling-dependent increase in the nucleophilicity of the N7 and N3

nitrogens of the purine bases.

Future studies with SN1 acting alkylating agents, i.e. methylnitrosourea (MNU), may be

able to distinguish between these two possibilities. SN1 alkylators, such as MNU, decompose

to the methyldiazonium ion which is the ultimate alkylating species for these agents.9 1 The

methyldiazonium ion is so highly reactive, that any difference in methylation quantities seen with

MNU would be strong evidence of altered base accessibility.

Another area of interest in this project would be the identification of products formed by

these agents in both DNA substrates. Preliminary studies in my laboratory have suggested that,

at least in the case of DMS reactivity, methylation is increased not only at the guanine-N7 but

also at the N3 position of adenine. Further characterization of the base alkylation products by

HPLC might provide insights into the structural or electronic changes caused by DNA

superhelical tension.

I have also shown that CAL y induces -50% more double-strand breaks in DNA with

negative superhelical tension when compared to positively supercoiled DNA. A closer

examination shows that the chemistry, sequence selectivity and binding affinity of the drug are

not different between the two substrates. Further study demonstrated that the observed

difference is a result of the reduced ability of glutathione to activate CAL y molecules bound to

the positively supercoiled substrate. This is evidence for a reduced accessibility of glutathione to

the minor groove of positively supercoiled DNA.

The direct implication of this work is that any damage process involving glutathione will

be inhibited in positively supercoiled DNA. This observation can be extended in the study of

other enediyne drugs with different binding modes. Perhaps esperamicin Al, an intercalative

enediyne, 108 will show even larger supercoiling effects. The combination of a binding preference

and increased access to GSH in negative superhelical DNA may result in a larger supercoiling bias

than one would expect on the basis of binding affinities alone. C-1027, an intercalating, thiol-

independent enediyne, 198,199 can serve as the non-thiol control in these studies, just as CAL 0

served as the control in the CAL y studies presented in Chapter 4.

In my opinion, the most exciting feature of this research does not lie in the effects of

supercoiling on small molecule-induced damage per se. Rather, it is the DNA structural

information derived from these simple biochemical assays that is important. Through the

measurement of damage quantities induced by Cu and DMS, we find evidence of supercoiling-

dependent guanine-N7 accessibility. Similarly, the simple measurements of strand breaks

induced by CAL y give evidence of a reduced accessibility of negatively charged molecules to the

minor groove of DNA.

I have demonstrated that two guanine-N7 selective agents with completely different

modes of action show the same behavior: overall higher reactivity in DNA with positive

superhelical tension. This suggests that supercoiling may play a role in the reaction of any agent

with has a selectivity for reacting with the N7 position of guanine; i.e. other DNA alkylating

species, 10 2,10 3,200 ,20 1 the pluramycin family of antitumor antibiotics, 202-204 aromatic amines, 20 5

polycyclic aromatic hydrocarbons, 20 6,20 7 and aflatoxins, 208 ,209 to name a few. When one

considers the fact that many of these compounds also intercalate in DNA, their interaction with

supercoiled DNA becomes even more complex.

This complexity is illustrated by aflatoxin B (AFB 1). This intercalative genotoxin forms

a covalent adduct with the N7 position of guanine following epoxidation at the 8 and 9

positions.210 The dilemma here is whether the intercalation activity will bias the molecule to

react with negatively supercoiled DNA or if the heightened reactivity of guanine in positively

supercoiled DNA will enhance adduct formation. AFBI adducts in the supercoiled substrates

can be quantitated by either conversion of the adducts to strand breaks using Fpg and Nth

enzymes or by a simple enzyme-linked immunosorbent assay coupled with immunoaffinity

chromatography. 211

There is an additional level of complexity introduced in this process, in that the covalent

modification induced by AFB 1 does not result in a strand break in the DNA. This will allow for

the investigation of the effect of supercoiling on the mutagenic potential of the guanine-N7 adduct

of AFB1 .2 12 There is the possibility that supercoiling will affect the adduct spectrum of AFB 1

as is seen in the case of Ac-4-HAQO. A differing adduct spectrum, if it did indeed exist, would

likely alter the mutational spectrum of AFB1 . Future experiments could explore this possibility

by damaging supercoiled plasmid DNA (e.g. pSP189 for the supF gene)2 13 with AFBI, isolating

the adducted plasmid molecules, transfecting cells with these fragments and measuring the

mutational spectrum.

My studies have also demonstrated a reduced accessibility of glutathione to the minor

groove of DNA. Other studies in our laboratory investigating the effect of thiol structure on the

partitioning of C4' radical chemistry (Figure 6) have shown that it is most probably the charge

and not the size of glutathione that limits its access to the minor groove. In fact, this work has

shown a variety of size/charge effects in the accessibility of thiols to the grooves of DNA that

may be amplified by DNA supercoiling. Future studies involving thiol-mediated chemistry

partitioning in supercoiled DNA may provide a wealth of information regarding not only DNA

structure but also simple chemical reactivity in DNA with varying superhelical densities. Other

work in this laboratory has shown that high resolution polyacrylamide gel electrophoresis can be

used to separate and identify the products of deoxyribose radical degradation.' 08 By the use of

an indirect end-labeling procedure, we can modify this technique to look at the effect of DNA

supercoiling on the thiol-mediated partitioning of deoxyribose radical chemistry induced by

oxidizing agents, such as the enediynes.

The effects of supercoiling in vivo become even more complicated when one considers the

number of different oxidizing and reducing species present in cells. This point is best illustrated

in the example of Cu and GSH, two chemicals used in the current studies. The combination of

Cu and GSH can induce DNA damage in the absence of H20 2. 18 1'182 The results of my studies

suggest that Cu may bind to positively supercoiled DNA with higher affinity. However, my

results also suggest that glutathione may have a decreased accessibility to positively supercoiled

DNA. GSH appears to be capable of mediating the reduction of Cu +2 to Cu+1 , so these two

supercoiling-dependent effects may act in concert in the Cu(II)-thiol system, adding another level

of complexity.

The real test of the work comes when the hypotheses generated from these in vitro

results are tested in vivo. Regions of superhelical tension have already been shown to exist in

transcriptionally active DNA sequences. 19,134 Will the agents studied here, and possibly other

agents, show marked differences in DNA damage quantities in the ends of active gene sequences?

The recent advances in ligation mediated PCR 16 1,2 14-216 can be utilized to address this question.

By using PCR primers specific to the 5' and 3' ends of active gene sequences, one can begin to

explore the question of differential damage quantities as a function of transcriptionally generated

supercoiling. The use of certain PCR stop assays may be helpful to modify this technique for

the analysis of DNA adducting chemicals as well as agents which cause direct strand breaks in the

DNA.

The effect of physiologic supercoiling on in vivo target selection remains to be seen.

Once we fully understand the impact of transcriptionally driven supercoiling in genotoxin target

selection, then we may be able to develop drugs which target active gene sequences inside cells.

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