Mutagenesis by the Primary Aflatoxin B,
DNA Adduct in Escherichia coli
by
Elisabeth Ann Bailey
B.A. Chemistry
Skidmore College, 1989
SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN BIOLOGICAL CHEMISTRY
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 1996
© 1996 The Massachusetts Institute of
Technology.
All rights reserved.
Signature of Author:uepartmeni oT unemistry
December 5, 1995
Certified by:.
\J
John M. Essigmann
Professor of Chemistry and Toxicology
Thesis Supervisor
Accepted by:
Dietmar Seyferth
Chairman, Departmental Committee on Graduate Students
OF TECHNOLOGY
MAR 04 1996
LBsRARIES
Thesis Committee
Professor Gerald N. Wogar
U
Chairperson
Professor John M. Essigmann
-xIj
I nesis oupl.
Professor Graham C. Walker_
Professor Peter C. Dedort
~CII
_I__
visor
Mutagenesis by the Primary Aflatoxin B,
DNA Adduct In Escherichia coli
by
Elisabeth Ann Bailey
Submitted to the Department of Chemistry on December 5, 1995 in Partial
Fulfillment of the Requirements for the Degree of Doctor of Philosophy
in Biological Chemistry
ABSTRACT
This dissertation describes the mutagenic consequences of the potent
liver carcinogen aflatoxin B, (AFB 1). The major DNA adduct of AFB, is 8,9dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B, (AFB,-N7-Gua). A problem that
has hindered the study of the biological properties of this lesion is its
extreme chemical lability, which results in either depurination of the modified
guanine or opening of the imidazole ring of the guanine residue to generate
the AFB 1-formamidopyrimidine adduct (AFB,-FAPY). The first aspect of this
work describes a procedure for the construction of a bacteriophage genome
containing either the chemically and thermally labile AFB,-N7-Gua adduct or
the apurinic (AP) site in a specific location within the genome. The second
aspect involves the use of these genomes to examine and compare the
genetic effects of AFB,-N7-Gua and the AP site in Escherichia coli.
A 13-mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react
with the exo-8,9-epoxide of AFB, to form an oligonucleotide containing a
single AFB 1-N7-Gua (at the underscored guanine). This modified 13-mer was
5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome
generated by annealing a 53-mer uracil-containing scaffold to M13mp7L2
linearized by EcoRl. Following ligation, the scaffold was enzymatically
removed with uracil DNA glycosylase and exonuclease IIl. The entire
genome construction was complete within 3 hours, and was carried out at
16 0C pH 6.6, conditions determined to be optimal for AFB,-N7-Gua stability.
A portion of the genome was subsequently heated to promote AFB,-N7-Gua
depurination to generate the AP site genome. Characterization procedures
indicated that the AFB 1-N7-Gua genome was -95% pure with a small
contamination of unmodified genome (5%), and an apurinic site genome
contamination of <0.2%.
Replication of the AFB,-N7-Gua genome in mucAB-containing SOSinduced E. coli yielded a mutation frequency of 4%. The predominant
mutations observed were G--T transversions, which were much more
strongly dependent on the activity of MucAB than UmuDC. The mutations
induced by the AP site (AP-*T), however, showed roughly equal dependence
on MucAB and UmuDC. Given the strong difference in the genetic
requirements for mutagenesis of the two lesions, it was concluded that
aflatoxin mutational spectra, derived in other studies from DNA globally
modified by activated forms of AFB1 and harboring a predominance of G->T
mutations, cannot be explained, for the most part, as resulting from
depurination of AFB,-N7-Gua adducts. The data are consistent, by contrast,
with AFB,-N7-Gua being the premutational lesion, although the involvement
of other adducts cannot be excluded at this time.
While most of the mutations from the specifically placed AFB,-N7-Gua
adduct were targeted to the site of the lesion, a significant fraction (13%) of
the mutations occurred at the base 5' to the modified guanine. In contrast,
the AP site containing genome gave rise to mutations targeted only to the
site of the lesion. The mutational asymmetry observed for AFB,-N7-Gua is
consistent with structural models indicating that the aflatoxin moiety of the
aflatoxin-guanine adduct intercalates on the 5' face of the guanine residue
[Gopalakrishnan, S., Harris, T. M., Stone, M. P. (1990) Biochemistry 29,
10438-10448].
Taken together, the results of this work suggest molecular mechanisms
that could explain important steps in the carcinogenicity of aflatoxin B, .
Thesis Supervisor: Professor John M. Essigmann
Title: Professor of Chemistry and Toxicology
Acknowledgements
First and foremost I would like to thank my thesis advisor, John
Essigmann, for his tremendous support and encouragement throughout my
time at MIT. There were many times when I became a bit disheartened with
my studies, and John was always there to lend moral support. John
provided a lab environment that, besides being supportive, encouraged a
great deal of independent thinking that enabled me to grow and develop as a
scientist. I would also like to thank both John and Dr. Wogan for their
personal contributions to the aflatoxin field. Their pioneering studies
provided the basis for my thesis work and I can only hope that someday I
will be respected as a scientist as much as they both are. I am grateful to
our collaborator, Tom Harris, and to his postdoctoral associate, Raj lyer, for
preparing the aflatoxin-containing oligonucleotides. I'd also like to thank
Tom for his encouragement and support in my work throughout this
collaboration. I thank my committee members, Dr. Wogan, Pete Dedon, and
Graham Walker.
I was very fortunate to have joined a lab with so many helpful,
supportive, and very bright individuals. I want to thank both Mike Wood and
Ashis Basu for my initial training in the lab. They were both very patient,
both great teachers, and Mike especially would always let me know if I was
not doing the "proper controls." I also thank Lisa, Susan, and Kevin for their
guidance; they were always willing to help with any problem I may have
encountered. I am grateful to my UROPs, Leila Tabibian and Maryann Smela
who were both very careful and efficient workers, and also to Anna Barrasso
who, along with Maryann, provided tremendous help with all of the
sequencing that was necessary toward the end of my project. I thank Kevin,
Becky, and Marjie for their careful reading of various sections of this
dissertation. I also want to thank Kim, Denise, and Laurie for their help and
guidance in the preparation of my manuscripts and my thesis. The people
that I will never forget are those who actually made grad school fun! I have
to thank the Muddy / Thirsty Ear crew - Susi, Mike, Brian, Dan, Kevin, and
Tanya. I thank Lisa and Barry for all of those spontaneous evenings that we
somehow always managed to turn into a party; you guys are awesome! I
thank Jill, a really great friend, for many fun shopping outings, trips to Au
Bon Pain for mocha blasts, trips to the beach that sometimes ended in
sunburns, and for always understanding the silly things that drove me crazy.
I thank Marjie for always remembering the little things, for celebrating
'mutants' with me, and for always being a great friend. I am grateful to
both Jill and Marjie for their caring and willingness to listen whenever I
needed a friend; I'll never forget you guys. I'II never forget Beth Berger, a
great person and an awesome friend; we had a lot of fun times.
I want to give special thanks to my family. Without my Mom, Dad, and
my sister MaryBeth I never would have made it through grad school. I
dedicate this dissertation to them. There were many times when I wanted to
quit, but with their constant love, support, and encouragement they gave me
the strength and the desire to continue. I thank them tremendously. Finally,
I want to thank my best friend and boyfriend, Dan. He always expressed his
confidence and belief in me and in my abilities as a scientist. He always
seemed to know when I was feeling unsure about myself, and always
managed to say the right things to encourage and motivate me through the
toughest of times. I could not have done this without his support and his
love, and for that I cannot thank him enough.
Table of Contents
Title Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
Com m ittee Page .......................................
2
A bstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
Acknow ledgem ents .....................................
5
Table of Contents ......................................
7
List of Figures .......................................
10
List of Tables ........................................
12
List of Abbreviations ...................................
I. Introduction ..................................
II. Literature Review ......................
........
A. Historical Perspective on Aflatoxin ..............
B. Aflatoxin Exposure and Human Disease ...........
13
14
26
27
28
1. The occurrence of aflatoxin in human foods
2. Human disease ........................
C. Metabolism of Aflatoxin .....................
1. Metabolic activation of AFB, . . . . . . . . .
2. AFB, detoxification ...................
D. Interactions of AFB, with DNA ..................
1. AFB,-DNA adducts .....................
2. Chemical stability of AFB,-N7-Gua ..........
. ..
. . . .
E. Biological Effects of Aflatoxin B, in Bacterial Cells ....
1. Repair of AFB,-induced DNA damage ........
2. SOS response in E. coli .................
3. Mutagenic consequences of AFB, in bacterial
cells ..................
...........
F. Biological Consequences of Aflatoxin B, in Mammalian
Systems ..............................
1. Repair of AFB,-induced DNA damage ........
2. Mutagenic consequences of AFB, in mammalian
systems ............
......
........
G. Sequence Specificity of Aflatoxin B, DNA
Modification and Mutagenesis ...............
H. Biology of the M13 Vector Used in this Work .......
III. Preparation of AFB,-N7-Gua and AP Site Singly-Modified
28
.
.
29
31
31
33
36
36
39
43
43
45
48
51
51
52
56
57
Bacteriophage Genomes .................
......
A. Introduction ..............................
1. Rationale for the use of a single-stranded
genom e ....................
.......
2. Choice of oligonucleotide sequence for AFB1
modification .......................
B. Materials and Methods .......................
1. M aterials ..........................
.
2. Preparation and purification of AFB, modified
oligonucleotides ...............
......
3. Preliminary AFB,-FAPY studies ............
4. Stability of the AFB,-N7-Gua adduct .........
5. Stability of AFB,-N7-Gua under genome
construction conditions ...............
6. Preparation of bacteriophage M13mp7L2
gapped genomes ...................
7. Construction of singly-modified genomes .....
8. Evaluation of the efficiency of uracil-containing
.
scaffold removal ...................
of
AP
of
removal
the
efficiency
9. Evaluation of
sites by exonuclease III from the AFB,-N7.............
Gua genome preparation
10. Characterization of the AFB,-N7-Gua and AP
..........
site singly-modified genomes
11. Quantitation of M13-G, M13-GA, M13-AFBj,
and M 13-AP .......................
C. Results .........................
.......
1. Strategy for construction of singly-modified
genomes ..........................
2. Stability of the AFB,-N7-Gua adduct ........
3. Stability studies and characterization of [AFB,N7-Gua d(CCTCTTCGAACTC)] ..........
4. Removal of the uracil-containing scaffold from
.. . . . . . . . . . . . . . . . . . . . . .
M 13-AFB,
5. Contaminating AP site genome can be
efficiently inactivated by its selective
sensitivity to exonuclease III ...........
6. Characterization of singly-modified genomes
7. Quantitation of M13-G, M13-GA, M13-AFBj,
and M13-AP .......................
D. Discussion ...............................
IV. Mutational Properties of AFB,-N7-Gua in Escherichia coil ...
A. Introduction ............................
B. Materials and Methods .......................
.
73
74
74
76
77
77
78
79
80
81
81
83
84
86
87
88
90
90
91
92
94
95
97
100
100
122
. 123
124
1. Materials
.......
...
124
of
singly2. Preparation and characterization
modified genomes ...............
...
124
3. Optimization of conditions for SOS induction ... 125
4. Transformation of E. co/i cells with singlymodified genomes ...............
...
128
5. Mutant enrichment procedure ............
129
..........
129
6. Mutation frequency determination
C. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
1. Optimal conditions for SOS induction ........
133
2. Mutation frequencies of AFB,-N7-Gua and the
AP site ........
................
134
3. Mutational specificity of AFB 1-N7-Gua and the
A P site . . . . . . . . . . . . . . . . . . . . . . . . . . 136
4. Genotoxicities of AFB,-N7-Gua and the AP site . 139
140
D. Discussion ..............................
170
V. Conclusions and Suggestions for Future Studies .........
References
.........................................
Biographical Sketch ....................................
178
194
List of Figures
Figure 1.
Structure of aflatoxin B1, (AFB
Figure 2.
HPLC chromatogram of AFB, DNA adducts .........
Figure 3.
Methods for construction of site-specifically modified
genom es
Figure 4.
)
. . . . . ...........
...
20
..
22
24
......................................
Structures of aflatoxin B1, aflatoxin B2, aflatoxin G1, and
aflatoxin G2 .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
61
Figure 5.
Metabolic activation of AFB, to the exo-epoxide
Figure 6.
Pathway for metabolic formation of the AFB,-dialcohol
Figure 7.
Structures of aflatoxin B,-induced DNA adducts ........
67
Figure 8.
Structures of aflatoxin M1 and aflatoxin P, ...........
69
Figure 9.
Rolling circle replication of bacteriophage M13
71
Figure 10.
Construction scheme for singly-modified genomes .....
Figure 11.
HPLC chromatogram of AFB,-FAPY containing
63
.......
..
........
oligonucleotide ..................................
Figure 12.
104
106
Agarose gel of M13-G, M13-GA, M13-AFB1 , and M13-AP
genom es
......................................
Figure 13.
AFB,-N7-Gua stability studies ....................
Figure 14.
HPLC chromatogram of AFB,-N7-Gua 13-mer after
incubation under genome construction conditions ...........
Figure 15.
65
Mass spectral data of AFB,-N7-Gua 13-mer ..........
10
108
110
112
114
Figure 16.
Efficiency of removal of uracil-containing scaffold ......
Figure 17.
Efficiency of removal of potentially contaminating M13-
AP from the M13-AFB, preparation .....................
Figure 18.
118
Characterization gel of M13-AFB1 , M13-AP, M13-G, and
M13-GA genomes ......................
.........
Figure 19.
Sful mutant enrichment procedure
Figure 20.
Type and percent distribution of AFB,-N7-Gua and AP
120
156
................
site induced mutations ..............................
Figure 21.
116
158
Mutation frequencies of AFB,-N7-Gua (G-T) and the AP
160
site (A P- T) .....................................
Figure 22.
A potential AFB,-N7-Gua mispair with syn dAMP
Figure 23.
A potential AFB,-N7-Gua zwitterionic mispair with dTMP . 164
Figure 24.
A potential energy minimized structure of the central
portion of the AFB,-N7-Gua 13-mer .....................
Figure 25.
......
162
166
Cartoon depicting the AFB,-N7-Gua adduct at the
moment of replication ..............................
168
List of Tables
Table 1.
SOS optimization; mutation frequencies of UV-irradiated
RF and ss DNA ....................................
Table 2.
152
Cell survival subsequent to UV-irradiation for SOS
induction . . . ...
...
...
...
.. .. .. .....
. .. . .. . .. . . . 153
Table 3.
Mutation frequencies of AFB,-N7-Gua and the AP site . . . . 154
Table 4.
Genotoxicities of AFB,-N7-Gua and the AP site .........
12
155
List of Abbreviations
AAF
AFB,
AFB,-N7-Gua
AFB,-FAPY
AP site
BSA
ds
DTT
E. co/i
Exo III
GSH
GST
HCC
A
HPLC
IPTG
LB
M13-AFB 1
M13-AP
M13-G
M13-GA
MF
MOPS
MSB
NER
nt
PAGE
RF
RRF
ss
S. typhimurium
TBE
UDG
UV
X-Gal
XP
N-(guanin-8-yl)-2(acetylamino)fluorene
aflatoxin B,
8,9-dihydro-8-(N7-guanyl)-9hydroxyaflatoxin B,
aflatoxin B,-formamidopyrimidine
apurinic site
bovine serum albumin
double-stranded
dithiothreitol
Escherichia coli
exonuclease III
glutathione
glutathione S-transferase
hepatocellular carcinoma
heated
high performance liquid
chromatography
isopropyl 8-D-thiogalactopyranoside
Luria bertani broth
M13-AFB,-N7-Gua containing genome
M13-AP site containing genome
M13 unmodified genome
heated M13 unmodified genome
mutation frequency
3-(N-morpholino)propanesulfonic acid
medium salt restriction endonuclease
buffer
nucleotide excision repair
nucleotide
polyacrylamide gel electrophoresis
replicative form
restriction resistant fraction
single-stranded
Salmone//a typhimurium
tris borate EDTA
uracil DNA glycosylase
ultraviolet light
5-bromo-4-chloro-3-indolyl i-Dgalactopyranoside
xeroderma pigmentosum
13
I. Introduction
The environment poses continual threats to our genetic material. There
are a multitude of chemical and physical agents to which we are exposed
every day. Certain environmental chemicals are known to be the cause of a
number of human genetic diseases, one being cancer. Over two centuries
have passed since it was first noted that there may be a link between human
carcinogenesis and exposure to chemicals and physical agents present in the
environment (Pott, 1775). The mechanisms by which these chemicals
induce cellular transformation are still, for the most part, not completely
understood. There is good evidence, however, that in many instances
chemical alterations of DNA play a requisite role in cancer development and,
indeed, most chemicals that are potent carcinogens bind very effectively to
DNA. Most chemical carcinogens are not inherently reactive, and thus some
form of metabolic activation is required to convert those carcinogens to
reactive, usually electrophilic derivatives that can form covalent adducts with
DNA (Miller, 1978).
The formation of carcinogen adducts in DNA is believed to induce
heritable alterations or mutations. Mutations may result from the
misreplication of damaged DNA that is either misinformational or
noninformational. However these mutations arise, they represent permanent
changes within the informational content of the cell. These changes may
affect the critical aspects of cell growth regulation and could conceivably be
the initiating events in carcinogenesis.
Aflatoxin B, (AFB,) (shown in Figure 1), is a metabolite of the fungus
Aspergillus flavus, and is a common contaminant of the food supply in
eastern Asia and sub-Saharan Africa. AFB, has been associated with an
increased incidence of hepatocellular carcinoma (HCC) in these areas of the
world (Busby and Wogan, 1984). The toxin requires metabolic conversion to
its exo-8,9-epoxide (Swenson et al., 1977; Essigmann et al., 1977) in order
to cause damage to DNA (Miller, 1978), the event that presumably initiates
the genetic changes resulting in HCC (Harris, 1991). The AFB, epoxide
reacts with guanine to form a population of adducts (Essigmann et al.,
1983), shown in Figure 2, the principal of which both in vitro (Essigmann et
al., 1977; Martin and Garner, 1977; Lin et al., 1977; Groopman et al., 1981)
and in vivo (Lin et al., 1977; Croy et al., 1978; Croy and Wogan, 1981a;
Croy and Wogan, 1981b) is 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B,
(AFB,-N7-Gua). The positively charged imidazole ring of this adduct
promotes depurination, resulting in apurinic (AP) site formation.
Alternatively, under slightly basic conditions the imidazole ring of AFB,-N7Gua can open to form the chemically and biologically stable AFB,formamidopyrimidine adduct (AFB,-FAPY); shown in Figure 2. In addition, a
very small percentage of AFM,-N7-Gua, AFP,-N7-Gua, and unmodified
guanine (indicated by the presence of the dihydrodiol) is also formed. An
16
AFB 1-FAPY intermediate structure is also present at a very low percent (see
Figure 2). The abundance of the initial AFB,-N7-Gua and AFB 1-FAPY
adducts indicates that these two lesions, along with the AP site, individually
or collectively, represent the likely chemical precursors to the genetic effects
of AFB,.
Mutations induced by electrophilic forms of AFB 1 have been examined in
Escherichia coli induced for the SOS response (Foster et al., 1983;
Sambamurti et al., 1988; Sahasrabudhe et al., 1989) and in a number of
mammalian systems (Trottier et al., 1992; Levy et al., 1992; Soman and
Wogan, 1993; McMahon et al., 1990; Aguilar et al., 1993). These genetic
studies indicate that several mutations, including GC- AT transitions and
GC--TA transversions, occur in DNA globally modified with AFB,.
The
predominant mutation observed, however, induced or selected in vivo,
appears to be the GC--TA transversion. There is speculation that the
premutagenic lesion responsible for this mutation is the AFB,-induced AP site
(Foster et al., 1983; Foster et al., 1988; Kaden et al., 1987), since dAMP is
the base most often inserted opposite AP sites in E. coli induced for the SOS
response (Loeb and Preston, 1986; Lawrence et al., 1990). It has always
been a possibility, however, that the parental adduct, AFB,-N7-Gua, or the
AFB,-FAPY adduct could also give rise to this mutation.
A thorough examination of the genetic effects induced by each of these
lesions requires that each lesion be studied, in parallel experiments, within
the same site of individual genomes. Site-specifically modified genomes
have been employed to study the mutational consequences of a number of
DNA adducts, such as 0 6-methylguanine (Loechler et al., 1984; Ellison et al.,
1989); 0 4-methylthymine (Dosanjh et al., 1991); T-T pyrimidine dimers
(Banerjee et al., 1988; Banerjee et al., 1990; LeClerc et al., 1991); thymine
glycol (Basu et al., 1989); N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF)
(Burnouf et al., 1989; Reid et al., 1990); apurinic sites (Lawrence et al.,
1990); 8-hydroxyguanine (Wood et al., 1990); the major benzo[a]pyrene
DNA adduct, (+)-anti-B[a]P-N 2-Gua (MacKay et al., 1992); vinyl chlorideinduced DNA lesions such as, 1,N6-ethenoadenine, 3,N 4-ethenocytosine, and
4-amino-5-(imidazol-2-yl)imidazole (Basu et al., 1993); and cisdiamminedichloroplatinum(ll) DNA adducts (Bradley et al., 1993; Yarema et
al., 1995). The use of this procedure was first illustrated in our laboratory
upon the mutational studies of 0 6-methylguanine (Essigmann et al., 1986).
A double-stranded (ds) vector, containing an 0 6-methylguanine within a
specific site was constructed. See Figure 3 for general construction
schemes of site-specifically modified genomes. The mutation frequency of
0 6-methylguanine, however, at the specific site of modification was only
0.04% in E. coil. In contrast, when the lesion was present in singlestranded (ss) DNA the mutation frequency increased by an order of
magnitude to 0.4% (Loechler et al., 1984). The reasons that DNA lesions
within singly-modified duplex DNA yield such low mutation frequencies are
believed to include 1) preferential repair of double-stranded DNA, and/or 2)
the specific loss of the damaged strand when polymerase blocking lesions
are present in one strand of a duplex genome ("strand-bias")
(Koffel-Schwartz et al., 1987; Thomas et al., 1994). Thus, most of the
mutational studies listed above have employed the use of single-stranded
site-specifically modified genomes. These genomes, however, were
prepared to contain chemically stable DNA adducts. Unfortunately, the
chemical lability of AFB,-N7-Gua has precluded the use of traditional
procedures (Yarema and Essigmann, 1995) for the construction of ss singlymodified genomes containing this adduct.
The work presented in this dissertation has focused on the development
of a strategy for the construction of a ss viral genome containing the
unstable AFB,-N7-Gua adduct in a defined position. Two genomes were
constructed: one containing an AFB,-N7-Gua adduct, and one containing an
AP site in a specific location within the M13mp7L2 bacteriophage genome.
These genomes were then used to determine the relative contributions of the
primary AFB,-N7-Gua adduct and the AP site to the mutagenic spectrum of
AFB, in E. coli.
Figure 1. The structure of aflatoxin B1 (AFBj).
20
Aflatoxin B1
W
Figure 2. Reversed-phase HPLC analysis of DNA adducts formed in rat liver
by AFB 1 . The chromatogram represents rat liver DNA hydrolysate 2 h after
administration of [3H] AFB 1 at a dose of 0.6 mg/kg. These data were
adapted from Croy and Wogan (1981a) with permission.
22
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Figure 3. Three methods used typically for the construction of sitespecifically modified genomes.
24
Eco RI
AK Site
viral or
plasmid
M13mp7L2
genome
viral genome
Adduct-containing
oligonucleotide
1)Eco RI
NI
DNA
2) Anneal
scaffold
single-stranded
viral genome
Anneal
DNA
ligase
ligase
DNA
polymerase
adN I S
DNA ligase
L1
denature
opposite strand
remove
scaffold
Ssingle-stranded
DNA
double-stranded
DNA
Examine
- Biological
Effects
25
II. Literature Review
26
In this section I shall review studies involving the discovery of the
aflatoxins, the interaction of aflatoxin B, (AFB 1) with DNA, and the genetic
consequences of AFB,-induced DNA lesions in both Escherichia coli and
mammalian systems.
A. Historical Perspective on Aflatoxin
The aflatoxins were first discovered in England in 1960 as a result of an
outbreak of a disease that killed thousands of young turkeys, pheasants, and
ducks (Goldblatt, 1969; Busby and Wogan, 1984). This disease was known
as "turkey X disease" and was characterized by extreme fatigue, loss of
appetite, and death within one week. Examination of these birds subsequent
to death revealed hepatic necrosis and hemmorhage and engorgement of the
kidneys. Since veterinary examination failed to detect biological
transmission of the disease, bacterial or viral infection was ruled out. It was
therefore thought that the feed was contaminated with a toxic chemical.
The contaminant was eventually traced to a shipment of peanut meal that
was shown to be highly toxic to chicks and ducklings, and eventually
carcinogenic to rats (Lancaster et al., 1961). Microscopic examination of
the toxic meal indicated that it contained a fungal contaminant, eventually
determined to be Aspergillus flavus (Sargeant et al., 1961). The toxic
metabolite produced by the fungus was designated "aflatoxin" from the
contraction of "A. flavus toxin."
27
B. Aflatoxin Exposure and Human Disease
1. The occurrence of aflatoxin in human foods
Human populations are exposed to aflatoxin by the consumption of foods
that have been directly contaminated by toxigenic strains of A. flavus.
Aflatoxin is most commonly found as a contaminant in agricultural
commodities that may be consumed by humans, such as corn, other grains,
peanuts, other legumes, fruits and vegetables, and spices; all of these are
most likely contaminated by A. flavus during growth, harvesting, or storage
(Busby and Wogan, 1984). Aflatoxin has also been found as a contaminant
of meat, milk, eggs, and other dairy products derived from animals that had
consumed aflatoxin-contaminated food stuffs (Busby and Wogan, 1984).
Another human health risk may be associated with occupational exposure to
dusts from aflatoxin-contaminated grains during harvest, storage, or
processing (Busby and Wogan, 1984).
The guidelines for aflatoxin contamination in human food products in the
United States were set in the 1970's at 20 pg aflatoxin/kg (20 ppb) (Busby
and Wogan, 1984). Investigations of food samples from Africa and Asia
during the late 1960's indicated aflatoxin in food stuffs at levels significantly
higher than the acceptable levels later determined for the United States.
Aflatoxin levels in peanuts and corn from Mozambique, Uganda, Thailand,
28
the Philippines, and Guatemala well exceeded 100 pg/kg (Busby and Wogan,
1984). It was believed that high temperature and humidity, and the lack of
proper storage conditions were major factors associated with aflatoxin
contamination in these areas of the world, since these conditions are
conducive to fungal growth.
2. Human disease
Dietary exposure to aflatoxin is associated with an increased incidence of
hepatocellular carcinoma (HCC) particularly in Southeast Asia and subSaharan Africa where climate and food storage conditions are often
favorable for fungal growth. In these areas, including Uganda, the
Philippines, Swaziland, Kenya, Thailand, and Mozambique, the incidence of
HCC has been statistically associated with the level of aflatoxin ingestion
(Wogan, 1975; Busby and Wogan, 1984). Aflatoxin ingestion has been
quantitatively measured in populations from these geographical regions, and
in each instance elevated cancer incidence was associated with high levels
of aflatoxin intake.
A strong correlation has also been established between HCC and
exposure to hepatitis B virus (HBV) infection (Peers et al., 1987; Beasley,
1988; Yeh et al., 1989). Areas of high incidence of HBV in Africa and Asia
coincide with areas where aflatoxin contamination of the human food supply
29
is elevated. These studies have suggested that upon a background of
uniformly high levels of HBV infection, the levels of exposure to aflatoxin
could be a critical determinant in the role of aflatoxin in the development of
HCC. A study was carried out by Cova et al. (1990) where a Pekin duck
model was used to examine the effect of congenital duck hepatitis B virus
(DHBV) infection and aflatoxin exposure in the induction and development of
liver cancer. Higher mortality and a higher incidence of liver tumor
development was observed in ducks infected with DHBV and treated with
aflatoxin than in noninfected ducks treated with aflatoxin. The correlation of
aflatoxin exposure and HBV infection to hepatocellular carcinoma indicates
that the elucidation of the pathogenesis of HCC must take into consideration
investigations involving the interrelationship of aflatoxin and HBV infection.
Recently there have been investigations suggesting that the "X protein"
produced by HBV interacts with the p53 tumor suppressor gene product,
possibly inhibiting normal functions of the protein, such as transcriptional
activation of specific genes and DNA repair processes (Unsal et al., 1994;
Wang et al., 1994). This interference may contribute to the induction of
HCC in AFB,-exposed individuals.
30
C. Metabolism of Aflatoxin
1. Metabolic activation of AFB,
The toxic substances produced by A. flavus were extracted and isolated
from the fungus, and the structures identified in the laboratory of G. N.
Wogan (Asao et al., 1963; Chang et al., 1963; Asao et al., 1965). Four
major aflatoxin compounds were produced (Figure 4): aflatoxins B1 and B2
(AFB 1 and AFB 2) were so named owing to their strong blue fluorescence
under ultraviolet light, whereas aflatoxins G, and G2 (AFG, and AFG 2)
fluoresced green under ultraviolet light (Asao et al., 1963). AFB, was shown
to be, by far, the most abundant of the four compounds in aflatoxin
contaminated food stuffs (Busby and Wogan, 1984). Approximately 75% of
the total aflatoxins in contaminated corn samples consisted of AFB 1. The
next most abundant was AFB 2 (-20%), followed by AFG, (-5%), and AFG 2
(<1%). AFB, and AFG, possess an unsaturated bond at the 8,9 position on
the terminal furan ring, whereas essentially inactive AFB 2 and AFG 2 are
saturated at this position.
Studies on AFB,-induced carcinogenesis have focused on its interaction
with cellular macromolecules, especially DNA. As with most xenobiotics,
AFB, requires metabolic conversion to an electrophilic intermediate in order
to cause chemical damage to DNA (Miller, 1978). Early work carried out in
the laboratory of E. C. Miller and J. A. Miller (University of Wisconsin),
where 8,9-dihydro-8,9-dihydroxy-AFB, (referred to as the dihydrodiol) was
isolated from an acid hydrolysate of the rRNA-aflatoxin adduct formed by
hepatic microsomal oxidation of AFB 1, indicated that oxidation of the 8,9
double bond of the terminal furan ring of AFB 1 was likely to be the critical
step in metabolic activation of AFB, (Swenson et al., 1973; Swenson et al.,
1974). It was later shown that oxidative metabolism of AFB 1 does indeed
occur at the 8,9 double bond of the terminal furan ring giving rise to its
reactive intermediate - the AFB,-8,9-epoxide (Swenson et al., 1977;
Essigmann et al., 1977) (Figure 5). The existence of the epoxide and
definition of its stereochemistry as the exo diastereomer was further
confirmed on the basis of structural studies carried out on the DNA-bound
forms of the carcinogen (Essigmann et al., 1977; Martin and Garner, 1977;
Lin et al., 1977; Croy et al., 1978) (see next section).
The metabolic activation of AFB, has been shown to require cytochrome
P450 enzymes. In studies performed by Aoyama et al. (1990), 12 forms of
human hepatic cytochrome P450 were expressed in hepatoma cells by
means of recombinant vaccinia viruses. Five forms, P450s IA2, IIA3, 1IB7,
IIIA3, and IIIA4, were shown to activate AFB, to mutagenic metabolites as
assessed by His revertants of Salmonella typhimurium, with IllA4 being the
most efficient. Recent studies carried out by Ueng et al. (1995), employed a
32
reconstituted system containing bacterial recombinant (human) P450 IllA4
and bacterial recombinant (human) P450 IA2 to examine the oxidation
products of AFB 1 . Their data are consistent with previous studies in which
the view is that P450 IIIA4 is more active than P450 IA2 in forming the
genotoxic AFB,-exo-8,9-epoxide (Shimada and Guengerich, 1989; lyer et al.,
1994); in fact P450 IIIA4 did not activate any AFB 1 to the endo-epoxide.
Studies have shown the endo epoxide to be essentially nongenotoxic in
bacterial mutagenicity assays (lyer et al., 1994). P450 IA2, however, was
shown to form both the exo- and the endo-8,9-epoxide. These data suggest
that P450 IIIA4 is a major human liver P450 enzyme involved in AFB 1
activation to its electrophilic intermediate, AFB,-exo-epoxide, prior to its
reaction with DNA.
2. AFB 1 detoxification
There is considerable evidence to suggest that conjugation of AFB,-exoepoxide with glutathione (GSH) is an important step in AFB 1 detoxification.
An important protective mechanism against AFB 1 cellular damage involves
the glutathione S-transferase (GST) catalyzed reaction of the 8,9-epoxide
with glutathione and the subsequent excretion of the conjugate in the bile
(Moss et al., 1985). Hence, a significant factor for assessing the overall
toxicological hazard to an organism subsequent to AFB 1 exposure is the
balance between the activation (P450 IIIA4) and detoxification (GST)
33
pathways (Gorelick, 1990). A comparison of AFB 1 exo- and endo-epoxide
glutathione conjugation by rat, mouse, and human glutathione S-transferases
was carried out by Raney et al. (1992).
In both rats and humans, the endo-
epoxide as well as the exo-epoxide were found to be substrates for GSH
conjugation. Mouse liver cytosol, however, conjugated the exo-epoxide
almost exclusively. This is consistent with another study in which high GST
activity toward AFB,-exo-epoxide in mice was observed (Monroe and Eaton,
1988). High GST activity toward AFB,-exo-epoxide is believed to be
correlated to the observation that mice are resistant to AFB,-induced
hepatocellular carcinomas (Akao et al., 1971). Recently a correlation has
been seen regarding a higher risk of HCC development in individuals
harboring mutations in GST and epoxide hydroxylase (EPHX) genes
(McGlynn et al., 1995); EPHX is another AFB 1 detoxification gene.
Increased AFB,-exo-epoxide-GSH conjugation has been postulated to
account for the ability of a compound known as oltipraz [5-(2-pyrazinyl)-4methyl-1,2-dithiol-3-one] to provide protection against AFB 1 toxicity and
carcinogenicity (Kensler et al., 1987). Oltipraz is a known chemoprotective
agent that inhibits carcinogenesis of the stomach, colon, bladder, skin, and
breast (Kensler et al., 1992). Its protective action is thought to involve the
induction of important electrophilic detoxifying enzymes, such as epoxide
hydrolase and GST (Bueding et al., 1982). Subsequent studies have
34
confirmed the chemoprotective activity of oltipraz and its unsubstituted
analog, 1,2-dithiole-3-thione, against AFB,-induced hepatocellular carcinomas
in male F344 rats (Groopman et al., 1992; Roebuck et al., 1991; Kensler et
al., 1992; Bolton et al., 1993). More recent studies (Egner et al., 1995)
indicated that long-term intervention with oltipraz produced a significant
reduction in aflatoxin-albumin biomarkers at all times during AFB1 exposure.
In contrast, this decrease was not observed in a delayed transient
intervention with oltipraz until the ninth day of intervention; this difference,
however, was maintained for the duration of AFB, exposure. In another
study it was shown that the chemopreventive efficacy of oltipraz in the
reduction of AFB,-induced tumorigenesis was correlated to a persistent
induction of GSTs in the liver following multiple and single dosing of the drug
(Primiano et al., 1995). Collectively, these results support the use of oltipraz
in chemopreventive interventions in individuals residing in high risk areas for
aflatoxin exposure and hepatocellular carcinoma development.
Another enzyme, aldehyde reductase, with known activity towards a
metabolite of AFB1 is expressed in rat liver during carcinogenesis (Hayes et
al., 1993; Judah et al., 1993). AFB,-aldehyde reductase (AFB,-AR) was
shown to convert AFB,-dihydrodiol to AFB,-dialcohol (Judah et al., 1993)
(Figure 6). AFB,-dihydrodiol is capable of binding to protein via Schiff-base
formation and it has been suggested that this reaction is involved in AFB 1
35
cytotoxicity (Neal et al., 1981). Thus, AFB 1-AR may have significant AFB 1
detoxifying potential by converting the dihydrodiol to its inactive dialcohol
form, thus preventing AFB,-protein adducts.
D. Interactions of AFB1 with DNA
1. AFB,-DNA adducts
As discussed above, the reactive intermediate of AFB 1 that is presumed
to be responsible for its interaction with DNA is AFB,-exo-8,9-epoxide
(Figure 5). Structural identification of the epoxide and the major DNA adduct
formed by aflatoxin B1 was determined in vitro by Essigmann et al. (1977).
In this study, rat liver microsomes were used for the metabolic activation of
[3H]AFB 1 which was allowed to react with calf thymus DNA. Upon acid
hydrolysis of the DNA, an aflatoxin guanine-derivative was efficiently
liberated. Analytical reversed-phase HPLC of the DNA hydrolysate revealed
that approximately 90% of the aflatoxin-bound DNA eluted as a single peak.
Subsequent spectral and chemical studies of the material represented in this
peak indicated that the major product of the interaction of metabolically
activated aflatoxin with DNA is 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin
B, (AFB,-N7-Gua) (Figure 7). This study also revealed that the guanine and
hydroxyl functions possessed a trans configuration, indicating that the AFB 1epoxide was the exo-epoxide (discussed above). Similar studies were carried
36
out in vitro by Lin et al. (1977), Martin and Garner (1977), and Wang and
Cerutti (1980), in which the major acid hydrolysate of DNA treated with
metabolically activated AFB 1 was AFB,-N7-Gua.
The in vivo interaction of activated AFB 1 with DNA was also examined in
a number of studies in which the products of covalent binding of AFB 1 were
isolated from rats given injections of the carcinogen. In one study (Croy et
al., 1978), male Fischer rats were dosed with 2.0 mg AFB,/kg body weight,
and several hours later their liver DNA isolated and acid hydrolyzed. The
principal covalent product formed in rat liver DNA was AFB,-N7-Gua,
identical to that produced in vitro in the presence of a rat liver microsomal
activating system (discussed above). This study also revealed a dosedependent relationship between the level of the occurrence of AFB,-N7-Gua
in liver DNA and AFB 1 dose (0.125 - 1.0 mg/kg body weight). Work carried
out by Lin et al. (1977), in parallel with in vitro studies, also showed that the
major acid hydrolysis product of AFB 1-DNA or -ribosomal RNA adducts, from
rats given injections of [3H]AFB,, cochromatographed on HPLC with the
major adduct that was formed in vitro with metabolically activated AFB,,
AFB,-N7-Gua. In additional studies, AFB 1-DNA adduct formation has been
examined in rat liver following exposure by aerosol inhalation (Zarba et al.,
1992). Rats were exposed to aerosol inhalation of AFB 1 for up to 120 min,
and their DNA isolated and analyzed for AFB 1-DNA adducts. A linear dose-
37
response relationship was observed between increasing length of exposure,
and the amount of AFB,-N7-Gua adducts formed per milligram of DNA. The
aforementioned studies indicate that AFB,-N7-Gua is the major AFB 1-induced
DNA adduct formed in rat liver DNA by both ingestion and inhalation.
Interestingly, the liver is the primary site of pathological effects of
aflatoxin in the rat (Newberne and Butler, 1969), whereas in the mouse, the
most sensitive tissue to aflatoxin toxicity is the kidney (Akao et al., 1971).
Comparative investigations of AFB 1-DNA adducts formed in rat and mouse
livers and kidneys were carried out by Croy and Wogan (1981b). In this
study, a positive correlation was found between levels of covalent AFB,DNA modification and organotropic effects of aflatoxin. Much higher levels
of AFB,-N7-Gua were observed in the livers of rats than in the kidneys. In
mice, however, higher levels of AFB,-N7-Gua were observed in the kidney
than in the liver. Additional in vitro kinetic studies indicated that mouse liver
microsomes are limited in their capacity to activate AFB 1 (Croy and Wogan,
1981b). Other studies, however, have indicated that the resistance of mice
to AFB,-induced liver carcinoma is correlated to a high GST activity toward
AFB,-exo-epoxide (Monroe and Eaton, 1988; Raney et al., 1992).
AFB 1-DNA binding and adduct formation have been studied in other
biological systems as well. Salmonella typhimurium were exposed to AFB 1 in
38
culture in the presence of rat liver postmitochondrial supernatant (Stark et
al., 1979). Results indicated that 70% of the AFB 1 bound to DNA was
chromatographically (HPLC) identical to AFB,-N7-Gua. The AFB,-N7-Gua
adduct is also the major DNA adduct generated in human cells exposed to
AFB 1, such as human bronchus and colon cells (Autrup et al., 1979), and
epithelioid human lung cells (Wang and Cerutti, 1979).
The interaction of AFB 1 with DNA has been shown to involve an initial
intercalation event. Spectroscopic 1H NMR studies have been utilized to
examine the conformation of AFB 1 within DNA (Gopalakrishnan et al., 1990).
NOE (Nuclear Overhauser Effect) contacts within oligonucleotides
d(ATCAFBGAT)-d(ATCGAT) and d(ATAFBGCAT) 2 revealed that the aflatoxin
moiety is intercalated on the 5'-face of the modified guanine. These data
provide evidence for a reaction mechanism that involves an intercalated
transition complex between aflatoxin B, 8,9-epoxide and B-DNA.
2. Chemical stability of AFB,-N7-Gua
The chemical stability of AFB, bound to DNA was investigated in vitro
(Wang and Cerutti, 1980; Groopman et al., 1981). Like other adducts
formed at the N7 position of guanine, AFB,-N7-Gua in DNA is expected to be
unstable due to the positive charge in the imidazole ring of the guanine
(Figure 7). In studies carried out by Wang and Cerutti (1980), the half-life of
39
AFB,-N7-Gua in DNA was examined at 37 C under pHs 6.7, 7.0, and 7.3.
[3H]AFB,-DNA was prepared by the reaction of [3H]AFB, with calf thymus
DNA in the presence of rat liver microsomes, and incubated under the above
conditions for various amounts of time. The half-life was determined by acid
hydrolysis of the DNA at various time points. The half-life of AFB,-N7-Gua
was determined to be 19 h at pH 6.7, 12 h at pH 7.0, and 8 h at pH 7.3.
The major reaction pathways found to be responsible for the decrease in
AFB,-N7-Gua bound to DNA under physiological conditions were 1) the
release of the AFB 1 dihydrodiol from DNA, 2) the release of AFB,-N7-Gua
from DNA, and 3) the appearance of the putative 8,9-dihydro-8-(2,6diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy AFB 1 (AFB,formamidopyrimidine adduct, or AFB 1-FAPY) in the acid hydrolysates of
AFB 1-DNA (see Figure 7). At this time, the structure of the imidazole ringopened AFB 1-FAPY adduct had been tentatively identified by Lin et al.
(1977); the structure was further established by studies performed by
Hertzog et al. (1982), but to this day a fully satisfactory structural
determination of AFB 1-FAPY has not been produced. The proposed AFB 1FAPY adduct was determined to be chemically more stable than AFB,-N7Gua, and the adduct was observed only at pHs greater than 7.0. Additional
studies carried out by Groopman et al. (1981) examined the stability of
[14C]AFB, and [3H]AFB1 bound to calf thymus DNA over a 48 h incubation
period in phosphate buffer at pH 6.8-8.0 (37 °C). This study examined AFB,-
40
N7-Gua stability in DNA modified with AFB, at two different levels: one
residue per 60 nucleotides, and one residue per 1500 nucleotides. Aliquots
of AFB,-modified DNA were removed at various timepoints, acid-hydrolyzed
and analyzed for the presence of the various AFB 1-N7-Gua-derived
structures. The quantitative distribution of the resulting products released
from the DNA was dependent upon the pH and the level of modification. At
neutral or slightly acidic pHs and at a low level of modification (one adduct
per 1500 bases) the AFB,-N7-Gua was considerably stable, with a half-life of
100 h. As the level of modification increased to one adduct per 60 bases
the half-life decreased to 48 h. Furthermore, as the alkalinity of the aqueous
solution increased so did the rates of formation of the AFB,-FAPY. The
AFB,-FAPY adduct, however, was generated to the same extent in DNAs
modified once in 60 bases and once in 1500 bases (- 70-80% of the acidhydrolyzed products). In addition, the spontaneous depurination rate (halflife) of AFB,-N7-Gua, examined at neutral pH with DNA modified at one
adduct per 1500 bases, was determined to be approximately 50 h.
AFB 1-DNA adduct stability was also examined in vivo. The patterns of
covalent AFB 1-DNA adducts in the livers of male Fischer rats were examined
after exposure to single and multiple doses of AFB, (Croy and Wogan,
1981a). AFB,-N7-Gua was shown to be the principal adduct formed after a
0.6 mg/kg dose was administered (see Chapter I, Figure 2); it represented
80% of the covalent products present 2 h after dosing. During the first 24
h, approximately 70% of AFB,-N7-Gua was removed from DNA with a halflife corresponding to 7.5 h. Twenty percent was converted to the more
stable AFB 1-FAPY derivative, which remained relatively constant over the
next 48 h. The other remaining minor adducts were due to activated AFB1
hydroxylated metabolites, aflatoxin M, (4-hydroxy-AFB,) and aflatoxin P, (the
O-demethylated derivative of AFB,) (Figure 8). The 8,9 double bond of these
metabolites is not altered, leaving it available for oxidation at that position.
The in vitro depurination rate of AFB,-N7-Gua, discussed above, was
determined to be 50 h (Groopman et al., 1981). The half-life of this lesion in
rat liver is much shorter (7.5 h), suggesting that its removal may be
facilitated enzymatically either by depurination or some other repair pathway.
Similar kinetics for the removal of AFB,-N7-Gua lesions from the DNA of
epithelioid human lung cells had been observed prior to this study (Wang and
Cerutti, 1979), where 60% of the initial AFB, adducts were rapidly removed
during the first 24 h of exposure, while only 15% of the adducts were
removed from AFB,-DNA incubated for the same time period under
physiological conditions in vitro.
The aforementioned studies indicate that the primary AFB1 DNA adduct
(AFB,-N7-Gua) is chemically unstable due to the positive charge in the
imidazole ring of the guanine. The adduct can breakdown via two primary
42
pathways (Figure 7), one involving depurination of the AFB,-N7-Gua residue
resulting in apurinic (AP) site formation; this reaction may be facilitated
enzymatically in vivo. Alternatively, under physiological or sightly basic
conditions the imidazole ring of AFB,-N7-Gua can open to form the
chemically and biologically stable AFB1 -formamidopyrimidine adduct (AFB 1FAPY). In addition, a small percentage of the AFB,-N7-Gua adducts are
released as the AFB1 dihydrodiol yielding an unmodified guanine residue; this
reaction is unlikely to pose a potential detriment to the integrity of the DNA.
Hence, the initial AFB,-N7-Gua adduct, the AFB 1-FAPY and the AP site,
individually or collectively, represent the likely chemical precursors to the
genetic effects of AFB, .
E. Biological Effects of Aflatoxin B, in Bacterial Cells
1. Repair of AFB,-induced DNA damage
Several studies in E. coil provide evidence that AFB1 adducts are
eliminated from DNA by a repair pathway known as nucleotide excision DNA
repair (NER). Nucleotide excision repair in E. coli requires the gene products
of uvrA, uvrB, and uvrC, see reviews (Sancar and Sancar, 1988; Van
Houten, 1990; Sancar, 1994; Friedberg et al., 1995). These genes are part
of the SOS response system, a system induced by E. coli to enable it to
escape the genotoxic effects of helix distorting DNA lesions (Walker, 1984;
43
Walker, 1985; Friedberg et al., 1995). The repair of helix distorting DNA
adducts is initiated by the UvrABC excision complex (UvrABC endonuclease).
This complex incises the lesion-containing DNA at sites flanking the lesion,
thus releasing an oligonucleotide of approximately 13 bases in length
containing the damage. UvrA and UvrB are involved in DNA damage
recognition, and UvrC is involved in the incision step. This repair pathway
has demonstrated the ability to excise a wide variety of bulky DNA lesions,
including psoralen (Sancar and Rupp, 1983), AAF (Tang et al., 1982),
mitomycin C (Otsuji and Murayama, 1972), cisplatin (Alazard et al., 1982),
and thymine dimers (Sancar and Rupp, 1983). In previous studies, Sarasin
et al. (1977) showed that E. coli treated with metabolically activated AFB1
required the gene products of uvrA and recA for survival. Additional studies
have demonstrated that AFB, is mutagenic in double stranded DNA in E. coli,
but only in the absence of the uvrB gene product (Foster et al., 1983), or the
uvrA gene product (Sambamurti et al., 1988; Sahasrabudhe et al., 1989). A
study carried out by Oleykowski et al. (1993) tested whether AFB,-N7-Gua
and AFB 1-FAPY lesions were removed equivalently by the UvrABC
endonuclease. Their results indicated that both the AFB,-N7-Gua and AFB,FAPY lesions were removed in an identical fashion by UvrABC. This removal
was shown to be similar to the removal of other bulky DNA lesions by
UvrABC; one incision was at the eighth phosphodiester moiety 5', and the
other was at the sixth phosphodiester moiety 3' of the modified nucleotide.
44
Although AFB,-N7-Gua is removed efficiently in rats treated with aflatoxin
(Croy and Wogan, 1981a), the AFB 1-FAPY lesion is known to persist in the
DNA. There is evidence to suggest that AFB,-FAPY and AFB,-N7-Gua
lesions are not repaired equally by mammalian nucleotide excision repair
systems as they apparently are by E. coli systems (Leadon et al., 1981).
This may partially explain the persistence of AFB 1-FAPY in mammalian DNA.
In addition to NER, another repair system is believed to be involved in the
removal of AFB,-FAPY lesions from E. coli DNA. AFB 1-FAPY has been
shown to be excised from DNA by E. co/i formamidopyrimidine (FAPY)-DNA
glycosylase activity (Chetsanga and Frenette, 1983). Its removal from the
DNA of mammalian cells by an analogous mechanism has not been
determined. A FAPY-DNA glycosylase is known to exist, however, in rodent
cells (Margison and Pegg, 1981), but its role in AFB,-FAPY repair as of yet is
unknown.
2. SOS response in E. coli
In studies carried out by Baertschi et al. (1989), treatment of E. coli with
metabolically activated AFB, was shown to induce expression of the umuC
gene product. Since umuC is a DNA damage-inducible gene that is part of
the SOS response system (Walker, 1984), its induction by AFB, strongly
suggested that other SOS gene products were involved in inducing the
45
genetic effects of AFB1 . The SOS response system is controlled by the gene
products of lexA and recA, see Walker (1985) and Walker (1984) for
reviews. The LexA protein serves as a repressor of the SOS gene products.
Exposure of cells to DNA damaging agents generates an inducing signal that
activates RecA molecules. When activated RecA interacts with LexA,
cleavage of the LexA molecule is promoted, thus inactivating its repressor
activity and allowing expression of the SOS gene products. The UmuDC
proteins are a set of proteins that are expressed upon induction of the SOS
response in E. coli (Walker, 1984; Walker, 1985). These proteins were
originally believed to be involved in an "error-prone repair pathway." The
existence of this type of processing was first observed upon genetic
experiments that indicated that certain cellular functions, induced by DNA
damage, were necessary in order to observe mutations as a result of certain
DNA damages. Mutations in umuD and umuC have been shown to abolish
the ability of E. col to be mutated by certain agents such as UV light, methyl
methanesulfonate, and neocarcinostatin. It is believed that these proteins
somehow enable DNA polymerases to bypass an otherwise blocking, or
lethal DNA lesion, but at the expense of possibly fixing a mutation at the
modified base.
Several naturally occurring plasmids, such as pKM101, contain analogs
of the umuDC genes and have been shown to restore mutability to strains
46
deficient in UmuDC (Walker, 1985). The umuDC gene analogs present on
plasmid pKM101 are known as mucA and mucB (Walker, 1984; Walker,
1985). The MucAB proteins have been shown to enhance the yield of
mutants in umuDC' strains and appear to be generally more active than
UmuDC in promoting SOS mutagenesis. The UmuC and MucB proteins are
- 55% homologous, and UmuD and MucA proteins are - 30% homologous. It is
not clear whether these differences reflect differences in the biochemical
functions of the two sets of proteins. Studies carried out on DNA globally
modified with AFB 1 have shown a strong MucAB dependence for
mutagenesis (Foster et al., 1988; Urios et al., 1994). The exact
mechanisms of mutagenic processing by UmuDC and MucAB are unclear,
but there do appear to be differences between the two that may be
correlated to different levels of mutagenic processing (Hauser et al., 1992;
Blanco et al., 1986). Activated RecA protein is known to promote
proteolytic cleavage of UmuD (Shinagawa et al., 1988; Burckhardt et al.,
1988; Nohmi et al., 1988) and MucA (Shiba et al., 1992). The interaction of
RecA with UmuD and MucA is believed to be responsible for promoting
certain mutagenic processes of these proteins. It has been suggested that
the functional differences between UmuDC and MucAB proteins might be
due to their different dependence on the role of RecA protease (Blanco et al.,
1986; Hauser et al., 1992; Shiba et al., 1992). Studies carried out by Shiba
et al. (1992) have shown that strains containing mutant MucA protein
47
(harboring a threonine instead of an alanine at the cleavage site) do not
process MucA to its active form MucA', but are still as proficient as strains
containing wild-type MucA in promoting UV mutagenesis. These results
have raised the possibility that both intact MucA and RecA-processed MucA
(MucA') are active in mutagenesis. On the other hand, studies carried out
by Hauser et al. (1992), in which a very sensitive method was employed for
the detection of the cleavage product of MucA (MucA'), have shown that
MucA is cleaved much more readily than UmuD, and that restoration of
mutagenesis functions to normally nonmutable RecA-deficient strains by
MucA protein is correlated with the appearance of the cleavage product,
MucA'. These results suggest that the differences in mutagenic processing
exhibited by MucAB and UmuDC are correlated to the efficiency of
posttranslational processing of MucA and UmuD rather than an intrinsic
mutagenic-processing phenotype of the intact MucA protein. Both studies,
however, indicate that MucAB proteins appear to be less dependent than
UmuDC on the activity of RecA protease. Thus, if MucAB proteins perform
a similar role to UmuDC in the mutagenic process, then in the presence of
MucAB proteins the less stringent requirement for RecA protease may allow
for a more efficient bypass of DNA lesions.
3. Mutagenic consequences of AFB1 in bacterial cells
The mutagenic activity of aflatoxin B1 has been studied in the bacterial
48
strain Salmonella typhimurium (McCann et al., 1975). In this study, the
naturally occurring plasmid pKM101 (discussed above), was introduced into
S. typhimurium strains containing a hisG46 mutation. These strains were
then allowed to grow on a petri dish, in the presence of AFB1 and
mammalian liver extract for carcinogen activation. A mutation induced by
AFB 1 in hisG46 that reverts a histidine requirement back to prototrophy
results in growth on plates lacking histidine. This mutation assay is known
as the Ames Test, and has been used to examine the mutagenic activity of
hundreds of carcinogens. AFB 1 was shown to induce - 2000-fold more
revertants in tester strains containing pKM101 than in strains lacking the
mucAB-containing plasmid. In another study (Stark et al., 1979), S.
typhimurium containing pKM101 were incubated with aflatoxin B, in liquid
suspension culture in the presence of rat liver postmitochondrial supernatant,
and reversion to 8-azaguanine resistance measured. A null mutation within
the XPRT (xanthine phosphoribosyltransferase) gene of S. typhimurium
confers resistance to 8-azaguanine. AFB 1 was shown to induce reversions
to 8-azaguanine resistance, which increased linearly with AFB,
concentration. In addition, approximately 70% of the AFB, bound to DNA
was determined to be AFB,-N7-Gua.
The nature of mutations induced by electrophilic derivatives of AFB, has
been studied in several forward mutation assays in E. coHi. AFB,
49
mutagenesis was examined in the endogenous lacl gene in an SOS-induced
E. coil strain lacking uvrB and containing the mucAB mutagenesis enhancing
operon (Foster et al., 1983). The lacl system consists of characterized
codons within which specific mutations yield amber and ochre nonsense
codons. By monitoring - 70 of these sites, metabolically activated AFB 1 was
shown to specifically induce GC- TA transversions. A contrasting mutation
pattern was observed in studies carried out by Sambamurti et al. (1988),
where replicative form (RF) M13 DNA was modified in vitro with AFB,-8,9dichloride, an electrophilic derivative of the AFB 1-exo-8,9-epoxide. Forward
mutations were identified in the M13 lacZ a-complementing gene segment in
SOS-induced E. coil lacking uvrA and containing the mucAB locus. Both
GC--TA and GC--AT mutations were induced with equal efficiency by the
AFB 1-8,9-dichloride; a small fraction of frameshift mutations was also
observed. In the same study, the primary aflatoxin-guanine lesions of the
modified DNA were chemically converted to stable ring-opened AFB 1-FAPY
lesions prior to transfection into E. coli. AFB 1-FAPY was shown to be nearly
equal to the primary adduct in its ability to induce mutations; the FAPY
lesion also appeared to give rise to the same types of mutations as the
primary adduct. A more complete analysis of AFB,-8,9-dichloride induced
frameshift mutations was carried out by the same group (Refolo et al.,
1987). In this study both -1 and + 1 frameshifts were induced with
comparable efficiencies, in runs of G-C base pairs. The majority of + 1
50
frameshift mutations was the result of the addition of an A-T base pair at
these sites. Similar mutational studies were carried out, in the same
laboratory, with RF M13 DNA modified by metabolically activated AFB
1
(Sahasrabudhe et al., 1989). Again, both GC--TA and GC--AT mutations
were observed with equal efficiency. In contrast to the mutational spectrum
induced by the AFB 1-8,9-dichloride (Sambamurti et al., 1988; Refolo et al.,
1987), however, metabolically activated AFB, did not give rise to a
significant number of frameshift mutations.
F. Biological Consequences of Aflatoxin B, in Mammalian Systems
1. Repair of AFB,-induced DNA damage
Many studies provide evidence that AFB,-DNA adducts are removed from
the DNA of mammalian cells by the same mechanism that is responsible for
their removal from E. coli, nucleotide excision repair. Leadon et al. (1981)
have shown that AFB,-N7-Gua adducts are removed both spontaneously and
enzymatically from normal human fibroblasts (NF), and that their removal
from excision repair-deficient xeroderma pigmentosum skin fibroblasts of
Complementation Group A (XPA) is likely to be only spontaneous. In the
same study, subsequent to incubation with AFB,, cultures were incubated
under high pH (pH 8.2) in order to convert the AFB,-N7-Gua adducts to
AFB,-FAPY adducts. Cultures were then incubated to allow for DNA repair
processes to occur. No decrease in the amount of AFB 1-FAPY was observed
in either XPA or NF cells, indicating that AFB 1-FAPY adducts are not repaired
by NER in mammalian DNA. This lack of repair, along with the inherent
chemical stability of this adduct may explain the persistence of AFB,-FAPY
adducts in rat liver DNA, described above. Similar results were obtained by
Mahoney et al. (1984), where XP cells were significantly more sensitive than
normal cells to the cytotoxic and mutagenic effects of AFB,-dichloride
induced DNA damage. Again, the removal of AFB,-N7-Gua was more
efficient from normal cells than XP cells, and the AFB 1-FAPY adduct
persisted on the DNA in both repair proficient and deficient cells. More
recent investigations (Waters et al., 1992; Martin and Waters, 1992) of the
removal of AFB1 DNA adducts from excision-repair proficient and deficient
human and Chinese hamster ovary cells support the view that NER is the
repair pathway responsible for the removal of AFB,-N7-Gua adducts in
mammalian systems.
2. Mutagenic consequences of AFB1 in mammalian systems
AFB1 mutagenesis has been examined in XPA and DNA repair proficient
human cells upon the replication of an AFB,-modified pS189 shuttle vector
containing the supF marker gene (Levy et al., 1992). The plasmid was
prepared by its reaction with the AFB 1-8,9-epoxide (Baertschi et al., 1988).
Results indicated that AFB,-modified plasmid survival was lower and the
52
mutation frequency higher in XPA cells. Fifty percent of the mutations were
GC->TA transversions, 20% were GC-AT transitions, and 18% GC->CG
transversions. In work carried out by Trottier et al. (1992), Ad293 cells
were cotransformed with an expression vector harboring a rat liver P450 IA2
cDNA and the pS189 shuttle vector. Cells were then incubated with AFB 1
and the mutant sequences analyzed. The predominant mutations observed
in the supF marker gene were similar to that observed by Levy et al. (1992):
61% of the mutations were GC--TA transversions. The other mutations
consisted of GC->CG transversions (25%); and GC--AT transitions (9%).
There is substantial evidence that the activation of cellular ras (c-ras)
genes by specific single-base mutations may be an important step involved
in the transformation of normal cells to malignant cells (Bos et al., 1987;
Vogelstein et al., 1988). An activated c-Ki-ras oncogene was identified in
rat liver tumors induced by AFB 1 (McMahon et al., 1986). The nature of this
activation has been determined to arise from mutations in codon 12 of the cKi-ras gene in DNA isolated from rat liver tumors (McMahon et al., 1987;
McMahon et al., 1990; Soman and Wogan, 1993). The predominant
mutation observed in these studies appears to be a GC- AT transition at the
second position of codon 12 (GGT to GAT). Also present, but at a lower
frequency, were GC-TA transversions at both the first and second positions
of the codon (GGT to TGT and GGT to GTT). The ras gene of rainbow trout
53
liver tumors induced by AFB 1 was also analyzed for mutations (Chang et al.,
1991). In contrast to the mutations observed in the c-Ki-ras gene of rat liver
tumors, the predominant mutations observed in this study were GC-*TA
transversions at the second position of codon 12 (GGA to GTA). No GC- AT
transitions were observed at this position.
Recently, mutations in the p53 tumor suppressor gene of HCCs have
been examined from humans residing in southern Africa (Bressac et al.,
1991) and Qidong, China (Hsu et al., 1991), areas of the world where AFB 1
exposure is a frequent occurrence. Approximately half of these HCCs
contained GC- TA transversions in the third position of codon 249 (AGG to
AG7). Metabolically activated AFB 1 was also shown to induce a high
frequency of GC--TA transversions at the third position of codon 249 of the
p53 genes of human hepatocytes grown in culture (Aguilar et al., 1993). In
addition, but at a lower frequency, AFB 1 induced GC->TA transversions and
CG--AT transversions at adjacent positions (AGG to ATG and AGGC to
AGGA). Also observed, in the same study, at lower frequencies were
CG->AT mutations in codons 247 and 250, and GC-*TA mutations in codon
248. In another study, where AFB,-induced liver tumor DNA from
nonhuman primates was examined for mutations, GC--TA transversions in
codon 249 were not observed (Fujimoto et al., 1992). These data suggest
that the induction of mutations in codon 249 of the p53 gene, in the HCCs
54
discussed above, may have required other environmental factors in addition
to AFB1 exposure, such as exposure to hepatitis B virus.
The mutational spectrum of aflatoxin B1 was also examined in vitro in the
human hypoxanthine guanine phosphoribosyltransferase gene (HPRT)
(Cariello et al., 1994). AFB, produced a strong mutational hotspot in exon 3
of HPRT, consisting of a single GC--TA substitution at base pair 209
(GGGGGG to GGTGGG).
The genetic studies cited above indicate that several mutations occur in
DNA globally modified by activated forms of AFB,.
The predominant
mutation induced or selected in vivo and in vitro appears to be the GC->TA
transversion. At least three DNA lesions (Figure 7) - AFB,-N7-Gua, AFB1 FAPY, and the AP site - are candidates as the precursor(s) to the mutations
induced by aflatoxin. It has been reasonably suggested that the
premutagenic lesion responsible for the observed GC--TA transversions may
be the AFB,-induced AP site (Foster et al., 1983; Foster et al., 1988; Kaden
et al., 1987), since dAMP is the most common base inserted opposite AP
sites in E. coil induced for the SOS response (Loeb and Preston, 1986;
Lawrence et al., 1990). It is a formal possibility, however, that the parent
adduct, AFB,-N7-Gua, or the ring-opened AFB 1-FAPY adduct could also give
rise to this mutation.
55
G. Sequence Specificity of Aflatoxin B1 DNA Modification and Mutagenesis
The sequence specificity of AFB 1-DNA interactions has been examined in
vitro by the analysis of piperidine-labile sites of plasmid DNA modified with
metabolically activated AFB 1 (Muench et al., 1983). In another study,
oligonucleotides harboring guanine residues within a variety of flanking
sequences were modified with the AFB 1-8,9-epoxide; the extent of AFB1
modification of guanines within the different oligonucleotides was analyzed
by piperidine-lability (Benasutti et al., 1988). Sequence context effects of
AFB,-DNA modification were also determined by examining replication blocks
to DNA polymerase (Refolo et al., 1985; Marien et al., 1989), and also
replication blocks to DNA-dependent RNA synthesis (Yu et al., 1990). In all
of these studies GC rich sequences were much stronger sites for AFB,
modification than AT rich sequences. This is best described in studies
carried out by Benasutti et al. (1988), where it was determined that the
influence of the base at the position 5' to potentially-modified guanines is 5'G (1.0) > C (0.8) > A (0.3) > T (0.2), while the influence of the base at
the 3' position is 3'-G (1.0) > T (0.8) > C (0.4) > A (0.3).
More recent studies have examined whether hotspots for AFB 1-DNA
modification correlate to AFB,-induced mutation hotspots in vivo
(Sambamurti et al., 1988; Levy et al., 1992; Courtemanche and Anderson,
1994). While it was clear, in all of these studies, that most mutations are
56
targeted to G-C base pairs that are favored for damage, not all damage
hotspots were mutational hotspots. In another study, the mutational
spectrum of AFB 1 was examined in the supF gene of both pSP189 and
pS189 shuttle vectors (Courtemanche and Anderson, 1994). Both vectors
carry the supF gene but within different surrounding sequences. The AFB,induced mutational spectrum in the supF genes from each vector exhibited
some notable differences, indicating that sequence context effects appear to
operate not only locally, but over distances of tens of bases. In another
investigation, replication blocks induced by AFB,-dichloride adducts were
examined in codon 12 of the c-ras oncogene in vitro (Marien et al., 1989).
Although a predominance of point mutations has been observed in this
codon in the c-ras gene of DNA from rat liver tumors induced by aflatoxin
(Soman and Wogan, 1993), this was not a preferential blockage site for DNA
polymerase. Thus, it seems likely that differences in repair and bypass
efficiencies at different damage hotspots in vivo account for varying
sequence context effects for AFB,-induced mutagenesis. The mechanisms
controlling these sequence effects in vivo are not currently understood.
H. Biology of the M13 Vector Used in this Work
This dissertation describes the study of the mutational properties of
AFB,-N7-Gua through the use of a bacteriophage M13 vector containing the
AFB,-N7-Gua adduct in a specific location. The following is a brief
57
description of the life cycle of M13 and of specific characteristics of the
phage which allow it to be used in mutational studies described in this
thesis.
The vector used in this dissertation to study the mutagenesis of AFB,induced DNA lesions is the M13 bacteriophage system developed by
Messing (1983). M13 is a single-stranded (ss) filamentous bacteriophage
that infects E. coli that harbor an F factor plasmid, which allows for F' pilus
formation. Upon infection (see Figure 9), the phage adsorb to the tip of an F
pilus on the E. coil cell, where it then releases its DNA directly into the
cytoplasm of its host. During this process the coat protein of the
bacteriophage is removed. The infecting ss DNA is immediately used as a
template to produce a double-stranded replicative form (RF) DNA molecule.
This RF DNA is replicated by a rolling circle mechanism to build a pool of RF
molecules. Host-encoded enzymes and the viral gene II product are
responsible for this process. Gene II is responsible for nicking the (+) strand
of the newly synthesized RF molecule so that a 3' terminal hydroxyl group is
available for replication by host cell machinery. The (+) strand is displaced
as rolling circle replication occurs, until the polymerase returns to the origin.
At this point the newly displaced (+) strand is cleaved and its 5' and 3'
ends ligated. The RF molecule is also sealed, and can now serve as a
template for another round of replication. In the early stages of
58
bacteriophage infection (first 15 - 20 min), newly displaced (+) strands are
complemented to RF molecules by host cell machinery, and serve as
additional templates for RF DNA synthesis. Later in the infection stage, the
viral protein product of gene V brings about a switch from RF replication to
ss synthesis by binding to the newly formed viral (+) strands. The protein
coated strands are translocated to the cell membrane, where the viral DNA is
packaged in a protein coat as it is extruded from the cell. Bacteriophage
M13 does not lyse its host, but infected cells do grow more slowly than noninfected cells.
The M13mp series of vectors developed by Messing contains a portion of
the E. coil lacZ gene within an intergenic region of the M13 genome. pGalactosidase is the product of the lacZ gene of E. coli. The gene is
comprised of two fragments, the alpha fragment and the omega fragment.
Neither fragment alone is sufficient for enzyme activity, but the two can
associate to produce functional protein. These M13mp genomes encode the
alpha complement of 8-galactosidase. Certain E. coil host cells, in which the
lacZ gene has been deleted, carry the omega fragment of the lacZ gene on
their F factor plasmid. Thus, when M13mp phage are plated with
complementing host cells in the presence of isopropyl /-Dthiogalactopyranoside (IPTG), an inducer of the operon, and 5-bromo-4chloro-3-indoyl Pf-D-galactopyranoside (X-Gal), a substrate for f-
59
galactosidase, active enzyme is formed which cleaves X-Gal and results in
deep blue plaque color.
M13mp bacteriophage DNA contain a small region within the alpha
complement of the lacZ' fragment known as the "polylinker region" into
which small fragments of DNA can be inserted without adverse effects to
phage viability. This region of the M13 genome is commonly manipulated by
genetic engineering techniques to accommodate particular genes or
sequences of interest so that they may be studied in the context of various
genetic backgrounds of different host E. coil strains.
60
Figure 4. The chemical structures of aflatoxin B1 (AFB 1 ), aflatoxin B (AFB ),
2
2
aflatoxin G, (AFG,), and aflatoxin G2 (AFG 2 ).
6d
CM
oim
x
0
C=
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umm
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62
Figure 5. Aflatoxin B, (AFB1 ) is activated in the liver by cytochrome P450 to
generate its exo-epoxide.
63
00 1
O
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E
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IPP
Cr
0
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Figure 6. Proposed pathway for metabolic formation of the AFB1 -dialcohol
(Judah et al., 1993).
65
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66
Figure 7. The epoxide reacts with DNA to form the primary AFB, adduct,
AFB,-N7-Gua, which can undergo depurination to the apurinic (AP) site,
opening of the imidazole ring to form AFB,-formamidopyrimidine (AFB,FAPY), or release of AFB,-dihydrodiol to yield an unmodified guanine.
67
I
'I.O'
ri5
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68
Figure 8. Hydroxylated metabolites of aflatoxin B13: aflatoxin M, and
aflatoxin P1 .
69
OT
CL
0
C
0
(U
70
Figure 9. The life cycle of M13. M13 replicates via rolling circle replication
(see text for details).
bacteriophage
M13 DNA
bacteriophage
M13
~I~
_CIC_
--C ·-
.
_
_
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(+)
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__
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I
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nCQUMO
early stage
late
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host cell
replication
apparatus
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I
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72
S
I
IIIl. Preparation of AFB,-N7-Gua and AP Site Singly-Modified
Bacteriophage Genomes
A. Introduction
A procedure is described for the construction of a biologically active
single-stranded (ss) M 13 genome containing the chemically and thermally
labile AFB,-N7-Gua DNA adduct (Figure 10). The work established the
optimal conditions for preventing the two principal modes of chemical
degradation of the DNA adduct: depurination and opening of the positively
charged imidazole ring.
1. Rationale for the use of a single-stranded genome
The establishment of this construction scheme required the development
of a protocol to generate completely ss genomes. DNA adducts within
singly-modified duplex (double-stranded, ds) genomes show very low
mutation frequencies; thus the use of singly-modified ds DNA makes it very
difficult to obtain mutational properties of any given DNA adduct. This point
is illustrated by studies on 0 6-methylguanine. A double-stranded vector,
containing an 0 6-methylguanine in a specific site yields a mutation frequency
of only 0.04% in E. coli (Essigmann et al., 1986). In contrast, when the
lesion is present in ss DNA the mutation frequency increases by an order of
magnitude (Loechler et al., 1984). Two reasons have been suggested to
explain why DNA lesions within singly-modified duplex DNA yield such low
mutation frequencies. First, DNA adducts within duplex genomes are more
likely to be repaired than when they are within ss genomes. This effect is
most likely due to the observation that many DNA repair systems, such as
nucleotide excision repair (NER), prefer duplex DNA substrates (Sancar and
Sancar, 1988; Sancar, 1994). Second, even when repair systems are
compromised, the mutation frequency of a ds vector is often less than that
of a ss vector (Essigmann et al., 1986). Work from the laboratory of R.
Fuchs indicates that there may be a strand bias in the replication of duplex
genomes harboring adducts in only one strand (Koffel-Schwartz et al., 1987;
Thomas et al., 1994). It has been suggested that when blocking lesions are
present in one strand of a duplex genome, they trigger the specific loss of
the damaged strand (Koffel-Schwartz et al., 1987). Thus, in order to
observe mutations resulting from a single AFB,-N7-Gua adduct, placed in a
specific site within a genome, at a frequency that will provide the most
mutational information possible, I have employed the use of an AFB,-N7-Gua
singly-modified single-stranded M 13 genome.
Procedures used for the preparation of singly-modified ss genomes
traditionally involve heat-denaturation to remove the DNA strand opposite
that containing the adduct (Wood et al., 1990; Basu et al., 1993). Due to
the thermal instability of AFB,-N7-Gua, heat removal of the opposing DNA
strand would assuredly distrupt the integrity of the adduct. Thus, a
procedure was developed in which the opposing DNA strand, a 53-mer
75
uracil-containing scaffold oligonucleotide (Figure 10), could be gently
removed by the enzymatic activities of uracil DNA glycosylase and
exonuclease III, leaving a completely ss AFB,-N7-Gua singly-modified M13
genome (see details below).
2. Choice of oligonucleotide sequence for AFB 1 modification
The choice of an oligonucleotide sequence for modification by AFB,-N7Gua to be used for the construction of singly-modified genomes took into
account the following considerations. 1) It was necessary to choose a
sequence within which a mutation would allow for the mutant sequence to
be selected away from the wild-type sequences. This is commonly
accomplished by choosing a restriction sequence, not present anywhere else
within the genome, within which mutations would render the sequence
refractory to digestion by the respective restriction enzyme. 2) The
sequence must only contain one guanine residue since the AFB, epoxide will
react with all guanines present within the oligonucleotide. 3) It is not
necessary, but desirable, to choose a sequence in which the AFB,-modified
guanine is situated within a relatively warm or hot spot for AFB, modification
or mutagenesis. The first oligonucleotide prepared for use in these studies
was [AFB,-N7-Gua d(ATGCAT)] (AFB,-hexamer), an Nsil restriction sequence
(underscored base indicates position of adduct). This sequence contained
the AFB,-N7-Gua within a cold spot for AFB, modification, 5'-TGC-3'
(Muench et al., 1983; Benasutti et al., 1988). Initial studies involving
ligation of the hexamer into a six-base gapped-heteroduplex molecule proved
troublesome. Hence, it was deemed necessary to prepare a longer
oligonucleotide to improve the ligation efficiency. A 13-mer oligonucleotide
[AFB,-N7-Gua d(CCTCTTCGAACTC)] (AFB,-13-mer) was prepared for
ligation into a 13-base gapped genome. The 13-mer contains an Sful
restriction recognition sequence (the six underscored bases), and harbors the
AFB,-N7-Gua within a warm spot for AFB, modification, 5'-CGA-3' (Muench
et al., 1983; Benasutti et al., 1988). In the following sections I will discuss
only the use of the AFB,-13-mer for preparation of singly-modified genomes.
B. Materials and Methods
1. Materials
EcoRI was obtained from Boehringer Mannheim, and Hinfl and Haelll
were from New England Biolabs. Bacteriophage T4 polynucleotide kinase,
T4 DNA ligase, and exonuclease III were from Pharmacia. Uracil DNA
glycosylase (UDG) was from Gibco BRL. [y-32p] ATP (6000 Ci/mmol) was
from New England Nuclear. 5-Bromo-4-chloro-3-indolyi P-Dgalactopyranoside (X-Gal) and isopropyl f-D-thiogalactopyranoside (IPTG)
were from Gold Biotechnology. Bacteriophage M13mp7L2 genome was a
gift of C. W. Lawrence (Banerjee et al., 1990); M13mp7L2 is identical to
77
M13mp7L1 [utilized in experiments described in Banerjee et al. (1990)]
within the polylinker region of the genome. Cell lines used were E. coil
GW5100 (JM103 P1-) from G. Walker at MIT, and MM294 (lac') from K.
Backman at Biotechnica. Oligonucleotides [AFB,-N7-Gua d(ATGCAT)],
[AFB,-FAPY d(ATGCAT)], and [AFB 1-N7-Gua d(CCTCTTCGAACTC)]
(underscored bases indicates position of adducts) were prepared by
collaborators Rajkumar S. lyer and Thomas M. Harris at Vanderbilt University
(synthesis described below).
2. Preparation and purification of AFB1 modified oligonucleotides
The exo-8,9-epoxide of AFB 1 was prepared as described by Baertschi et
al. (1988) and the purity was established by 'H NMR. [AFB,-N7-Gua
d(ATGCAT)] and [AFB 1-N7-Gua d(CCTCTTCGAACTC)] were prepared
essentially as described by Gopalakrishnan et al. (1989). Briefly, 60 OD 2 60 of
oligonucleotide were dissolved in 1.23 mL of 0.01 M sodium phosphate
5 M disodium EDTA. The
buffer (pH 7.0) containing 0.1 M NaCI and 5 x 10'
solution was treated twice with 4 mg of AFB,-epoxide in CH2CI2 solution
with vigorous shaking for 10 min at 000C. The aqueous layer was washed
twice with CH2CI 2 to remove the AFB 1 dihydrodiol. Oligonucleotides were
purified by reversed-phase HPLC on a C-18 semi-preparative column (AIItech
Associates) by using an acetonitrile-water linear gradient (5-20% CH3CN in
0.01 M phosphate buffer, pH 7.0, 3.0 mL/min; 20 min). The AFB,-FAPY
78
derivative of the hexamer was prepared by treatment of the AFB,-N7-Guamodified oligonucleotide with sodium phosphate buffer, pH 9.0, for 4-5 hr at
37 0 C. The solution was adjusted to pH 7, lyophilized, desalted on a Sep-Pak
cartridge, and the product was purified by HPLC under the conditions given
above. All oligonucleotides were further purified by reversed-phase HPLC on
a Beckman Ultrasphere C-18 analytical column by using an acetonitrile-water
linear gradient (0-20% CH3 CN in 0.1 M NH4OAc buffer, pH 6.8, 1 mL/min;
60 min). The column effluent was monitored by a Hewlett Packard 1040A
diode-array UV absorbance detector at 260 nm and 360 nm. The AFB 1modified oligonucleotide was identified by its characteristic absorbance at
360 nm. The latter conditions were used for all HPLC studies unless
otherwise noted. The purity of each oligonucleotide was evaluated by both
HPLC and denaturing 20% polyacrylamide [19:1 acrylamide:bis(acrylamide)]
gel electrophoresis.
3. Preliminary AFB 1-FAPY studies
Before actually beginning studies on the AFB,-N7-Gua adduct, initial work
involved studies with the AFB 1-FAPY adduct [AFB 1-FAPY d(ATGCAT)].
Preliminary studies on AFB 1-FAPY involved HPLC of the oligonucleotide for
purification purposes, which revealed two AFB 1-FAPY oligonucleotide peaks
that appeared to be isomers of each other (see Figure 11). It was unclear at
this point whether both AFB 1-FAPY species (potential rotamers about the
79
N7,C5 bond of the modified guanine; personal communication T. Harris)
existed in chromosomal DNA under normal physiological conditions, or
whether they were characteristic only of the oligonucleotide. Thus, studies
were pursued with the AFB,-N7-Gua adduct within the same oligonucleotide
sequence [AFB,-N7-Gua d(ATGCAT)].
Ultimately, subsequent to preparation
of the AFB,-N7-Gua genome the AFB,-FAPY genome will be generated by
chemically opening the positively-charged imidazole ring of AFB,-N7-Gua
with mild alkaline conditions; this approach will hopefully yield an AFB,FAPY DNA adduct more closely resembling that formed in vivo.
4. Stability of the AFB,-N7-Gua adduct
[AFB 1-N7-Gua d(ATGCAT)] was incubated in 50 mM MOPS [3-(Nmorpholino)propanesulfonic acid] (pH 6.6, 7.0, 7.4, or 7.8), 50 pg/mL BSA,
10 mM MgCl 2 and 20 mM DTT at 160C, 250C, and 370C for 30 min, 1 h, and
24 h. The stability of the hexamer under these conditions was monitored by
reversed-phase HPLC. The percentages of intact oligonucleotide and any
breakdown products were calculated by integrating peaks within the HPLC
chromatogram. The percentage of AFB 1-FAPY hexamer was calculated by
integrating the peak(s) corresponding to an [AFB,-FAPY d(ATGCAT)]
standard oligonucleotide. The percentage of AP site oligonucleotide was
calculated by integrating the peak corresponding to AFB,-N7-Gua hexamer
that had been heated at 800 C, pH 6.6, for 15 min; these conditions
80
efficiently generate the AP site.
5. Stability of AFB,-N7-Gua under genome construction conditions
The stability of the AFB,-N7-Gua 13-mer [AFB,-N7-Gua
d(CCTCTTCGAACTC)] was examined under the exact conditions of the
genome construction, with the exception that exonuclease Ill was omitted.
The 13-mer (1.2 pjg) was incubated at 160 C in kinase/ligase buffer (K/L
buffer) (50 mM MOPS pH 6.6, 10 mM MgCI 2 , 20 mM DTT, 50 pg/mL BSA),
in a total volume of 200 pL, in the presence of T4 polynucleotide kinase (39
units) for 15 min. T4 DNA ligase (6 Weiss units) was added and the
reaction incubated for 1 h, followed by the addition of UDG (8 units) and
incubation for 90 min. The reaction was stopped by the addition of EDTA
(20 mM) and stored at -80 0 C. The stability of AFB,-N7-Gua under these
conditions was monitored by reversed-phase HPLC.
Additional structural information on the AFB,-N7-Gua 13-mer was
provided by electrospray mass spectrometry performed at Vanderbilt
University.
6. Preparation of bacteriophage M13mp7L2 gapped genomes
a. Preparation of scaffold oligonucleotides
Scaffold oligonucleotides were designed such that they were
complementary to twenty bases on both the 5' and 3' ends of an EcoRI
linearized M13mp7L2 genome. The scaffold contained a uracil in place of
every thymine, and harbored a 13-base sequence between the two twentybase complements that was complementary to the 13-mer oligonucleotide
containing the AFB,-N7-Gua adduct (see below). Both the uracilated
scaffold oligonucleotide and its nonuracilated counterpart were prepared
using an ABI PCRmate DNA Synthesizer. The scaffolds were gel purified and
their purity analyzed by denaturing polyacrylamide gel electrophoresis. The
sequence of the uracilated scaffold 53-mer is shown below; the 13-mer
oligonucleotide complement is underscored (the non-uracilated scaffold 53mer is the identical sequence except that a thymine is substituted for every
uracil).
5'-AAAACGACGGCCAGUGAAUUGAGUUCGAAGAGGCACUGAAUCAUGGUCAUAGC-3'
b. Construction of 13-base gapped genomes
Single-stranded M13mp7L2 DNA (130 ng/pL), prepared according to
procedures described in Lasko et al. (1987), was digested with EcoRI (1.7
units/pL) in 50 mM NaCI, 100 mM Tris-HCI pH 7.5, 5 mM MgCI 2, 100 pg/mL
BSA for 2 h at 230C. The linearized genome was diluted 1.5-fold with H2 0
(90 ng/pL), and heated at 80'C for 5 min with a two-fold molar excess of the
53-mer uracilated scaffold. The mixture was annealed by cooling slowly
82
overnight. This procedure yielded a circular M13 genome containing a 13base gap complementary to the oligonucleotide d(CCTCTTCGAACTC); the
underscored bases are the recognition sequence for Sful (Figure 10).
Gapped-duplex formation was confirmed by the conversion of ss linear DNA
to ss circular DNA as analyzed by electrophoresis on a 14 x 20 cm 1%
agarose gel at 120 V for 5 h in TBE buffer. The gel was stained in ethidium
bromide (1 pg/pL) for 1 h and photographed. The circular gapped-duplex
yield was estimated by visual inspection.
7. Construction of singly-modified genomes
In separate reactions, 39 pmol of either unmodified 13-mer or AFB,-N7Gua 13-mer were
32P-5'-phosphorylated
with [y_32p] ATP (30 pCi), in a total
volume of 15 pL, by incubation with T4 polynucleotide kinase (10 units) at
16 0C for 5 min. Unlabeled ATP was added to a concentration of 1 mM, and
the incubation was continued for 10 min. The oligonucleotides were ligated
with T4 DNA ligase (0.05 Weiss units/pL) for 1 h at 160 C into an equimolar
amount of the freshly prepared 13-base gapped molecules (8 pmol/mL)
(Note: For reasons that are not clear, higher concentrations of DNA ligase
appeared to greatly reduce the transfection efficiencies of the constructed
genomes into E. cogi cells). The 53-mer uracilated scaffold was removed by
further incubation at 160C for 90 min with UDG (0.04 units/pL) and
exonuclease III (0.4 units/pL). See Figure 10 for genome construction
83
scheme. Phosphorylation, ligation, and scaffold removal were all done in K/L
buffer (50 mM MOPS pH 6.6, 10 mM MgCI 2, 20 mM DTT, 50 pg/mL BSA).
A portion of the AFB,-N7-Gua genome (M13-AFB,) was heated at 800 C for
15 min in order to release the AFB,-N7-Gua from the DNA to generate an AP
site genome (M13-AP); these conditions efficiently generate the AP site.
The unmodified genome (M13-G) was heated in parallel as a control (M13GA). Unincorporated oligonucleotides, ATP, enzymes, and salts were
removed from the radiolabeled genomes by passing the ligation mixture
through a Sepharose CL-4B column (15 x 0.75 cm) preequilibrated with 10
mM MOPS (pH 6.6), 100 mM NaCI, and 1 mM EDTA. Columns were run at
40C; the genome eluted in the void volume.
8. Evaluation of the efficiency of uracil-containing scaffold removal
A new ss M13 genome, designated M13+, was prepared by ligation of
the unmodified hexamer oligonucleotide into the six-base gappedheteroduplex molecule as described above, except that a 46-mer scaffold
oligonucleotide was used instead of the 53-mer oligonucleotide. The 46-mer
was identical to the 53-mer except that the internal 13-base sequence,
complementary to the 13-mer oligonucleotide, was substituted with a sixbase sequence complementary to the hexamer oligonucleotide 5'-ATGCAT3'. The ligation mixture was transfected into E. coli MM294 cells that were
prepared for transformation according to the CaCl 2 transformation procedure
84
(Maniatis et al., 1989). After transformation the mixture was incubated at
37 0 C for 2-3 h to produce progeny phage, which were isolated from the
supernatant and plated with GW5100 cells in the presence of IPTG and XGal (Messing, 1983). Plates were incubated overnight at 370C for plaque
formation. Phage M13+ was isolated and sequenced (Sanger et al., 1977);
M13+ had a six-base insertion into the EcoRI site, as expected.
Single-stranded M13+ DNA was prepared and annealed (100 mM NaCI,
10 mM Tris-HCI pH 7.8) to an equimolar amount of
32P-5'-phosphorylated
uracilated scaffold. The annealed DNA was dialyzed into 10 mM Tris-HCI
(pH 7.5) and 1 mM EDTA. The efficiency of UDG was examined by
incubating 1 pg of scaffold-annealed M13+ with 1 unit of UDG in a total
volume of 30 pL at 160C for 1 h, 1.5 h, and 2 h in K/L buffer. The UDG
digests were incubated in 1 M piperidine for 1 h at 900C and the products
electrophoresed through a denaturing 12% polyacrylamide gel. The extent
of UDG digestion was determined by comparison to a Maxam and Gilbert
C + T(U) sequencing standard (Maxam and Gilbert, 1980).
The efficiency with which the uracilated scaffold was removed by the
combined activities of UDG and exonuclease III was determined by
incubating 500 ng of
32 P-scaffold-annealed
M13 + in a total volume of 20 pL
in the presence of both enzymes UDG (0.5 units) and exonuclease III (5
85
units), for either 90 min or 2 h in K/L buffer. The DNA was then
electrophoresed through a 1% agarose gel to separate ss circular and ss
linear forms. The efficiency of the scaffold removal was determined by
measuring the disappearance of
32p.
A Molecular Dynamics Phosphorlmager
was used for this analysis and all quantitation described in the following
sections.
9. Evaluation of the efficiency of removal of AP sites by exonuclease III
from the AFB,-N7-Gua genome preparation
AFB,-N7-Gua 13-mer was
32P-5'-phosphorylated
and heated at 80 0 C for
1 5 min to generate an AP site 13-mer that was ligated into the 13-base
gapped molecule containing the uracilated scaffold. The AP site containing
genome was characterized by incubating the ligation products in 0.1 M
NaOH for 10 min at 1000 C to cleave the AP site. The reaction products
were digested with ss DNA restriction enzymes Hinfl (100 units) and Haelll
(75 units) and the products were separated on a 20% denaturing
polyacrylamide gel (procedure described below). AP site genome formation
was confirmed by generation of a 15nt band as a result of alkaline cleavage
of the AP site prior to restriction digestion (band sizes are described in
Results).
In order to test the efficiency of AP site removal, another portion of the
86
ligation mixture was incubated with UDG and exonuclease III as described in
the genome construction section in order to remove the scaffold and
inactivate (linearize) the AP site genome. The reaction products were
electrophoresed through a 1% agarose gel to separate ss circular and ss
linear DNA. The efficiency of AP site genome inactivation was determined
by measuring the disappearance of
32 P from
the circular band on the agarose
gel by autoradiography and Phosphorlmager analysis.
10. Characterization of the AFB,-N7-Gua and AP site singly-modified
genomes
The integrity of the AFB,-N7-Gua adduct within the M13 genome (base
6240) was determined as follows. A portion of
32 P-labeled
M13-AFB1 was
heated at 80 0C, pH 6.6, for 15 min to generate M13-AP. Subsequently, the
genome was treated with 0.1 M NaOH at 100oC for 10 min to cleave the AP
site. These conditions were shown to maintain the integrity of the
unmodified and FAPY containing genomes. The reaction was neutralized
with HCI, and the DNA was incubated in 1 M piperidine at 90 0 C for 1 h to
cleave any potentially contaminating AFB,-FAPY molecules (M13-FAPY)
(Maxam and Gilbert, 1980). The DNA was ethanol precipitated,
resuspended in 25 pL H20, and digested at 370C for 1 h with ss restriction
enzymes Hintf (100 units) and Haelll (75 units) in MSB restriction
endonuclease buffer [50 mM NaCI, 10 mM Tris-HCI (pH 7.5), 10 mM MgCI 2,
87
and 1mM DTT]. These enzymes cleave the ss genome at sites flanking the
5' and 3' ends of the ligation site. The samples were electrophoresed
through a denaturing 20% polyacrylamide gel. The gel was
autoradiographed at -80 0 C with an intensifying screen.
11. Quantitation of M 13-G, M 13-GA, M 13-AFB,, and M 13-AP
Exonuclease III treatment, used in the final step of the genome
construction for removal of the scaffold, was expected to degrade, to a
certain extent, the M13 genome in a nonspecific fashion. Therefore, it was
necessary to determine the final yields of M13-G and M13-AFB1 after
scaffold removal, and M13-GA and M13-AP after removal of the scaffold
and heating. Because AFB,-N7-Gua is labile, the strategy used for DNA
quantitation was to determine the yield of M13-G directly and then to
determine the yield of M13-AFB, relative to that of M13-G. The yields of
M13-GA and M13-AP were also determined relative to that of M13-G. This
was accomplished by comparative Phosphorlmager analysis of the ss circular
band representing M 13-G to those representing M13-GA, M 13-AFB, , and
M13-AP, on an agarose gel.
The yield of M13-G was determined as follows. First, the ligation
efficiency of the unmodified 13-mer into the 13-base gapped genome was
determined. An aliquot (0.059 pmol) of the ligation reaction with unmodified
88
13-mer (without scaffold removal) was heated at 800 C for 15 min to
inactivate the ligase. The heated ligation mixture was cooled slowly
overnight in MSB restriction endonuclease buffer to make sure that the
scaffold, most of which presumably denatured during the heating step, was
completely reannealed; duplex DNA (scaffold-containing DNA) was used to
ensure complete digestion by restriction enzymes. The genome was
subsequently digested with Hinfl (40 units) and Haelll (50 units) and the
entire sample (0.059 pmol) electrophoresed through a 20% denaturing
polyacrylamide gel. A known amount of
32 P-5'-phosphorylated
unmodified
13-mer was included on the gel as an internal standard. The percentage of
completely ligated material (i.e., that representing circular M13-G that had
not yet been incubated with UDG and exonuclease III
for scaffold removal,
denoted "M13-GS") was determined by comparative Phosphorlmager
analysis of the 31nt band, which represents completely ligated HinfllHaelll
digested genome, to the internal oligonucleotide standard (band sizes are
described in Results).
In order to determine the final yield of M13-G, an equimolar amount of
M13-GS and M13-G (scaffold removed), were electrophoresed through a 1%
agarose gel to separate ss circular from ss linear DNA. The total amount of
M13-G was determined upon quantitative comparison (Phosphorlmager
analysis) of the radioactivity present in the ss circular band to that present in
89
the ss circular band of M13-GS, which represented a known amount of
circular genome (determined above). Electrophoresed on the same gel were
equimolar amounts of M13-AFB1 , M13-AP, and M13-GA. The total amount
of DNA present in each ss genome was determined by direct comparison of
the radioactivity present in the M13-G circular band to that present in the
circular bands of each genome, M13-GA, M13-AFB1 , and M13-AP. DNA
quantitations were done in triplicate. The specific activity of the AFB,-N7Gua oligonucleotide, relative to the unmodified oligonucleotide, was taken
into account when calculating the yield for the M13-AFB, and M13-AP
genomes.
C. Results
1. Strategy for construction of singly-modified genomes
The ends of a linearized ss M13mp7L2 genome were brought to within
13 nucleotides of one another by a uracil-containing scaffold to yield 13base gapped genomes (Figure 10). The gapped genomes were analyzed by
agarose gel electrophoresis, and a typical yield for gapped-genome formation
was 50%, with 50% linear M13mp7L2 remaining. Subsequently, the gap
was bridged by the 5'-phosphorylated AFB,-N7-Gua 13-mer. Removal of the
complementary 53-mer scaffold by UDG and exonuclease III yielded the ss
M13-AFB, genome. When unmodified 13-mer was employed the product
90
was the M13-G genome. Subsequent heating of a portion of each ligation at
800C pH 6.6 for 15 min yielded M13-AP and M13-GA, respectively.
Electrophoretic analysis of the genomic constructs is shown in Figure 12.
2. Stability of the AFB,-N7-Gua adduct
In order to determine the conditions most suitable for genome
construction it was first necessary to assess the stability of AFB,-N7-Gua
within DNA. The AFB,-N7-Gua hexamer was subjected to a range of pHs
and temperatures, for various amounts of time. The stability of the hexamer
was monitored by reversed-phase HPLC. The relative amounts of starting
material and breakdown product(s) were calculated by integrating
chromatographic peaks, and the data were graphed accordingly (Figure 13).
These data emphasize that low temperature is critical for AFB,-N7-Gua
stability. After incubation for 24 h at 370C, at all four pHs, AFB,-N7-Gua
was degraded extensively (Figure 13, A-D). Degradation was considerably
less after 24 h at 250C at all four pHs (Figure 13, E-H). In contrast, AFB 1N7-Gua was fairly stable after incubation at 160C at all four pHs, even after
24 h (Figure 13, I-L). As expected there was an increase in the formation of
the AFB 1-FAPY derivative as the pH increased. The largest percentage of
AFB 1-FAPY (-50%) was present after 24 h at 370C, pH 7.8 (Figure 13D). By
contrast, AFB 1-FAPY was present at < 1% after incubation at pH 6.6 at all
three temperatures for 24 h (Figure 13A, E, I). Since my goal was to
optimize conditions for the preparation of as pure an AFB,-N7-Gua genome
as possible, and the genome construction conditions provided a method for
elimination of contaminating AP site, I chose conditions that generated the
least amount of AFB 1-FAPY while retaining the most AFB,-N7-Gua. The data
indicated that the optimal temperature and pH combination for AFB,-N7-Gua
stability was 160 C and pH 6.6 (Figure 131). After 24 h under these
conditions 81% of the AFB,-N7-Gua hexamer remained, no significant level
of AFB 1-FAPY was formed, and 19% AP site was formed. AP site formation
was judged tolerable since the final step in the genome construction involved
treatment with the AP endonuclease exonuclease Ill. Exonuclease III was
shown to be highly efficient in the removal of AP site genomes (see below).
3. Stability studies and characterization of [AFB,-N7-Gua
d(CCTCTTCGAACTC)]
Preliminary studies indicated a very low extent of ligation (- 10%) of the
AFB,-N7-Gua hexamer, described above, into an M13mp7L2-derived six-base
gapped genome. To increase the yield of the AFB,-N7-Gua genome a longer
singly-adducted AFB,-N7-Gua oligonucleotide (13-mer) was synthesized.
The purity of the AFB,-N7-Gua 13-mer was assessed by HPLC (Figure 14A)
and further established by the presence of a single band on a denaturing
polyacrylamide gel. HPLC data indicated an AP site contamination of 2%.
Electrospray mass spectrometry of the monoanion (Figure 15) indicated a
92
large peak, eluting at 14.6 min, that consisted of an intense signal at 1390.0
(M-3H)/3z, and a weak one at 2084.9 (M-2H)/2z; observed MW = 4172.4,
and calculated MW = 4173.8. A much smaller peak preceded the large
peak, at 12.3 min. Although the spectrum for this peak is not shown in
Figure 15, it appeared to consist of a mixture of unmodified and AP site 13mer. Peaks observed at 1281.5 (M-3H)/3z and 1921.5 (M-2H)/2z likely
represent unmodified 13-mer; observed MW = 3846.6, and calculated MW
= 3845.6, and peaks at 1236.5 (M-3H)/3z and 1854.5 (M-2H)/2z likely
represent AP site 13-mer; observed MW = 3711.8, calculated MW =
3712.5.
The stability of the AFB,-N7-Gua 13-mer was examined under conditions
of genome construction (Figure 14B), except in the absence of exonuclease
III, which slightly degraded both control and modified oligonucleotides.
Importantly, although the phosphodiester bonds yielded to exonuclease III
treatment, the AFB,-N7-Gua moiety remained intact, i.e., the peak
representing the AFB,-N7-Gua containing oligonucleotide eluted at a slightly
earlier time but still maintained its characteristic absorbance at 360 nm for
AFB,-N7-Gua. HPLC analysis revealed that under conditions of the genome
construction < 1% of the AFB,-N7-Gua was degraded to the AP site, yielding
a total AP site contamination of - 3%. Evidence that the AFB,-N7-Gua moiety
remained intact within the oligonucleotide was indicated by its characteristic
93
chromophore absorbing at 360 nm (inset, Figure 14). It was thus concluded
that the AFB,-N7-Gua oligonucleotide was adequately stable to the genome
construction conditions. Furthermore, these data suggested that essentially
pure AFB,-N7-Gua genome could be constructed, presuming that the small
amount of contaminating AP site would be efficiently inactivated in the final
step of the genome construction upon treatment with exonuclease ill.
4. Removal of the uracil-containing scaffold from M13-AFB 1
In order to use M 13-AFB, for genetic studies, it was essential that the
scaffold (Figure 10) be completely removed in a manner that preserved the
integrity of the AFB,-N7-Gua moiety. This was accomplished by introducing
a uracil in place of every thymine in the scaffold such that the scaffold could
be gently removed by the complementary activities of UDG and exonuclease
Ill. I first investigated the efficiency with which the scaffold could be
digested with UDG. A
32 P-5'-phosphorylated
scaffold, annealed to ss M13+
DNA, was shown to be completely digested with UDG in 90 min at 16 0C, pH
6.6, as determined by piperidine treatment of the UDG-digested scaffold and
comparison to a Maxam and Gilbert C+T(U) reaction (i.e., all radioactivity
was present in a 14nt fragment representing the smallest 5'-labeled fragment
possible after UDG digestion, see scaffold sequence under section B6 of this
chapter). Next, I examined the efficiency with which exonuclease III, in
combination with UDG, could remove the uracilated scaffold. The scaffold
94
was shown to be completely removed in 90 min at 160C, pH 6.6, by the
combined activities of UDG and exonuclease III (Figure 16). The results
indicated that both UDG and exonuclease III were necessary in order to
achieve complete removal of the scaffold (Figure 16, lane 4); i.e.,
exonuclease III alone was not sufficient (Figure 16, lane 3). Ethidium
bromide stains of these gels revealed circular and linear DNA in all lanes,
indicating that the disappearance of
32P
was owed to removal of the scaffold
and not to nonspecific exonuclease III degradation.
Since the 53-mer scaffold oligonucleotide was identical to the 46-mer,
except that it harbored an internal 13-base complement to the 13-mer
oligonucleotide, instead of a six-base complement to the hexamer sequence,
it was assumed that the 53-mer scaffold would be removed as efficiently as
the 46-mer upon incubation under the above conditions.
5. Contaminating AP site genome can be efficiently inactivated by its
selective sensitivity to exonuclease III
It was my belief that the AP endonuclease activity of exonuclease III
would efficiently inactivate any contaminating AP site genome generated
during the synthesis of M13-AFB1 . To test this hypothesis, an AP site 13mer was produced by heating the AFB,-N7-Gua 13-mer at 800C, pH 6.6 for
15 min; the resulting 13-mer containing an AP site was ligated into the 13-
95
base gapped genome. Complete ligation was confirmed by the presence of a
31nt band upon electrophoresis, through a denaturing polyacrylamide gel,
following digestion of a portion of the ligation reaction with Hinfl and Haelll.
Characterization of the AP site involved pre-treatment of the digest with 0.1
M NaOH, which cleaved all but 3% of the AP site containing 31nt fragment
to a 15nt fragment, as determined by Phosphorlmager analysis (band sizes
are described below). This 3% was believed to be unmodified contamination
(i.e., guanine at position 6240) since it comigrated with the 31nt fragment of
the unmodified control, and was not sensitive to alkaline treatment.
In order to test the efficiency of AP site inactivation by exonuclease Ill,
another portion of the ligation mixture was incubated with UDG and
exonuclease III to remove the scaffold and linearize the AP site genome.
Electrophoresis of the reaction mixtures through a 1% agarose gel indicated
that 92% of the material was not only linearized, but digested so efficiently
that the 3'->5' exonuclease activity removed most of the
17). Since -8%
32P
label (Figure
of the circular material remained (determined by
Phosphorlmager analysis), and 3% represented unmodified genome
(discussed above), 5% was estimated to represent undigested AP site
genome. It was therefore estimated that -95% of the contaminating AP site
genome generated during M13-AFB1 construction should have been
destroyed by exonuclease II. The nonspecific exonuclease III degradation of
96
the unmodified control genome was determined to be approximately 30%.
This 30% reduction in circular genome is presumably attributable to the
action of exonuclease III on spontaneously formed AP sites about the 7kb
M13 genome. The unmodifiedA and AFB,-N7-Gua genome degradations
(30% and 20%, respectively) were equal to or lower than that of the
unmodified control, and thus were also determined to be nonspecific. Based
upon the data above, if we assume that 95% of a potential 3% AP site
contamination was inactivated by exonuclease III, it can be predicted that
only 0.15% AP site genome should remain. This extent of AP site
contamination was found to be insignificant based on biological studies with
M13-AFB1 (Chapter IV).
6. Characterization of singly-modified genomes
Oligonucleotide stability and AP site removal studies suggested that less
than 0.2% of M13-AFB, was contaminated with AP site genome. Further
studies were necessary, however, in order to determine the extent of
contamination by unmodified or AFB 1-FAPY genomes.
Genome characterization involved restriction digestion of the genomes
with ss restriction enzymes Hinfl and Haelll, which cleave at sites flanking
the 5' and 3' ends of the ligated oligonucleotide. As indicated in Figure
18A, HinflHaelll digestion potentially yields a 31nt fragment representing
97
completely ligated 13-mer, a 21nt fragment representing oligonucleotide
ligation only on the 5' end of the gap, and a 23nt fragment representing
oligonucleotide ligation only on the 3' end of the gap. To examine the
integrity of the AFB,-N7-Gua adduct within the genome (base 6240), the
genome was heated, prior to restriction digestion, to convert the AFB,-N7Gua moiety to an AP site. It was then treated with alkali to cleave the AP
site and annealed to the scaffold oligonucleotide (10x molar excess) in order
to ensure complete digestion with Hinfl and Haelll. As indicated in Figure
1 8B, a 32 P-labeled 15nt fragment would be generated only if there were
cleavage at the original site of modification. Such a band would indicate the
original presence of an AFB,-N7-Gua adduct, assuming the absence of any
AP site. Unmodified and AFB 1-FAPY genomes were shown to be stable
under these conditions, as indicated by studies performed on the respective
oligonucleotides involving the subjection of each to the genome
characterization procedures. The products of each reaction were separated
by denaturing PAGE (Figure 18C). The data indicate that heating M13-AFB1
generated M13-AP as observed by a shift in the "32nt" fragment (Figure 18,
lane 1), representing completely ligated AFB,-N7-Gua genome, to a faster
migrating 31nt fragment (Figure 18, lane 2), representing completely ligated
AP site genome. I have observed that on denaturing polyacrylamide gels,
AFB,-N7-Gua and AFB 1-FAPY containing hexamers, and the AFB,-N7-Gua
containing 13-mer migrate approximately one nucleotide slower than their
98
unmodified or AP site containing counterparts. This slower migration is most
likely a result of the extra mass of the oligonucleotide due to the aflatoxin
moiety (MW = 329.8). The faint smear immediately below the "32nt"
fragment is due to unmodified contamination (discussed below) and probably
to a small amount of AFB,-N7-Gua degradation to the AP site during
restriction digestion and electrophoresis. Subsequent alkaline treatment
resulted in cleavage of the 31nt AP site DNA fragment, as represented by its
conversion to a 15nt fragment (Figure 18, lane 3). In three separate
experiments, approximately 5% of the completely ligated M13-AFB 1 genome
preparation remained circular after heat/alkaline treatment as determined by
Phosphorlmager analysis (Figure 18, lane 3). These data suggested that
M13-AFB1 was greater than 95% pure, again assuming the absence of any
AP site. In order to determine, however, if the remaining 5% were M13FAPY as a contaminant, a portion of the heat/alkali treated DNA was treated
with piperidine prior to restriction digestion to cleave any AFB 1-FAPY
moieties (Maxam and Gilbert, 1980). As indicated in lane 4 of Figure 18
there was no additional cleavage upon piperidine treatment, indicating the
absence of M13-FAPY contamination. Additional evidence that the
remaining 5% was not M13-FAPY was its electrophoretic mobility,
consistent with a 31nt fragment. As described above, the AFB,-FAPY
moiety retards oligonucleotide migration similarly to the AFB,-N7-Gua
moiety, and thus we would expect it to migrate as a "32nt" fragment.
99
These results, in combination with AP site removal studies, are consistent
with the conclusion that the M13-AFB, was 95% pure with an unmodified
contamination of 5%. The unmodified control was unaffected by these
conditions (Figure 18, lane 6). See Figure legend for descriptions of the other
bands.
7. Quantitation of M13-G, M13-GA, M13-AFB,, and M13-AP
The ligation efficiency of the unmodified oligonucleotide into the 13-base
gapped genome was 88%. After removal of the scaffold, 52% of the ss
circular material remained, giving an overall M13-G yield of 46%. As
indicated above, the decrease in DNA was likely owed to nonspecific
exonuclease III degradation. The amounts of M13-GA, M13-AFB1 , and M13AP were determined by quantitative comparison, on an agarose gel, of ss
circular DNA represented by each genome to ss circular M13-G DNA,
assuming that the latter was present at a 46% yield. The final yields were
38% for M13-GA, 25% for M13-AFB1 and 18% for M13-AP.
D. Discussion
Evaluation of the genetic effects of DNA lesions is an essential step
toward understanding the molecular etiology of chemically induced
carcinogenesis. Although the study of the genetic effects of single DNA
100
lesions has been underway for more than a decade, preparation of singlymodified genomes containing chemically labile lesions such as AFB,-N7-Gua
for use in genetic studies has been ignored for the most part. The many
enzymatic and chemical steps needed to prepare site-specifically modified
genomes containing such lesions employ conditions that can alter the
structure of these important adducts.
This chapter describes the development a procedure with which to
construct a ss M 13 genome containing the chemically and thermally unstable
AFB,-N7-Gua adduct and its AP site counterpart (Figure 10). 5'Phosphorylated AFB,-N7-Gua 13-mer was ligated into a gap produced by
manipulation of the M13mp7L2 bacteriophage genome; the procedure
employed is similar to that of Banerjee et al. (1988), except the scaffold to
which the modified oligonucleotide eventually anneals harbors uracil for each
thymine. The scaffold was removed by uracil DNA glycosylase and
exonuclease III to yield ss M13-AFB,.
The entire genome construction was
completed in less than 3 h and was carried out under slightly acidic
conditions and low temperature determined to be suitable for AFB,-N7-Gua
stability (Figures 13 and 14). Subsequently, a portion of M13-AFB, was
heated to generate M13-AP. Data indicate that M13-AFB, was - 95% pure;
the only detectable impurities were M13-AP (<0.2%) and M13-G (5%).
Since M13-G should not give rise to targeted mutations and M13-AP
101
contamination is extremely small, these contaminants did not interfere
significantly with the use of these genomes for in vivo mutational studies
(Chapter IV).
This construction approach yielded genomes containing either the AFB,N7-Gua or the AP site within the Sful recognition sequence (5'-TTCGAA-3').
Situation of the lesion in a restriction site provided a method for mutant
selection during in vivo studies, since mutant DNA was refractory to
digestion by Sful. Another noteworthy feature of the genome construction
protocol is that ligation of the 13-mer into the 13-base gap restored the lacZ
reading frame, which is out of frame by +2 in the parental M13mp7L2
genome. Thus, completely ligated material yields blue plaques in the
presence of X-Gal and IPTG. The AFB,-N7-Gua adduct is within an in-frame
5'-GAA-3' codon (5'-CCTCTTCGAACTC-3').
A G to T transversion within
this codon yields an in-frame ochre (5'-TAA-3') that is partially suppressed in
E. coli SupB hosts, thus yielding light blue plaques. Consequently, only
plaques resulting from targeted G to T transversions are light blue, thus
providing facile detection of that mutation type. G to T transversions are the
principal mutations observed in AFB,-treated organisms (Foster et al., 1983;
Trottier et al., 1992; Levy et al., 1992; Aguilar et al., 1993), and account for
75% of the mutations observed for AFB,-N7-Gua in parallel in vivo
investigations (Chapter IV).
102
The system described in this work should be applicable for the
construction of many other singly-modified genomes containing structurally
labile DNA adducts, such as other adducts at the N7 positions of guanine,
i.e, N7-methylguanine (Ezaz-Nikpay and Verdine, 1992), dibenzo[a,/]pyrene
N7 purine adducts (Li et al., 1995), (chloroethyl)nitrosourea adducts such as
N7-(2-hydroxyethyl)guanine and N7-(2-chloroethyl)guanine (Bodell and
Pongracz, 1993), and nitrogen mustard N7 guanine adducts (Povirk and
Shuker, 1994). The ability to produce DNA substrates containing uniquely
situated unstable DNA adducts will facilitate the investigation of the genetic
effects of these lesions in both prokaryotic and eukaryotic systems. This
type of analysis is crucial toward understanding, at a mechanistic level, the
molecular events involved in the early stages of induction of genetic
diseases.
103
Figure 10. Construction scheme of singly-modified ss genomes containing
AFB,-N7-Gua or the AP site (AFB,-N7-Gua structure in inset). See text for
details.
104
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Figure 11.
HPLC chromatogram of A) [AFB,-FAPY d(ATGCAT)]; the AFB,-
FAPY species is present within peaks at both 41 min and 46 min as
indicated in the chromatogram shown in B) [AFB 1-FAPY d(ATGCAT) after
incubation for 24 h at 25 0 C. The peak at -41
min converts to the peak at -46
min under these conditions. In an additional experiment, both peaks were
collected and incubated further at 370 C for a total of 72 h. Each peak gave
rise to an equilibration of the two peaks, with the peak at - 46 min being - 5fold more in each case after the 72 h time course. Potential rotational
isomers of the species are shown in inset.
106
A.
2'
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time (m8
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time (min)
--
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AFB,-FAPY d(/
o
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E
4
40
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time (min)
time (min)
O-H
Figure 12. Single-stranded M13mp7L2-derived genome migration on a 1%
agarose gel showing both ss circular and ss linear DNA. The 13-mer
oligonucleotides were
32 P-5'-phosphorylated
prior to genome construction.
The gel was dried and autoradiographed. The circular DNA represents
potentially viable genome. Linear DNA should not be viable and is the result
of either non-specific exonuclease III degradation of the circular DNA or 13mer oligonucleotide ligation to either the 5' or 3' side of the gappedheteroduplex molecule.
108
Circular
Linear
109
Figure 13. [AFB 1-N7-Gua d(ATGCAT)I was incubated at the indicated
temperatures and pHs. AFB,-N7-Gua stability within the oligonucleotide
hexamer was monitored by reversed-phase HPLC (see text for details), and
the resulting data graphed accordingly. Black bars represent the AFB,-N7Gua hexamer, open bars represent the AFB 1-FAPY hexamer, and shaded bars
represent the AP site hexamer. The conditions determined to be optimal for
AFB,-N7-Gua stability are depicted in I (pH 6.6, 160 C).
110
37 0C
160C
250C
100
I:
50
0
100
\I
50
0
100
I
50
0
100
50
C1
00
a
0
0.5
1.0
24
0
0.5
1.0
Hours
111
0
0.5
1.0
24
Figure 14. Reversed-phase HPLC profiles and UV spectrum (inset) of A)
[AFB 1-N7-Gua d(CCTCTTCGAACTC)I
and B) the same oligonucleotide after
incubation under the genome construction conditions (pH 6.6, 160C, for 2 h
and 45 min). Peaks denoted "*" represent buffer components.
112
oE
cm (
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0
a,
0
0
CY)
C\o
(wu o9z)
113
rvw
Figure 15. Mass spectral data of [AFB 1-N7-Gua d(CCTCTTCGAACTC)].
A)
Chromatogram of species eluting from the column attached to the mass
spectrometer. B) Mass spectrum of the peak at 14.6 min which represents
the AFB,-13 mer (note: add 9 min on to time to retention time to determine
the true elution time). See text for details and for description of peak at
12.3 min.
114
100
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60
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115
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Figure 16. Autoradiogram showing the efficiency of removal of the uracilcontaining scaffold. UDG = uracil DNA glycosylase; Exo III = exonuclease
ill. A
32P-labeled
uracilated scaffold (A), or a 32P-labeled non-uracilated
scaffold (B) were annealed to M13+ ss DNA (note the presence of some
linear ss DNA that, as expected, also annealed to the scaffold). Both the
uracilated and the non-uracilated constructs were incubated for 90 min at pH
6.6 (see text for details) without enzyme (lanes 1 and 5, respectively), with
UDG (lanes 2 and 6, respectively), with Exo III (lanes 3 and 7, respectively),
or with both UDG and Exo III (lanes 4 and 8, respectively). Reaction
products were electrophoresed on a 1% agarose gel to separate ss circular
and ss linear genomes; the gel was dried and autoradiographed. Removal of
the uracilated scaffold by the combined activities of UDG and exonuclease III
was confirmed by the disappearance of
116
32P
label (lane 4).
++
1+
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Figure 17. Autoradiogram showing the efficiency of removal of potentially
contaminating AP site genome from the M13-AFB1 preparation. A
32 P-5'-
phosphorylated AP site 13-mer was used to construct an AP site genome
containing the uracilated 53-mer scaffold. Prepared in parallel were genomes
containing an unmodified 13-mer, an unmodified heated (A) 13-mer, and an
AFB,-N7-Gua 13-mer. Subsequent to ligation of the 13-mers, each genome
was incubated for 90 min at pH 6.6 (see text for details) with either no
enzyme ("scaffold on"), or with UDG and exonuclease III ("U & E").
Reaction products were electrophoresed through a 1% agarose gel, the gel
was dried and autoradiographed and shows the separation of ss circular and
ss linear genomes. Inactivation of the AP site genome by exonuclease III
was confirmed by the disappearance of
removal of the scaffold.
118
32P
from the circular band after
e
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cc
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LM
1
Figure 18. Characterization of M13-AFB1 , M13-AP, and M13-G. A: Map of
the Hinfl/Haelll restriction fragments containing the ligated 13-mers (black
bars). Complete 5' and 3' ligation of the 13-mers into the gapped molecule
yielded a 32P-labeled 31nt fragment, whereas ligation on either the 5' or 3'
side produced
32P-labeled
fragments of 21nt or 23nt, respectively. B:
Incubation of M13-AFB, at 80 0C, pH 6.6 for 15 min, followed by alkaline
treatment at 1000 C for 10 min cleaved the genome at the site of the
modification. Subsequent digestion by Hinfl/Haelll yielded a 32 P-labeled 15nt
fragment. G* = AFB,-N7-Gua. C: Autoradiogram of the reaction products
electrophoresed on a 20% denaturing polyacrylamide gel. The gel shows
radioactive fragments obtained from either M13-AFB1 (G*) (lane 1), M13-AP
(AP) (lane 2), or M13-G (G) (lane 5). Lane 3 is the result of heat/alkaline
treatment of M1 3-AFB, . Lanes 4 and 6 are after heat/alkaline/piperidine
treatment of M13-AFB1 and M13-G, respectively. Lanes headed "M" are 832 oligonucleotide size markers. Lanes 1 and 2, and 3-6 are from two
separate gels.
aThe
bands at -18-19nt in lane 1 are believed to represent
Hinfl/Haelll digested genome, ligated at only the 5' end of the
oligonucleotide where the 3'->5' exonuclease activity of exonuclease III was
inhibited by the AFB,-N7-Gua moiety. bThe bands at - 16-17nt in lane 2 are
believed to represent material resulting from depurination of the material
described in a (lane 1).
120
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IV. Mutational Properties of AFB,-N7-Gua in Escherichia coli
122
A. Introduction
The work discussed in this chapter is focused on determining the relative
contributions of the primary AFB,-N7-Gua adduct and the AP site to the
mutagenic spectrum of AFB,.
Singly-modified genomes were constructed as
described in Chapter III, each containing either an AFB,-N7-Gua adduct or an
AP site in a defined position within a viral genome. These genomes were
then used to compare the in vivo mutagenic potential and mutagenic
specificity of each lesion upon replication in E. coil.
Mutation frequencies (MF) were determined in E. coil that had, or had
not, been treated with UV irradiation to induce the SOS response, in order to
examine the effect of the normal E. coli SOS mutagenic processing analog,
umuDC. Previous data, however, have suggested that AFB1 -induced base
substitution mutations in E. coli are dependent on the presence of the
mucAB mutagenesis enhancing gene products, and that the normal E. coil
analog, umuDC, is not sufficient (Foster et al., 1988; Urios et al., 1994).
Thus, the mutation frequency of AFB,-N7-Gua was determined in E. col cells
(DL7) containing pGW16, a point mutant derived from the naturally occurring
plasmid pKM101 that carries mucAB (Walker, 1978; McNally et al., 1990).
The mucAB operon of pGW16 encodes enhanced SOS mutagenic processing
compared to its progenitor.
123
B. Materials and Methods
1. Materials
Restriction enzymes and Sephadex G-50 Quick Spin columns were
purchased from Boehringer Mannheim. Bacteriophage T4 polynucleotide
kinase, T4 DNA ligase, and exonuclease 111 were from Pharmacia. Uracil
DNA glycosylase was from Gibco BRL. 5-Bromo-4-chloro-3-indolyl P-Dgalactopyranoside (X-Gal) and isopropyl P-D-thiogalactopyranoside (IPTG)
were from Gold Biotechnology. [a-3 5S] dATP and Sequenase Version 2.0
sequencing reagents were from Amersham. Plasmid preparation kits were
from Qiagen. Bacteriophage M13mp7L2 genome was a gift from C. W.
Lawrence of the University of Rochester (Banerjee et al., 1990). The cell
lines used were DL7 (AB1157; lacAU169) (Lasko et al., 1988), DL7/pGW16
(DL7; pGW16 mucAB) (Lasko et al., 1988), GW5100 (JM103, P1-) from G.
Walker, MIT, and NR9050 (F'prolaclZAM15, Aprolac, supB) from R. M.
Schaaper at NIEHS.
2. Preparation and characterization of singly-modified genomes
Single-stranded (ss) M13 genomes containing a unique AFB,-N7-Gua
adduct (M13-AFB,), AP site (M13-AP), unmodified guanine (M13-G), or
unmodified guanine "heated" (M13-GA) were generated as described in
Chapter III, except that unlabeled ATP (1 mM) was used for phosphorylation
124
of the respective oligonucleotides.
3. Optimization of conditions for SOS induction of E. coli
Before transforming E. coli DL7/pGW16 cells with the site-specifically
modified genomes it was necessary to optimize the conditions for UV
induction of SOS-processed mutagenesis in these cells. UV-irradiated (50
J/m2 ), replicative form (RF) M13mp 18 DNA (10 ng/pL) was used to
determine the levels of SOS induction of UV-irradiated (UV +) DL7/pGW16
cells (0, 15, 30, 45, 60, and 75 J/m 2 ). The extent of SOS induction of
these cells was measured on the basis of their ability to induce mutations in
UV-damaged M13mp18 DNA. Both DNA and cells were UV-irradiated using
a 15 W General Electric germicidal lamp at a dose rate of 1.2 J/m 2/s at 254
nm as measured by an Ultraviolet Products radiometer. Luria Bertani (LB)
media (Maniatis et al., 1989) (100 mL) was inoculated with 2 mL of a fresh
overnight culture of DL7/pGW16 cells. The culture was grown to mid/late
log phase, 10" cells/mL (OD600 = 0.6; Klett a 100), centrifuged at 4000 g
for 10 min at 40 C, and the cells resuspended in 50 mL of ice cold 10 mM
MgSO 4 . The resuspended cells (25 mL) were dispensed into 150 mm x 15
mm plastic petri dishes and UV irradiated at the fluences indicated above.
Following UV-irradiation each culture was added to 50 mL of prewarmed
2xLB media, incubated at 37 0 C for 30 min to express the SOS mutagenic
processing functions maximally (Defais et al., 1976), and made competent
125
for electroporation according to procedures described (Yarema et al., 1994).
Briefly, cells were centrifuged at 4,000 g for 10 min, the pellet resuspended
in an equal volume of ice cold sterile H20, and the cells recentrifuged at
8,000 g for 10 min. The cell pellet was resuspended in one half volume H2 0
and recentrifuged at 13,000 g for 10 min. From each 100 mL of cell
culture, 400-600 pL of UV-treated cells (brought up to volume with cold
sterile H2 0) was prepared for transformation (i.e., 2-3 samples/100 mL
culture at 190 pL of cell suspension per transformation). One hundred mL of
culture was used to prepare 800 pL - 1.2 mL of non-UV-treated cells for
transformation (i.e., 4-6 samples/100 mL). Prior to electroporation, cells
were plated on LB plates and incubated at 37 0 C for colony formation in order
to determine cell survival at the various UV fluences.
Next, UV-irradiated RF M13mp18 DNA (50 ng) or unirradiated control
DNA (10 ng) was mixed with 190 pL of each cell suspension (UV-irradiated
at 0, 15, 30, 45, 60, or 75 J/m 2). Each mixture was transferred to a cold
Bio-Rad Gene Pulser cuvette (0.2 cm) and electroporations performed with a
BTX Electro Cell Manipulator 600 system set at 50 yF and 129 Q. The
electroporation field strength determined to be optimal for cell survival and
transformation efficiency was 7.5 kV/cm for UV + DL7/pGW16 cells
(optimized with cells irradiated at a UV fluence of 50 J/m 2), and 12.5 kV/cm
for UV- DL7/pGW16 cells. Immediately following electroporation, 1 ml of
126
SOC medium (Hanahan, 1985) was added and a portion of the bacterial
suspension was plated in the presence of GW5100 cells, X-Gal, and IPTG
(Messing, 1983) to determine the transformation efficiency. Additional SOC
(2 mL) was added, and the mixture incubated for 1.5 to 2 h at 37 0 C for
progeny phage production. Each mixture was centrifuged at 17,000 g for
10 min to pellet the cells. The phage-containing supernatant was
recentrifuged and stored at 40C. Mutational analysis of the progeny was
based on the ability of the M13mpl18 a-donor lacZ' peptide fragment to
complement the E. coli a-acceptor M15 protein to produce functional Pgalactosidase. Null mutations within lacZ' yield clear or light blue plaques
upon plating in the presence of GW5100 cells, X-Gal, and IPTG. Thus, the
mutation frequencies from each transformation were calculated by
determining the fraction of clear and light blue plaques within the total
number of clear, light blue, and dark blue plaques. Dark blue plaques consist
mostly of wild-type phage; phage containing mutations that do not give rise
to a null phenotype for P-galactosidase activity, however, are also dark blue.
SOS optimization was also carried out with ss M13mp18 DNA.
Procedures were as described above for RF DNA except that the cells were
irradiated at 0, 30, 45, 60, and 75 J/m 2
127
4. Transformation of E. coil cells with singly-modified genomes
Immediately following preparation, each genome was centrifuged for 4
min at 2,000 g at 4°C through a Sephadex G-50 Quick Spin column
preequilibrated in H20. A portion (30 pL) of each reaction mixture was
examined by gel electrophoresis and the presence of completely ligated
genomes was confirmed by comigration with ss circular DNA. The
remainder of the DNA was maintained on ice until transfected into E. co/i.
Transfections were always performed within 3 h of genome preparation.
E. coli DL7 and DL7/pGW16 cells were prepared for transformation by
electroporation as described above. Half of each culture was UV irradiated
at 45 J/m2 , a fluence that proved optimal for induction of SOS-dependent
mutagenesis (see Results). For each transformation, 190 pL of the cell
suspension was added to 5 pL of the DNA solution. The total amount of
potentially viable DNA represented in this volume was determined according
to procedures described in Chapter III for each genome, and represents
approximately 44 ng of unmodified, 36 ng of unmodified A, 24 ng of M13AFB 1 , and 17 ng of M13-AP genome. Electroporation and plating procedures
were carried out as described above except that NR9050 was used as the
plating bacteria instead of GW5100.
128
5. Mutant enrichment procedure
E. coli DL7/pGW16 cells (UV + and UV-) were transformed as described
above with M13-AFB 1 , M13-AP, and control genomes. In each case
approximately 106 progeny phage, representing roughly 2 x 104 individual
transformation events (infective centers), were pooled from 8-16
independent transformations and used to produce RF DNA by using the
QIAGEN midi plasmid purification system. Mutations within the Sful
restriction site, resulting from either AFB,-N7-Gua or the AP site, render the
sequence refractory to digestion by Sful. Thus, the mutant population was
enriched by digesting agarose gel-purified RF DNA (250 ng) with Sful (30
units) at 37 0 C for 2 h in the restriction buffer supplied with the enzyme. As
controls, genomes were incubated in buffer lacking the enzyme. The
reaction mixtures were diluted 100-fold in H20, and 100 pL (5 ng of DNA)
was used to transform 100 pL of DL7 cells. Electroporation, plating, and
phage production were as described above.
6. Mutation frequency determination
In order to compare directly the genetic effects of AFB1 and the AP site,
the mutation frequency and specificity for each lesion was calculated in
exactly the same manner. Therefore, mutation frequencies for both lesions
in DL7/pGW16 cells represent the fraction of surviving genomes that results
from the insertion of nucleotides dAMP, dGMP, dTMP, but not dCMP,
129
opposite the lesion. Genomes resulting from the insertion of dCMP opposite
the AP site would be selected against by the restriction enzyme Sful during
the mutant enrichment procedure. Furthermore, the fraction of genomes
containing the wild-type sequence may result, only in part, from insertion of
dCMP opposite the AP site. M13-AFB1 characterization studies indicate an
unmodified contamination of 5% (see Chapter III). Thus the M13-AP,
derived from M13-AFB 1 , should have the same level of M13-G
contamination. Contaminating M13-G likely contributes to the fraction of
progeny that appears to be the result of dCMP insertion opposite the AP-site,
especially since they are likely to survive much better than M13-AP
genomes.
DNA used in the above transformations gave rise to plaques of three
color types: colorless, dark blue, and light blue. Dark blue plaques resulted
from restoration of the M13mp7L2 lacZ reading frame, which is out of frame
by + 2, upon ligation of the 13-mer oligonucleotide. Light blue plaques
resulted from a targeted G--T transversion at the site of the adduct. The 13mer oligonucleotide contains the AFB 1-N7-Gua within a 5'-GAA-3' codon,
and a G->T transversion within this codon results in an in-frame ochre (5'TAA-3') that is partially suppressed in E. coli NR9050 cells; partial
suppression yields light blue plaques. Plaques resulting from all other
adduct-induced base substitutions were dark blue. Colorless plaques
130
resulted from large deletions in the lacZ gene as a result of genetic
engineering procedures (discussed below), or were due to contaminating,
undigested parental M13mp7L2 DNA. Frameshift mutations within the
target sequence would also have resulted in colorless plaques.
RF DNA, prepared from DL7/pGW16 transformations, was treated (Sfu +)
or not (Sfu-) with Sful, transformed into DL7 cells, and plated for plaque
formation. The restriction resistant fraction RRF (ratio of plaques from Sfu +
to those from Sfu-) was higher than the true mutant fraction of the lesion
under study because the Sfu + sample included some wild type DNA that
had evaded digestion and some large deletions attributable to genetic
engineering procedures (these deletions were due to exonuclease III
digestion on the 3' side of the gap and ligation across the gap, in the
absence of 13-mer oligonucleotide ligation; digestion was such that the lacZ
frame remained intact). To calculate the true mutant fraction, a sampling of
dark blue and light blue plaques was sequenced (Meeker et al., 1993) from
Sfu + transformations. The fraction of each class that contained mutations
targeted within the six base Sful site was determined. The mutation
frequency (%) of the lesion was 102(RRF)(fraction of plaques in the RRF
determined be true mutants by sequencing). See Figure 19 for enrichment
procedure and MF determination. In some cases (i.e. M13-G and M13-GA
control transformations) two rounds of mutant enrichment were required.
131
Thus, the first RRF (RRF,) was used to prepare RF DNA that was then
subjected to another round of Sful digestion and transfection into E. coli.
The fraction of resistant DNA at this point (RRF 2) was determined as the
ratio of plaques from Sfu + to those from Sfu- of RRF 1 DNA. Mutation
frequencies were calculated as 102(RRF 1 x RRF 2)(fraction of plaques in RRF 2
harboring mutations within the Sful sequence).
Given that the predominant mutations observed for both AFB,-N7-Gua
and the AP site, in DL7/pGW1 6 cells, were phenotypically scorable "light
blue" mutants (G-*T and AP->T, respectively), comparative mutation
frequencies resulting from transformations of M1 3-AFB 1 and M1 3-AP into
DL7 cells (cells lacking pGW16) represent only the frequency of "light blue"
G--T and AP-,T mutations, respectively, relative to the total number of light
blue plus dark blue plaques. Given that the G-T frequency was so low from
M13-AFB1 DL7 transformations (<0.05% and 0.5%), Sful mutant
enrichment of the progeny phage was carried out to facilitate calculation of
the frequency of light blue plaques. Sful enrichment was not necessary,
however, for M13-AP transformations since the AP->T frequencies were
sufficiently high (0.6% and 18%) for accurate MFs to be obtained from light
blue plaque numbers. Experiments performed with M13-AFB1 and M13-AP
progeny, where MFs were determined by calculating light blue frequencies
both prior to, and subsequent to Sful mutant enrichment, indicated that the
132
mutant enrichment procedure does not affect the final calculated mutation
frequency. Mutation frequencies were determined from Sful mutant-enriched
pooled phage for M13-AFB1 (from three ligations and subsequent
transformations). Mutation frequencies represent an average of three
ligations and subsequent transformations for M13-AP. In SOS(-) cells light
blue frequencies (AP--T) were 0.8% ±0.3%; MF was 0.4% when determined
from plating pooled phage from the three transformations. In SOS(+) cells
light blue frequencies (AP--T) were 15% ± 5%, and 18% when determined
from plating pooled phage. Mutant sequences from DL7 transformations of
each genome were confirmed by sequence analysis of 24 light blue plaques.
C. Results
1. Optimal conditions for SOS induction
The optimal UV fluence for induction of SOS-processed mutagenesis in
DL7/pGW16 cells was determined by transfecting UV-damaged M13mp18
RF or ss DNA into cells irradiated by UV with a range of fluences (0, 1 5, 30,
45, 60, and 75 J/m 2). The mutation frequencies for both RF and ss DNA
experiments are indicated in Table 1. The mutation frequency for the RF
transformations increased (- 25-fold) in a UV dose-dependent manner, from
0.03% at 0 J/m 2 up to 0.75% at 30 J/m 2 where it leveled off until 75 J/m 2;
at this fluence the MF dropped to 0.12%. A similar pattern was observed
133
with UV-damaged ss DNA. The mutation frequency for ss transformations
increased - six-fold, from 0.8% at 0 J/m 2 to 5.1% at 30 J/m 2 where it leveled
off until 75 J/m 2. As indicated in Table 2, cell survival begins to decrease
above 20 J/m 2. The optimal fluence for SOS induction was determined to be
45 j/m 2; at this fluence mutagenicity was well above background and was
well within a range of UV doses that appears to induce similar mutation
frequencies in M13mp18 DNA (30 to 60 J/m 2). At this fluence cell survival
was still relatively high (-40%).
It was assumed that these conditions would also be optimal for induction
of the SOS response in E. coli DL7 cells, which are different from
DL7/pGW16 cells only in that they lack the mucAB-encoding pGW16
plasmid.
2. Mutation frequencies of AFB,-N7-Gua and the AP site
Single-stranded AFB 1 and AP site containing genomes were prepared
(Chapter III) and introduced by electroporation into E. coli DL7/pGW16 cells
as described above. As indicated in Table 3, the total mutation frequency
for AFB,-N7-Gua in UV + DL7/pGW16 cells was 4.0%, including 2.9% G--T.
This frequency represents an average calculated from two independent
pooled phage analyses (phage were pooled from eight individual
transformations) and was confirmed by the analysis of two individual
134
ligations and subsequent transformations in order to ensure that the average
mutation frequency and specificity of "pooled phage" reflected that of an
individual ligation/transformation event. The mutation frequency of AFB,-N7Gua in UV- DL7/pGW16 cells was 2.9%, including 2.1% G->T. This
frequency was also calculated from pooled phage, representing eight
individual transformations, and the frequency was confirmed by the analysis
of two individual ligations and subsequent transformations.
The AFB,-N7-Gua data do not indicate a significant difference in the
mutation frequencies between UV + and UV- DL7/pGW1 6 cells. In order to
determine if DL7/pGW1 6 cells were expressing the mucAB mutagenesis
enhancing gene products even without induction by UV light, and also to
compare the specificity of AFB,-induced to AP site-induced mutations
(discussed below), I examined in parallel the mutation frequency and
specificity of an AP site, generated by heating the AFB1 genome to release
the modified base. AP sites are known SOS-dependent mutagens (Loeb and
Preston, 1986; Lawrence et al., 1990). The data in Table 3 indicate that the
AP site control (phage were pooled from nine individual transformations) was
mutagenic in both UV- and UV+ DL7/pGW16 cells: 31% including 16%
AP->T (UV-), and 50% including 37% AP->T (UV +). By contrast, the
mutation frequencies of the AP site in UV- and UV + DL7 cells (i.e., cells
lacking mucAB) were 0.6% and 18% AP-*T, respectively. The mutation
135
frequencies of AFB,-N7-Gua in UV- and UV + DL7 cells were <0.05% and
0.5% G->T, respectively. These data indicate that, in contrast to
DL7/pGW16 cells, UV induction of SOS functions in DL7 cells markedly
enhances (10-30 fold) AFB,-N7-Gua and AP site mutagenesis. The evident
expression of the SOS mutagenic processing phenotype in DL7/pGW16 cells
without preinduction with UV is possibly due to a point mutation in pGW1 6
within the LexA repressor-binding site of the mucAB operator (McNally et al.,
1990). This mutation weakens LexA protein binding to the mucAB operator
resulting in an elevated level of mucAB expression (McNally et al., 1990),
seemingly sufficient to effect an SOS processing phenotype in these
unirradiated cells. This model is supported by the observation that a small
amount of MucA protein can be spontaneously cleaved to its activated form
MucA'. Apparently this cleavage occurs in the absence of activated RecA
(RecA*) (Hauser et al., 1992).
3. Mutational specificity of AFB,-N7-Gua and the AP site
The mutational specificity of AFB,-N7-Gua and the AP site were
examined in both UV- and UV+ DL7/pGW16 cells. The distributions are
indicated in Figure 20. The mutations observed for AFB 1 were predominantly
G--T transversions targeted to the original site of the adduct (base 6240) in
both UV- cells (74% of all mutations) and UV + cells (73% of all mutations).
G->A transitions were less frequent at 18% in UV- cells and 13% in UV +
136
cells. G--C transversions were infrequent at 1.4% in UV- cells and 2.5% in
UV + cells.
The most striking of all the mutations observed were base substitutions
at the base 5' to the modified guanine (base 6239): C--*T (7.0% of all
mutations) in UV- cells, and C--T (10.0%) and C-->A (2.5%) in UV + cells.
Evidence that these non-targeted 5' mutations were due to the aflatoxin
moiety was obtained by examining the mutational specificity of the AP site.
As indicated in Figure 20, the predominant mutations observed were AP--T:
52% of all mutations in UV- cells, and 74% in UV + cells, followed by AP--A
(34% in UV- cells, and 20% in UV+ cells) and AP--C (14.5% in UV- cells,
and 6.0% in UV + cells). Only two tandem mutations (not shown in Figure
20) involving base 6239 were observed for the AP site in UV + cells:
Cp(AP)--ApC (0.4%) and Cp(AP)--TpC (0.4%). One tandem mutation was
observed in UV- cells: Cp(AP)- TpC (0.6%). Thus, unlike the results
obtained with AFB,-N7-Gua, no significant number of mutations was
observed at base 6239. These results suggest that the mutations induced at
base 6239 are a signature mutation of the aflatoxin moiety and not due to
an AP site.
Earlier work has shown that frameshift mutations occur in DNA modified
globally by reactive derivatives of AFB 1 (Sambamurti et al., 1988; Refolo et
137
al., 1987). It was therefore of interest to determine to what extent these
mutations may have been caused by AFB,-N7-Gua in my system. This
analysis was complicated, however, owing to a high background of "genetic
engineering" mutations that had the same phenotype (colorless) as the
putative frameshifts. Sequence analysis of these colorless plaques indicated
that a significant proportion contained large deletions originating from
exonuclease III digestion on the 3' side of the gapped genome and
subsequent ligation across the gap; as described above these "genetic
engineering" mutants were not selected against during the mutant
enrichment process. Therefore, in an attempt to determine the level of AFB,induced frameshift mutations, a sufficient number of colorless plaques was
sequenced so that it was possible to set an upper limit of their occurrence.
Upon sequencing 300 clear infective center plaques, only one
frameshift/base substitution mutation was observed in UV + transformed
DL7/pGW1 6 cells (TTCGAA to TTTAA); these were present at the same
frequency, however, within the clears from M13-G transformations and were
therefore determined to not be due to the AFB,-N7-Gua adduct. The upper
limit of occurrence of AFB,-N7-Gua-induced frameshifts was determined as
follows. Since clear plaques represented -30% of the total number of
infective centers, subsequent to M13-AFB, transfection and plating, 300
clear plaques were present among - 700 dark blue and light blue plaques.
True frameshifts would have been generated from AFB,-N7-Gua containing
138
genomes; the majority of these genomes gave rise, either by base
substitution mutations or correct lesion bypass, to dark blue and light blue
plaques. Thus, the number of AFB,-N7-Gua-induced frameshifts within the
total number of blue plaques represents the real frameshift mutation
frequency. Therefore, the absence of a true frameshift within 300 clears is
the absence of a true frameshift in 700 blue plaques. Thus, the upper limit
of frameshift occurrence was determined to be 1/700(102) = 0.15%. These
data indicate that at least in this system AFB,-N7-Gua-induced frameshifts
are very infrequent events.
4. Genotoxicities of AFB,-N7-Gua and the AP site
The survival of the AFB,-N7-Gua and AP site containing genomes were
examined in parallel with mutation frequencies. Studies have indicated that
induction of the SOS response increases not only the mutation frequency of
transfected modified DNA but also the survival of the DNA relative to the
unmodified control (LeClerc and Istock, 1984). Since the mutation
frequency of M13-AFB 1 and M13-AP in DL7/pGW16 cells did not increase
significantly with UV-irradiation of the cells, survival was not expected to
increase either. As indicated in Table 4 survival of M13-AP did not increase
with DL7/pGW16 UV treatment. The survival of M13-AFB 1 , however,
unexpectedly decreased with host UV treatment. In addition, although
survival does not decrease, infective center numbers for M13-AFB 1 did not
139
indicate an increase in survival subsequent to transfection into DL7 cells
induced for the SOS response, whereas the mutation frequency does
increase with host UV irradiation. However, survival did increase (three-fold)
for M13-AP in SOS induced DL7 cells; this was most likely due to the fact
that the mutation frequency for M13-AP in SOS-induced compared to SOSuninduced DL7 cells was large (-30-fold).
It is possible that the transformation efficiencies did not reflect the actual
genotoxicities of AFB,-N7-Gua or the AP site because the total amounts of
transfected genome were too high. Cells were transformed with 20-50 ng of
DNA, an amount that has been shown to be beyond the linear range
observed for plaque formation as a function of the amount of DNA
transfected (K. J. Yarema, personal communication). Therefore, the
observed number of plaques may not accurately reflect the actual
genotoxicities of these DNA lesions.
D. Discussion
DNA globally modified with activated forms of AFB 1 contains at least
three DNA lesions: AFB,-N7-Gua, AFB 1-FAPY, and the AP site. The aspect
of my work was to determine the relative contributions of AFB,-N7-Gua and
the AP site to the mutagenic spectrum of AFB 1 . Both lesions were situated
140
at a specific site within the M13 bacteriophage genome (Chapter llI) and the
mutation types and frequencies directed by each were determined.
The predominant mutations caused by AFB,-N7-Gua (Figure 20) were
G--T transversions (- 75% of all mutations), similar to that observed previously
with metabolically activated AFB, in E. co/i induced for the SOS response
and containing the mucAB mutagenesis enhancing gene products (Foster et
al., 1983), and in mammalian systems (Trottier et al., 1992; Aguilar et al.,
1993). A possible premutagenic lesion responsible for this mutation is the
AP site that results from the facile depurination of the AFB,-N7-Gua. In the
experimental system described here there are several possible ways that an
AP site may be responsible for these mutations. It is possible, but unlikely,
that the M 13-AFB1 genomes used for these studies were contaminated with
a small amount of M13-AP genome generated during genome construction
procedures. Genome characterization data (Chapter III) suggest that an
enzymatic step (exonuclease III treatment) applied at the final stage of
genome construction efficiently inactivated any contaminating AP site
genome, leaving M13-AFB, 295% pure with a 5% contamination of
unmodified genome. A more likely mechanism by which the AP site could
be responsible for the observed mutations is through spontaneous or enzyme
mediated in vivo depurination of AFB,-N7-Gua prior to DNA replication past
the site of the adduct. AFB,-N7-Gua is removed from rat liver DNA with a
141
half-life of 7.5 h (Croy and Wogan, 1981a), suggesting that the adduct is
intact during the short time span of replication of M13-AFB1 in E. coil
(Messing, 1983). Although we may predict, based on the aforementioned
considerations, that the AP site does not effect the observed G->T
transversions, we cannot entirely rule out the possibility that depurination of
the primary AFB 1 DNA adduct is a precursor to these mutations. Hence, it
was deemed necessary to compare the mutational spectra of AFB,-N7-Gua
and the AP site in the context of both the mutational specificity and the
genetic backgrounds required to produce the observed mutations.
The mutational specificity of the AP site (Figure 20) is similar to what
others have observed in E. coil induced for the SOS response (Loeb and
Preston, 1986; Lawrence et al., 1990). Thus, the predominant mutation
effected by both AFB, and the AP site appears to result from insertion of
dAMP opposite the site of the adduct. For both lesions the principal
mutations required some form of SOS mutagenic processing, but the relative
contributions of UmuDC and MucAB to these mutations were different for
the two lesions (Table 3). The G--T and AP-.T mutation frequencies in Table
3 are depicted in Figure 21. The data represent three SOS mutagenic
environments: largely umuDC-mediated mutagenesis (UV+ DL7), largely
mucAB-mediated mutagenesis (UV- DL7/pGW1 6), and combined umuDCand mucAB-mediated mutagenesis (UV + DL7/pGW16). The data suggest
142
that the umuDC gene products enable the insertion of dAMP opposite the AP
site (AP--T) at a frequency similar to that resulting from the mucAB gene
products alone (18% and 16%, respectively). Furthermore, the combination
of umuDC and mucAB expression, in UV + DL7/pGW16 cells, doubles the
AP->T mutation frequency, indicating that both UmuDC and MucAB
contribute significantly to the frequency of AP- T mutations. However, in
contrast to AP site mutagenesis, AFB, -N7-Gua mutagenesis relies much
more heavily on the gene products of mucAB than umuDC to effect G->T
transversions; the umuDC and mucAB gene products induce G->T
transversions at frequencies 0.5% vs. 2.1%, respectively. In addition, and
in contrast to AP site mutagenesis, the combined expression of umuDC and
mucAB (UV+
DL7/pGW16) resulted in only a small increase in AFB, induced
G->T transversions (from 2.1% to 2.9%).
The AFB,-N7-Gua data are consistent with studies carried out on DNA
globally modified with AFB, showing a strong MucAB dependence for
mutagenesis (Foster et al., 1988; Urios et al., 1994). The exact
mechanisms of mutagenic processing by UmuDC and MucAB are unclear,
but there do appear to be differences between the two that may be
correlated to different levels of mutagenic processing (Hauser et al., 1992;
Blanco et al., 1986). Activated RecA protein promotes proteolytic cleavage
of UmuD (Shinagawa et al., 1988; Burckhardt et al., 1988; Nohmi et al.,
143
1988) and MucA (Shiba et al., 1992). The interaction of RecA with UmuD
and MucA is believed to be responsible for promoting certain mutagenic
processes of these proteins. It has been suggested that the functional
differences between UmuDC and MucAB proteins might be due to their
different dependence on the role of RecA protease (Blanco et al., 1986;
Hauser et al., 1992; Shiba et al., 1992). Studies carried out by Shiba et al.
(1992), involving site-directed mutagenesis of the putative cleavage site of
MucA have shown that strains containing mutant MucA protein (harboring a
threonine instead of an alanine at the cleavage site) do not process MucA to
its active form MucA', but are still as proficient as strains containing wildtype MucA in promoting UV mutagenesis. These results have raised the
possibility that both intact MucA and RecA-processed MucA (MucA') are
active in mutagenesis. On the other hand, studies carried out by Hauser et
al. (1992), in which a very sensitive method was employed for the detection
of the cleavage product of MucA (MucA'), have shown that MucA is cleaved
much more readily than UmuD, and that restoration of mutagenesis functions
to normally nonmutable RecA-deficient strains by MucA protein is correlated
with the appearance of the cleavage product, MucA'. These results suggest
that the differences in mutagenic processing exhibited by MucAB and
UmuDC are correlated to the efficiency of posttranslational processing of
MucA and UmuD rather than an intrinsic mutagenic-processing phenotype of
the intact MucA protein. Both studies, however, indicate that MucAB
144
proteins appear to be less dependent than UmuDC on the activity of RecA
protease. Previous work has shown that there is an interaction between
RecA and UmuDC proteins targeted at DNA lesions (Lu et al., 1986). It has
also been suggested that RecA is involved in targeting UmuD, UmuD', and
MucA' to the DNA (Frank et al., 1993). Furthermore, previous studies have
indicated that UmuDC may actually block DNA replication (Marsh and
Walker, 1985) and that RecA protease activity may be involved in alleviating
the UmuDC-dependent replication inhibition to allow DNA elongation and,
therefore, mutagenesis (Blanco et al., 1986). It is likely that the MucAB
proteins perform a similar role to UmuDC in the mutagenic process.
Therefore, the higher mutability of certain DNA lesions in the presence of
MucAB proteins may reflect the less stringent requirement of the MucAB
proteins for RecA mediated proteolytic activation. Hence, in a MucAB
environment these lesions would be bypassed more efficiently and
consequently more mutagenic.
The data in this study indicate a direct difference in the dependence on
MucAB compared to UmuDC for the mutagenic bypass of the AP site and
AFB,-N7-Gua. AFB,-N7-Gua appears to rely more heavily on MucAB than
UmuDC to effect G-"T mutations, whereas the AP site seems to effect AP--T
mutations equally well in the presence of either set of proteins. If the
requirement for RecA is less stringent in the presence of MucAB proteins, a
145
MucAB-blocked AFB,-N7-Gua may be alleviated more efficiently than a
UmuDC-blocked AFB,-N7-Gua. Thus, the differences in the genetic
requirements for the mutagenesis of AP sites (AP--T) and AFB,-N7-Gua
(G--T) may be owed to a UmuDC-blocked AP site that simply does not
require as high a level of UmuD cleavage for lesion bypass as does a
UmuDC-blocked AFB,-N7-Gua; only in the presence of a MucAB block,
where RecA protease activity is not as crucial for MucA cleavage, can AFB,N7-Gua lesions be bypassed. Moreover, although the AP site and AFB,-N7Gua give rise ultimately to the same mutation, the apparent differences in
the genetic requirements for mutagenic bypass of the two lesions suggest
that AFB,-induced G--T transversions are promoted through mechanisms not
involving, in a predominant manner, the simple depurination of the AFB,-N7Gua adduct.
Since it is predicted that AFB,-N7-Gua, and not the AP site, gives rise to
a significant proportion of the observed G--T transversions then what might
be the mechanism of this mutagenic bypass? It is very possible that AFB,N7-Gua is a noninformational lesion. In E. coli induced for the SOS response
the base most often inserted opposite noninformational lesions is dAMP; this
mechanism would ultimately give rise, in the case of AFB,-N7-Gua, to a G-*T
transversion. Another possibility that may result in AFB,-N7-Gua induced
G--T transversions involves a potentially misinformational AFB,-N7-Gua that
146
mispairs with an incoming dATP where the base and pentose moieties are in
the syn conformation (Figure 22) (the basis for this model, however, has not
been established experimentally). AFB,-N7-Gua was also shown to give rise
to a significant proportion of G-A transitions, - 13% of the total mutations
(total mutation frequency of -0.5%).
Again, it is possible that this mutation
is due to insertion of dTMP opposite a noninformational AFB,-N7-Gua. As
we might expect, based on polymerase preference favoring incorporation of
adenine opposite noninformational lesions (Strauss, 1991), dTMP would be
inserted less frequently and therefore would give rise to a smaller percent of
the total mutations (13% in these studies vs. 75% dAMP incorporation). An
alternative possibility for G-!A transitions is based on the observation that
N7 alkylation is known to change the pKa value of guanine from 9.2 to 7.1,
resulting in deprotonation at the N1 position. The resulting zwitterionic form
of guanine was hypothesized to be capable of participating in G:T mispairing
(Lawley and Brooks, 1961). Crystallographic evidence supports
deprotonation and altered base pairing of N7 alkylated guanines (Yamagata
et al., 1983). Therefore, as depicted in Figure 23, the zwitterionic form of
AFB,-N7-Gua may mispair with thymine, giving rise ultimately to a G-A
transition. Hence, AFB,-N7-Gua may be a misinformational lesion, a
noninformational lesion, or both. Consequently, the mutagenic bypass of
AFB,-N7-Gua may comprise several mechanisms.
147
The most striking difference between the mutational specificity of AFB,N7-Gua and the AP site is represented by a significant proportion (13%) of
mutations targeted to M13 base 6239 (5' to the AFB1 modified guanine)
(Figure 20). Data indicate that these mutations are not induced by the AP
site. Given that the total mutation frequency for AFB,-N7-Gua in this system
is high (4.0%) compared to many helix-distorting DNA adducts (Wood et al.,
1990; MacKay et al., 1992; Basu et al., 1993), 13% of the mutations
represents a significant mutant population at 0.5%. The observed
mutational asymmetry disposed to the 5' side of the adduct is in accord with
the molecular architecture of AFB,-N7-Gua, established by NMR
(Gopalakrishnan et al., 1990) showing the aflatoxin moiety of the aflatoxinguanine adduct intercalated on the 5' face of guanine (Figure 24). It is
unknown to what extent the intercalative state exists at the moment of
replication, but it is reasonable to speculate that the aflatoxin moiety remains
proximal to the 5' base during DNA synthesis, thus potentially interfering
with 5' base pairing. As shown in Figures 24 and 25, the C-G base pair at
position 6240 appears to be distorted, most likely due to the AFB, moiety. It
is proposed that the AFB 1 moiety may shift in the 5' direction, perhaps as a
consequence of normalization of the C-G pair. As a result, C6239 may rotate
out of the helix to accommodate the AFB, moiety, and thus not be available
for base pairing. The C->T transition at position 6239 might then occur
owing to the insertion of adenine across from the AFB, moiety. However,
148
examination of a DNA molecular model indicates only one potential hydrogen
bond between that the aflatoxin moiety and the incoming dATP, i.e.,
between the protons at the N6 position of adenine and the oxygen of the
carbonyl group (position C1) of the cyclopentanone ring of the aflatoxin.
Another possibility is that dATP may stack with the AFB1 moiety in such a
way that it might then be incorporated into the newly synthesized DNA
strand during DNA replication (Johnston and Stone, 1995). It is also
possible that the 'A rule' may be involved in the mutations observed at the
base 5' to AFB,-N7-Gua. The aflatoxin moiety may be encountered by the
polymerase and read as a noninformational base at the 5' position. In
addition, the aflatoxin moiety may render the 5' base noninformational
simply due to their relative proximities (in this case the 5' base would not
rotate out of the helix). In this case, however, one might expect to observe
an insertion mutation owing to the polymerase possibly encountering both
the 5' cytosine and the aflatoxin moiety as "non-informational" bases. Since
this was not observed, rotation of the 5' base out of the DNA helix is an
attractive model for the mechanism of this particular mutation.
The observation that mutations occur at base 6239 suggests that
mutational spectra obtained in previous studies from DNA globally modified
with AFB 1 may harbor mutations at bases 5' to potentially modified
guanines. Since guanine residues that are flanked by AT rich sequences are
149
poor targets for modification by AFB 1 (Muench et al., 1983; Benasutti et al.,
1988) it is conceivable that the majority of 5' mutations would reside in 5'
cytosines or guanines; these would mistakenly have been scored as resulting
from either an additional AFB,-modified guanine in the same strand (GGX) or
an AFB,-modified guanine in the opposite strand (CGX) (underscored base is
potentially modified base; italicized base is potentially mutated 5' base). It is
noteworthy that only through the use of a specifically placed AFB,-N7-Gua
adduct, as in the present studies, is it possible to acquire data suggesting a
mechanism for AFB1 mutagenesis that involves not only the modified base
but also the base 5' to the intercalated aflatoxin moiety (a cytosine in this
study). Future work will examine how bases other than cytosine 5' to the
adduct influence mutagenesis at this site. Such studies may shed light on
the so far inexplicable observation that AFB 1 gives rise to a small percent of
mutations at A:T pairs (Foster et al., 1983; Sahasrabudhe et al., 1989).
This work strongly suggests that the aflatoxin moiety of the primary
aflatoxin adduct, AFB,-N7-Gua, gives rise to a significant proportion of the
observed AFB,-induced mutations. The data indicate that the aflatoxin
moiety gives rise to 5' non-targeted mutations at base 6239, and to targeted
G--T transversions at base 6240. Since the half-life of AFB,-N7-Gua in vivo,
7.5 h (Croy and Wogan, 1981a), is shorter than the time required for DNA
replication in human cells, one might predict that the AFB,-N7-Gua is
150
removed from DNA before a mutation can be fixed by the DNA polymerase.
However, human populations that are constantly exposed to aflatoxin in their
diet may have a steady state level of AFB,-N7-Gua DNA adducts that might
be responsible, in part, for the mutations observed in human hepatocellular
carcinomas. It will be of interest to examine the mutagenic potential of the
more chemically stable AFB,-induced adduct, AFB,-FAPY, in comparison to
the mutational spectra obtained in this thesis work for AFB,-N7-Gua and the
AP site.
151
Table 1: SOS Optimization. Mutation frequencies (%) of UVirradiated RF and single-stranded (SS) M13mp18 DNA in
DL7/pGW16 cells irradiated under various UV fluences.
Dose to DNA (J/m 2)
Dose to Cells (J/m 2)
0
15
30
45
60
75
RF
0.04 0.03 0.06 0.05 0.15 0.05
50
0.03 0.03 0.75
1.0
1.2
0.12
0.4
ND
1.6
0.6
0.5
0.8
0.8
ND
5.1
5.2
3.8
4.0
SS
50
152
Table 2: DL7/pGW16 survival (%) subsequent to UV-irradation
under various fluences.
Dose to Cells (J/m 2 )
% survival
0
10
20
30
40
50
60
100
89
89
45
44
32
25
153
Table 3: Mutation frequencies (%) of AFB,-N7-Gua and the
AP site.
UV Treated
AFB 1-N7-Gua
Unmod
<0.05
0.5
0.04
0.06
DL7 a
DL7/pGW1 6 b
-
2.9 (2.1)
+
4.0 (2.9)
UV Treated
AP Site
0.06
0.20
Unmod A
DL7 a
0.6
18.0
DL7/pGW1 6 b
31.0 (16.0) 0.13
50.0 (37.0) 0.24
0.04
0.05
aMutation frequencies in DL7 cells represent the fraction of targeted
G--T mutations or AP--T mutations from AFB,-N7-Gua or the AP site,
respectively (i.e. fraction of light blue plaques relative to the total
number of dark blue plus light blue plaques).
bMutation frequencies in DL7/pGW16 cells represent all mutants.
Numbers in parentheses represent the fraction of targeted G--T or
AP--T from AFB,-N7-Gua or the AP site, respectively, as described
in'.
154
Table 4: Genotoxicities of AFB,-N7-Gua and the
AP site; percent (%) survival relative to control.
UV-
UV+
42
47
18
54
DL7/pGW16
M13-AFB,
M13-AP
DL7
M13-AFB,
M13-AP
44
11
155
Figure 19. Sful mutant enrichment procedure and mutation frequency
calculation. RF DNA was prepared from progeny phage, comprised of both
mutant and wild-type sequences, produced after transformations of cells
with M13-AFB 1, M13-AP, M13-G, or M13-GA (see text for details). The
DNA was subjected to digestion with restriction enzyme Sful; mutations
within the Sful restriction site of the RF DNA render the sequence refractory
to digestion by the enzyme. Thus, the mutant population was enriched
given that Sful-digested RF III DNA (linear) is not viable upon transfection
into E. coli. The restriction resistant fraction (RRF) was calculated by
determining the relative survivals of the digested and buffer control samples.
Sequence analysis of individual plaques from transfections of Sful digested
DNA revealed the fraction of true mutants within the RRF. The mutation
frequencies were calculated as shown. See text for details.
156
Progeny
Phage
0M
RF DNA
preps
Mutant
RF genome
(mixture of mutant
and wild-type DNA)
0 0 0
Wild-type RF
genomes
1)Buffer
1) R
Dige
Control
ransfect
) E. coli
M
2) Transfect
into E. coli
0
0 0
O
Determine
Restriction Resistant
Fraction (RRF)
Sequence Analysis
- Wild-type
- Genetic engineering mutations
Ru
(large and small deletions)
Determine Fraction
of Adduct-Induced
Mutations
- Aflatoxin specific mutations
Mutation Frequency
(102)RRF
=
(fraction of plaques
in the RRF determined to be
true mutants by sequencing)
157
100%
Figure 20. Type and percent distribution of AFB,-N7-Gua and AP site
induced mutations in E. co/i DL7/pGW16. G* is the position of the lesion. A
total of 926 "dark blue" sequences was examined from M13-AFB1 and M13AP transformations, collectively. The number of dark blue mutants
sequenced with mutations in the Sful restriction site was 96 for AFB,-N7Gua (UV +), 59 for AFB,-N7-Gua (UV-), 80 for AP site (UV +), and 85 for AP
site (UV-). The number of light blue mutants sequenced (G-'T or AP->T) was
96 for AFB,-N7-Gua (UV+), 48 for AFB,-N7-Gua (UV-), 24 for AP site (UV-),
and 24 for AP site (UV +). The number sequenced for each individual
mutation is shown in parentheses, and is represented as the number
sequenced / total number of dark blue or light blue true mutants sequenced
(#/Total). Mutants at <0.5% of the total number of mutants are not
reported.
158
AP Site
#/Total % Dist.
AFB,-N7-Gua
#/Total % Dist.
UV Treatment
of Cells
T------ (15/59) --- 7.0 -------------- -C-----(3/59)--- 1.4 -- -(25/85)--14.5 --
A ---- (36/59)-- 17.6 -- -(59/85)-- 34.0 -51.5
--
--
-
(24/24)--
8/48)--74.0
5'-TTC
T ----A
---C+---A ---T ----
'
(35/96)--10.0 -------------- 0 (7/96)--- 2.5 --------------- 0 (11/96)--- 2.5 -- - (17/80)--- 6.0-(40/96)-- 12.5 --- (61/80)-- 20.0 -(96/96)-- 72.5 - - - (24/24)-- 74.0 - -
159
Figure 21.
Mutation frequencies of A) AFB,-N7-Gua (G- T) and B) the AP
site (AP--T) in both DL7 and DL7/pGW16 cells. The histogram depicts the
differences in the genetic requirements for the mutagenic bypass of the two
lesions (see text for details).
1 60
I_
_
__
_
A
_
_
1C_
~_
___I
_
__
_I
AFB,-N7-Gua
130
TT
t3`
P
O
a)
4-
3
, -
UV
L-------------
B
-
+
+
-
DL7
DL7/pGW16
- ~- ~~C-----------
__
--
--
L-----·l~-----
lAP Site
50
e
r- ri
0
40
U)
30
%AP ->T
C-
C
0
.4*1-
20
10
n
3"
~pr
Q
r
1
uv
UV
~I~--
--
DDL7
-I
D+
DL7/pGW16
---I~--·I~··---·---·
--
161
T~
~II_
_
Figure 22. A potential AFB,-N7-Gua mispair with dAMP in the syn
conformation.
162
~0·1
II···
~I
II
L
I·I
II·_~
·~
ý01
-- 0O
'syn' dAMP
AFB 1 -N7-Gua
(Template strand)
GAFB1
:A mispair
163
Figure 23. A potential AFB,-N7-Gua zwitterionic mispair with dTMP. The
hypothesis for zwitterion formation is based on the observation that the pKa
at the N1 position of N7-alkylated guanines is 7.1 (versus 9.2 for a normal
guanine) (Lawley and Brooks, 1961).
164
A
IF-=IIm
1
""aa"aa
E
o,. €
v
AFB,-N7-Gua
dTMP
zwitterion
(Template strand)
GAFB1
: T mispair
165
Figure 24. A potential energy minimized structure of the central portion of
[AFB,-N7-Gua d(CCTCTTCGAACTC)-d(GAGTTCGAAGAGG)].
The model
was derived from and is consistent with NMR data for [AFB,-N7-Guad(ATCGAT)-d(ATCGAT)I], where the AFB 1 moiety is intercalated above the
5'-face of the modified guanine (Gopalakrishnan et al., 1990).
166
3'
5'
GC
TA
TA
5240
- CG
6239
3'
167
5'
Figure 25. Cartoon based upon the model shown in Figure 24, depicting the
AFB,-N7-Gua adduct at the moment of replication. It is proposed that the
AFB, moiety shifts in the 5' direction, perhaps as a consequence of
normalization of the C-G pair at position 6240. C6239 may rotate out of the
helix to accommodate the AFB, moiety. A C->T transition is the observed
mutation at position 6239, and may occur owing to the insertion of adenine
across from the AFB1 moiety.
168
5'
(
3'
3'-OH
I.
isCtoT
169
V. Conclusions and Suggestions for Future Studies
170
The studies presented in this dissertation were focused on elucidating the
mutagenic mechanism of the potent liver carcinogen aflatoxin B1. Three
AFB,-induced DNA adducts - the primary adduct, AFB,-N7-Gua; the ringopened adduct, AFB,-FAPY; and the AP site - are the likely precursors to the
mutagenic effects of aflatoxin. The examination of the mutagenic potential
of each of these lesions, each in a unique site within a single-stranded viral
genome, was determined to be the best approach toward deciphering which
lesion(s) give rise to the mutations observed in hepatocellular carcinomas
believed to be induced by AFB,.
Thus, the first segment of my thesis
involved the development of a procedure for construction of a singlestranded bacteriophage M13 genome containing the chemically and
thermally labile AFB,-N7-Gua adduct or the AP site in a unique site. The
construction protocol was designed such that the integrity of the AFB,-N7Gua adduct was maintained. These genomes were then used to examine
and compare the mutagenic potential of both AFB,-N7-Gua and the AP site
in E. coli. Based on the results obtained from these mutational studies, the
following are suggestions for future experiments.
1) The next logical step is to examine the mutational properties of the ringopened AFB,-FAPY adduct in the same system. Initial chromatography
(HPLC) studies on the AFB,-FAPY adduct within the hexamer sequence
d(ATGCAT) indicated that, at least in the oligonucleotide, two potential
171
rotamers of the adduct may exist. Hence, it was determined that the best
way to prepare the AFB 1-FAPY genome would be to first construct the AFBjN7-Gua genome as described in this thesis, and to then open the imidazole
ring of the guanine with mild alkali, using published conditions for ringopening of the primary adduct (Sambamurti et al., 1988). This procedure
will likely generate an AFB,-FAPY adduct that most closely resembles that
formed in vivo. The elucidation of the mutagenic potential of this lesion will
be of great value towards a thorough understanding of the mutagenic
mechanism of aflatoxin B, .
2) The E. co/i DNA repair enzyme FAPY DNA glycosylase has been shown in
one study (Chetsanga and Frenette, 1983) to excise AFB,-FAPY adducts
from DNA. Hence, it would be of value to examine the mutagenic potential
of AFB,-N7-Gua and AFB 1-FAPY within E. co/i strains lacking this repair
enzyme.
3) In light of the mutations observed at the base 5' to the modified guanine
(base 6239), it will be very interesting to examine the effect of changing the
5' base from a cytosine to either A,T, or G. Currently, AFB,-N7-Gua
oligonucleotides are being prepared that are similar to the 13-mer used in
this dissertation, except that the internal sequence (Sful sequence) harbors
an A or a T at the base 5' to the modified guanine. The two 13-mers are
172
shown below (the internal six-base sequence is underscored; the AFB,-N7Gua is bold; the 5' base is italicized).
(1) 5'-TCTTTGAACTCTC-3'
and
(2) 5'-CTCTTAGAACTCC-3'
Since there is no longer a restriction recognition sequence for mutant
enrichment procedures, these sequences were designed to contain the AFB,N7-Gua within a phenotypically scorable sequence. Each sequence harbors
the AFB,-N7-Gua adduct within a stop codon. Oligonucleotide (1) contains
the adduct within an opal stop codon (5'-TGA-3'). Thus, the wild-type
sequence will plate light blue on an opal suppressor plating strain, such as
NR8044, and all mutations, except G--A transitions, will yield dark blue
plaques. A G--A transition will produce an ochre stop (5'-TAA-3') that will
yield clear plaques on NR8044. Similarly, oligonucleotide (2) contains the
AFB 1-N7-Gua within an amber stop codon (5'-TAG-3'). Thus, wild-type
sequences will plate light blue on the amber suppressor strain GW5100, and
all other mutations, except G-.A transitions which also give rise to an ochre
stop in this sequence, will give rise to dark blue plaques. In order to
distinguish G-->A mutations apart from the large percentage of genetic
engineering mutations (clear plaques) that will likely present themselves at a
similar frequency to that observed in this study, it will be necessary to plate
the progeny phage, produced from genomes prepared from both sequences,
173
on an additional strain. On an ochre suppressor strain, such as NR9050,
G--A transitions within both of these sequences will give rise to light blue
plaques. Hence, by using a combination of bacterial plating strains each
type of mutation induced by AFB1 in these sequences can be selected
phenotypically. The examination of the mutagenic potential of bases other
than cytosine or guanine 5' to the AFB,-N7-Gua adduct may shed light on
the hitherto inexplicable observation that AFB, gives rise to a small
percentage of mutations at A:T pairs (Foster et al., 1983; Sahasrabudhe et
al., 1989).
Another reason that it will be necessary to study these particular
sequences is in light of the spontaneous frameshift mutations that were
observed in the Sful recognition sequence of the 13-mer oligonucleotide. As
discussed in Chapter IV, spontaneous frameshift/base substitution mutations
were detected (TTCGAA to TTTAA) in both the AFB,-N7-Gua sequence and
in the unmodified control. This mutation appeared to be real since it was
present within the modified sequence and the unmodified control at a
frequency of -0.2%.
This mutation may be the result of an inherent effect on
DNA replication of this particular oligonucleotide sequence. The possibility
that this spontaneous mutation manifests itself as a single base substitution
in the presence of a modified guanine (i.e., TTCGAA to TTTGAA), instead of
a frameshift/base substitution, is unlikely but not inconceivable. Thus, it is
174
necessary to examine the mutational specificity of AFB,-N7-Gua within
several other sequences, including the two sequences shown above, and
also within sequences that are considerably different from the one used in
this study. The acquisition of these data would hopefully support the
current hypothesis, proposed based on the data from this dissertation,
suggesting that AFB,-N7-Gua gives rise to mutations at the base 5' to the
modified guanine.
4) The experiments described in (2) involve changing the 5' base to either a
T or an A. At this point it is not possible to produce an oligonucleotide that
harbors a guanine residue 5' to an AFB,-modified guanine. This is due to the
fact that all guanines within the oligonucleotide will react with the AFB,epoxide upon oligonucleotide preparation. However, since the AFB,-epoxide
tends to react more readily with GC rich sequences, mutations induced by
AFB, appear to be present at higher frequencies in these sequences than in
AT rich sequences. Therefore, it would be very useful to be able to examine
the mutagenic potential of AFB,-N7-Gua within sequences that are GC rich.
In addition, codon 249 (5'-AGG-3') of the p53 gene is a known hotspot for
mutations in DNA from liver tumors believed to be induced by aflatoxin.
Thus, it would be very interesting to be able to examine the mutational
consequences of an AFB,-N7-Gua adduct within this exact sequence in a
site-specific fashion.
175
William Kobertz, in our laboratory, has proposed a method for production
of AFB,-N7-Gua oligonucleotides that contain more than one guanine
residue. The idea is based on the knowledge that intercalation of aflatoxin
within the DNA helix is a key step prior to its covalent interaction with
guanine residues (Gopalakrishnan et al., 1990). The basic concept involves
blocking potential intercalation sites on the 5' face of all guanine residues
that we wish to shield from reaction with the AFB,-epoxide. It is proposed
that psoralen adducts, which intercalate on the 3' face of thymine,
specifically situated in the opposing strand (duplex DNA is required for AFB,epoxide reactivity), will block AFB 1 intercalation, and thus prevent AFB,epoxide reactivity at selected guanines. The basis for this hypothesis is
being investigated presently.
5) Since the data presented in this thesis suggest that AP sites do not give
rise to the majority of AFB,-induced G-*T transversions, it would be useful to
examine the frequency of these mutations in E. coil strains that overproduce
an AP endonuclease. In the event that the frequency of AFB,-N7-Gua
induced G--T transversions remains constant in these overproducing strains,
it would further support the view that AP sites are not responsible for the
observed G-+T mutations. This type of experiment was attempted by first
producing an E. co/i strain that was believed to slightly overproduce the AP
endonuclease T4 endonuclease V; this AP endonuclease is active on ss DNA
176
(Wallace, 1988). This strain was then used to compare the frequencies of
G--T or AP- T mutations from AFB,-N7-Gua or AP site containing genomes,
respectively. Unfortunately, the data from these preliminary experiments
were inconclusive.
6) The work presented in this thesis involved, in part, a comparative
examination of induction of the major mutation, G--T transversions, by both
AFB,-N7-Gua and the AP site (AP--T). These studies were performed in both
DL7 E. coli and in DL7 cells containing the mucAB mutagenesis-enhancing
gene products. Data indicated that G--T transversions induced by AFB,-N7Gua were much more dependent on MucAB than UmuDC (see Figure 21,
Chapter IV). It was not determined, however, whether the other AFB,-N7Gua induced mutations were also highly dependent on MucAB (such as the
5' mutations). It would be interesting to examine the entire mutational
spectra of AFB,-N7-Gua in cells lacking the MucAB proteins.
7) The methodology presented in this dissertation, describing conditions for
the construction of singly-modified M13 genomes containing either an AFB 1N7-Gua, an AP site, or an AFB 1-FAPY adduct (proposed procedure only),
should be applicable toward the preparation of singly-modified plasmids to be
used in mutational studies in mammalian systems. This would be the next
logical step in elucidating the mutational properties of aflatoxin B,.
177
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Biographical Sketch
I was born on July 2, 1967 in Glen Cove, Long Island, New York. I
spent my first twelve years in Farmingville, New York, where I attended
Techumseh Elementary School. The summer before the Lake Placid 1980
Winter Olympics my parents decided to move the family to Keene, a small
town in upstate New York. I lived in Keene for six years where I attended
Keene Central School that had all of 200 students from kindergarten through
twelfth grade. After graduating from Keene Central in 1985 I entered
Skidmore College in Saratoga Springs, NY. I majored in chemistry and in
1989 received my B. A. with honors in chemistry. I decided to pursue my
interest in biochemistry upon entering the Department of Chemistry at MIT in
the Fall of 1989. I became a member of the laboratory of Dr. John
Essigmann. My doctoral research focused on examination of the mutational
properties of the potent liver carcinogen, aflatoxin B1. This project led to
two publications, one was still in preparation at the end of my doctoral
studies, and the other, "Mutational Properties of the Primary Aflatoxin B1
DNA Adduct in Escherichia co/i", will be published in the Proceedings of the
National Academy of Sciences. Upon completion of my doctoral studies on
December 5, 1995, I joined the laboratory of Dr. Bruce Demple, in the
Division of Molecular and Cellular Toxicology at the Harvard School of Public
Health, as a postdoctoral associate, where I will continue to pursue a career
in cancer research.
194