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:
",I. /
I
Division of Toxicology
June, 1998
Certified by:
Peter C. Dedon
Associate Professor of Toxicology
Thesis Supervisor
Accepted by:
Peter C. Dedon
Chairman, Committee on Graduate Students
d
MASSACHUSETTS INSTITUTE
OF TECHNOLOGY
SJlUNo8~
U9RARHe~
Division of Toxicology
This doctoral thesis has been examined by a committee of the Division of Toxicology as follows:
-
A
__
Professor John M. Essigmann
\
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I
Chairman
Associate Professor Peter C. Dedon
/
Professor William G. Thilly
Professor Steven R. Tannenbaum
Associate Professor Ram Sasisekharan
.---
,Sunervisor
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-hydroxyadenine
8-oxoG
8-hydroxyguanine
Ac-4-HAQO
4-acetoxy-aminoquinoline- 1-oxide
AFB 1
aflatoxin B
CAL y
calicheamicin yjl
CAL 0
calicheamicin 011
CBE
chicken blood extract
Cu
copper
Cyt Glycol
cytosine glycol
DMS
dimethyl sulfate
DNA
deoxyribonucleic acid
e-aq
solvated electron
E. coli
Escherichiacoli
EDTA
ethylenediaminetetraacetic acid
EtBr
ethidium bromide
Fe
iron
Fpg
formamidopyrimidine-DNA glycosylase
y-rad
y-radiation
'H
hydrogen atom
H20
2
hydrogen peroxide
Lk
linking number
MNU
methylnitrosourea
NO'
nitric oxide
Nth
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 2 0 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 calicheamicinilI/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, Proceedingsof the NationalAcademy 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,
Proceedingsof the American Association of Cancer Research, 38, 127.
LaMarr, W.A.; Sandman, K.M.; Reeve, J.N.; Dedon, P.C. (1996) Supercoiling and radicalmediated 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, DepartmentofResearch andDevelopment,
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
EnglandPharmacologists,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 Drosophilamelanogasterand 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 ions 30 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
32 37
been implicated in many cellular processes such as nucleosome assembly, - 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-4HAQO are 3-(deoxyguanosin-N2-yl)-4-aminoquinoline- 1-oxide, N-(deoxyguanosin-C8-yl)-4aminoquinoline- 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
62 64
oxygen species: -
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. 7 3-75 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 damage 79 -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'-PhosphoGlycoaldehyde
B
B
P (H
1te
Q'00Propenoic
Base
Acid
reactive
oxygen
OOH
species
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
N.
N
N
1'n2
H
IH
N"O
OH
N
2N
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
H2 N
N
8-oxoG
NNO
O
H2 N
2
N
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
96,97
mutagenicity, 92 -94 carcinogenicity 95 and cytotoxicity.
The specific DNA alkylating agent used in the following studies is the SN2 acting DNA
methylator, 9 1 dimethyl sulfate (DMS).
H3 C-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
86,101
methylation at the N3 position of adenine, which constitutes <20 % of the total alkylation.
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. '10 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)
WRITHE (Wr)
B-DNA
-10.5 base
pairs
Twist the number of
helical turns per
molecule
Writhe: the number
of times the double
helix crosses itself
Figure 1: The energy imparted to DNA by superhelical tension can be relieved in two different ways. Twist is a change in
the helical repeat of DNA. Writhe is a crossing of the double helix over itself. Negatively supercoiled DNA is underwound
and positively supercoiled DNA is overwound. The third way that DNA can releive superhelical tension is by the
etc.). To date, these
formation of unusual secondary structures ( i.e. cruciforms, hairpins, Z-DNA, unpaired regions,
structures have only been observed in negatively supercoiled DNA.
Tw = -3
Wr = 0
Tw = -2
Wr = -1
A Lk
Tw = -1
Wr = -2
Tw = 0
Wr = -3
-3
Figure 2: In any closed system, twist and writhe can interconvert as long as the linking number change (ALk) remains
constant. The energy of superhelical tension can be expressed as all twist, all writhe, or a mixture of the two. Several
studies suggest that up to 30% of superhelical energy can be expressed as changes in twist. This is important since
changes in DNA twist will have a greater effect on local DNA conformation, such as the width and depth of the grooves,
than writhing.
Negative underwound
A Lk<O
Positive overwound
A Lk>O
POLYMERASE
Figure 3: As first proposed in the twin supercoiling domain model of Liu and Wang, 15 superhelical tension is
generated in vivo by the act of transcription. The polymerase machinery tracking along the DNA helix generates
negative supercoiling at the 5' end of the gene and positive supercoiling at the 3' end of the gene. A rigorous
model system for the study of supercoiling should therefore include both negatively and positively supercoiled
substrates.
Figure 4: The enediyne family of antitumor antibiotics. Though these molecules have
a variety of structures, they all have a common mode of activation and DNA damage
induction.
Dihydrothiophene
SSCH 3
Q
R
R~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 diradical
species. When this diradical is positioned in the minor groove of DNA, it abstracts deoxyribose
hydrogen atoms. The sugars then undergo oxygen-dependent breakdown reactions that result in
DNA strand breaks or abasic sites. Calicheamicin forms >95% double strand breaks by exclusive
abstraction of the 4' hydrogen on one strand and the 5' hydrogen on the other.
B
-0
O
4'
rCH
0
R
-O
+
B
RB
,O
CCR
\
,
P
0
B
P-O
B
0=_'
O
5,
P-1
P
J
P-
H
P
P
+
B
_10-
R
Figure 6: Deoxyribose radical breakdown pathways. The enediynes abstract minor groove hydrogen
atoms from deoyxribose (1', 4' & 5') . The resulting sugar radicals then undergo a series of oxygendependent reactions that yield a variety of lesions, including strand breaks and abasic sites.
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
PreparationofrHMfB. 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).
Preparationof chicken blood extract (CBE) that relaxes superhelicaltension. 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.
Productionofpositively supercoiledpUC19 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.
2nd Dimension
A)
B)
ALk
0
Relaxed
C
O
(000P
00O
a)
C
E
.5
+2
S.C~
+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 twodimensional 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 death 96,132 by methylating DNA, predominantly at
98 00
the N7 position of guanine. -1
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 Escherichiacoli genome has a a = - -0.05, the genomes of Drosophila
melanogasterand 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
22 34
the 3'-ends. 19,2 1, ,1
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 DNAdamaging 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
5,119
levels of supercoiling are similar to those estimated to exist in vivo.
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. 5 8' 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 + 1:139141
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 8oxoG 142 ' 143 and strand breaks 59,14 4 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 'OHlike 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
52,53,153
tension on radiation-induced DNA damage has been controversial.
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.
PreparationofsupercoiledpUC19substrates.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 ofsupercoiledpUC19with 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 ofsupercoiledpUC19with 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-borateEDTA) 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 60 0 C.
QuantitationofPlasmid 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 'OHlike 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 'OHmediated 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 /H2 0 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.
15
O
- supercoiled
*
+ supercoiled
O
- supercoiled
*
+ supercoiled
00)
0
0
0
E
0
0-
o( 5
o
0
0
o
,
5
10
15
y-radiation, Gray
0
5
0
1
2
Fe(II)/EDTA, gM
- supercoiled
+ supercoiled
)
00
E
o-0
5
0
0R
0
0
25
50
ONOO, IM
75
Figure 8: Strand breaks produced by y-radiation, peroxynitrite and Fe+2/EDTA/H 2 0 2 are not
sensitive to DNA supercoiling. Samples of negatively and positively supercoiled pUC19 were treated
with y-radiation, peroxynitrite, Fe+2/EDTA/H 2 0 2 , as described in Materials and Methods, and gelresolved plasmid topoisomers were quantified by phosphorimager analysis of probed gels (n = 3-5).
40
O)
60
0
O - supercoiled
*
+ supercoiled
O
50
30040
0
E
E 20
30
20
10
10
20
40
60
Fe(ll), jIM
80
100
5
10
15
Cu(ll), IM
Figure 9: Strand breaks produced by Cu+2 /H2 02, but not Fe+2 /H20 2 , are sensitive to DNA
supercoiling. Samples of negatively and positively supercoiled pUC19 were treated with Cu+2 /H2 02
and Fe+ 2/H2 0 2 , as described in Experimental Procedures, and gel-resolved plasmid topoisomers were
quantified by phosphorimager analysis of probed gels (n = 3-5).
Table 1: Supercoiling-dependent differences in methylation
of DNA by [3H]-dimethyl sulfate
Radioactivity (cpm) bound to DNA
(20 uM)
High [DMS]
(100 IrM)
Negatively
Supercoiled DNA
586 +145
1607 + 134
Positively
Supercoiled DNA
733 + 67
2100 + 204
0.799
0.765
Low [DMS]
Ratio -/+
DMS shows a supercoiling bias, methylating the positively supercoiled DNA more
than DNA with negative superhelical tension. Supercoiled substrates were treated
with [3 H]-DMS and then the DNA was purified. The negatively and positively
supercoiled substrates are indicated by - and +, respectively. The amount of methyl
transfer was quantitated by scintillation counting. DMS causes -25% more
methylation in the positive substrate at two different DMS concentrations.
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 Drosophilamelanogaster 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'-ends 17 -22 ,134 --which is consistent with the twindomain 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
19,137
unwinding that is dissipated as global overwinding of the helix.
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 sequenceselectivity, 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 supercoiledsubstrate. 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 Methanothermusfervidusas 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 topoisomerasecontaining 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. 01 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 370 C. 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] endlabeled 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 glycerolbased 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 supercoiledDNA. 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, 01 8 no change in
supercoiling is observed for esperamicin C, an analog of CAL y missing the terminal sugararomatic 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 binding 121,175 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 Oproduces 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 supercoilingbias. 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
SH
O
O
HSJ
+H3 N
NH
o,CH 3
O
OO
Methyl thioglycolate
O
O
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 sequenceselectivity 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 supercoilingdependent 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 druginduced 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
19,22
genes that are subjected to high levels of both positive and negative supercoiling.
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 supercoilinginduced 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
o
10
E
0
% damage in - supercoiled DNA
% damage in + supercoiled DNA
0.0
0.2
0.4
0.6
0.8
1.5
0.1
1.0
CAL y,nM
Figure 10: Calicheamicin y produces 50% more damage in negatively supercoiled DNA than in
positively supercoiled DNA when activated by glutathione. Samples of negatively and positively
supercoiled pUC19 were treated with calicheamicin y and 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-strand breaks in negatively supercoiled DNA to double-strand breaks in positively
supercoiled DNA is 1.5 + 0.1 (n = 5).
I
Highly
Supercoiled
or +)
CAL y i(-
.2-
S250,000g
Plasmi
+ drug
Form II
Srel
Supercoiled
(- or +)
ESP C
Form I
ESP AlRelaxed
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 effect
of binding of calicheamicin y (squares), esperamicin C (triangles) and esperamicin Al
(circles) on the topology of negatively supercoiled pUC19 was determined by sucrose
gradient centrifugation as described in Materials and Methods. Esperamicin Al is
included to show the characteristic behavior of an intercalating molecule which is
consistent with net positive writhing of the plasmid molecule. The sedimentation of
supercoiled pUC19 DNA was determined relative to nicked, open-circular pUC19.
0.5
0.4
0
O
Q
O
0
0.2
0.0
0.0
0.5
1.0
1.5
1
2.0
Supercoiled DNA, mg/ml
Figure 12: Calicheamicin y binds with equal affinity to negatively and positively supercoiled pUC19. The
relative affinity of calicheamicin yfor negatively (open circles) and positively (closed circles) supercoiled
pUC19 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 presence
of varying concentrations of unlabeled, supercoiled pUC19. The reduction of damage in the labeled
DNA correlates directly with the overall binding constant for calicheamicin with the supercoiled DNA
competitor. Representative experimental results are shown in the graph; the average ratio of damage
in the presence of negatively supercoiled DNA to that with positively supercoiled DNA is 1.0 ± 0.1 (n=8).
15-
O - supercoiled
* + supercoiled
I
)
10-
E
0
0
5-
20
40
60
80
CAL 0, nM
Figure 13: Calicheamicin 0 is not affected by DNA supercoiling. Samples of
negatively (open circles) and positively (closed circles) supercoiled pUC19 were
treated with calicheamicin 0 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-strand breaks in negatively supercoiled DNA to double-strand breaks in
positively supercoiled DNA is 0.95 ± 0.08 (n = 5).
Methyl Thioglycolate Activation
Glutathione Activation
O
O -supercoiled
* +supercoiled
O
- supercoiled
*
+ supercoiled
10]
10
O
5-
O *0
0
0.2
0.4
0.6
CAL y, nM
0.8
0.2
0.4
0.6
CAL y, nM
Figure 14: Activation of calicheamicin yby methyl thioglycolate abolishes supercoilingdependent 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 mM
methyl thioglycolate or 10 mM glutathione as described in Materials and Methods and gelresolved plasmid topoisomers were quantified by phosphorimager analysis of probed gels;
representative experimental results are shown in the graph. The average ratio of double-strand
breaks in negatively supercoiled DNA to double-strand breaks in positively supercoiled DNA for
methyl thioglycolate is 0.97 ± 0.08 (n = 5); the average ratio for glutathione is 1.5 ± 0.1 (n=5).
0.8
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 supercoilinginduced 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/H 20 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/H 20 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 Cuinduced 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/H2 0 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, thiolindependent 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 supercoilingdependent 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. 21 1
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 AFB 1 .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 AFB 1 . 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 H2 0 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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