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Volume 272, Number 52, Issue of December 26, 1997
pp. 33211-33219
(Received for publication, August 19, 1997, and in revised form, September 3, 1997)
From the ABSTRACT Like other polycyclic aromatic hydrocarbons,
certain metabolites of benz[a]anthracene have been
implicated as potent carcinogens. These effects are thought to be
caused by the covalent binding of these species to nucleophilic groups
on the bases of DNA. To address the molecular mechanisms by which these
molecules induce mutations, this study employed oligonucleotides
containing four site-specific N6
adenine-benz[a]anthracene diol epoxide adducts. Using a
prokaryotic in vivo replication system, we have shown that
both non-bay region anti-trans-benz[a]anthracene adducts are
essentially nonmutagenic. In contrast, the bay region
anti-trans-benz[a]anthracene lesions do
induce point mutations at the adduct site. The mutagenic frequency of
these bay region lesions is dependent on the stereochemistry about the
adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated. The ability of the bacterial replication machinery to bypass the lesions does not correlate with the
differences observed in their mutagenesis. While both non-bay region
adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree. In vitro studies of the interactions of E. coli DNA
polymerase III with these adducts have also been undertaken to further
dissect the relationship between adduct structure and biological
activity.
INTRODUCTION Under normal growth conditions, Escherichia coli DNA
polymerase III and its accessory factors constitute an extremely
efficient enzyme system. The holoenzyme complex can incorporate up to
1000 nucleotides/s, and estimates of replication fidelity are in the range of 1 mistake/1010 bases copied (1). This extremely
high fidelity is a result of multiple individual reactions, all of
which have intrinsically high fidelity: the polymerization step,
3 Benz[a]anthracene (BA) is one of the many polycyclic
aromatic hydrocarbons which are formed during the incomplete combustion of organic materials, including gasoline, and tobacco. It is found in
particularly high concentrations in coal tar and the wood preservative creosote, and it has been shown that levels of BA increase 7- to 8-fold
in meat that is grilled over charcoal (2).
Like other PAHs, BA undergoes metabolic activation to form vicinal diol
epoxides. The mutagenicity of numerous BA metabolites has been
determined (3-5), and it is widely accepted that the biological
effects of BA as well as other PAHs result from the reaction of bay
region diol epoxides with the bases of DNA. A bay region diol epoxide
is defined as a vicinal diol epoxide in which the epoxide ring
encompasses one of the bay region carbon atoms (see Fig. 1). In
comparison with other diol epoxides of BA, the bay region diol epoxides
are 10-35 times more mutagenic in certain bacterial strains and
65-125 times more mutagenic in V79-6 Chinese hamster lung cells
(4-6).
Animal studies implicate BA as a mild carcinogen, especially in
comparison with such compounds as benzo[a]pyrene and
dibenz[a,h]anthracene (3, 7). The low rate of
tumorigenicity of BA is thought to be due, at least in part, to the low
level of metabolism of BA to the bay region diol epoxide (8-11).
Exactly what it is about the bay region diol epoxides that leads to the
biological effects seen is still unclear. The initial bay region theory
of carcinogenesis was based on calculations which showed that the
epoxide was particularly reactive at this position and could easily
open to form a strongly electrophilic carbocation that would then be
attacked by the nucleophilic groups in DNA. It was suggested that this
increased reactivity of the bay region diol epoxides was the basis for
the increased mutagenicity/carcinogenicity of these compounds (12). As
has been pointed out, however, increased reactivity alone cannot
account for the increased mutagenicity of these compounds since other
(non-bay region) diol epoxides also form covalent DNA adducts with
approximately the same efficiency (1). Further, some of the most potent
bay region diol epoxides turn out to be some of the least reactive.
An alternative explanation for the observed dichotomy between the bay
region and the non-bay region diol epoxides of PAHs is that the adducts
which they form adopt conformations that are recognized in different
ways by the cellular repair/replication machinery and that these
differences account for the varied mutagenicity of the adducts (13).
Clearly, these questions remain to be answered, and we consider site-
and stereo-specific BA-DNA lesions to be ideal for determining
structure-function relationships of adducts resulting from mutagenic
versus non-mutagenic PAH metabolites.
We initiated a project to study the biological effects of site-specific
DNA adducts that form when certain PAH metabolites interact with DNA. A
primary goal of this research, in conjunction with the structural
studies which are being carried out by Dr. Michael Stone at Vanderbilt
University, is to determine the basis for the different mutagenic
effects of similar compounds, all of which bind to DNA in the same
basic manner.
In this study, we examined four benz[a]anthracene diol
epoxide-DNA adducts (two bay region and two non-bay region). These adducts were synthesized chemically by methodology developed by Kim
et al. (14, 15) (see Fig. 1), but the same structures would
result from trans opening of each of the following BA
anti-epoxides by the exocyclic nitrogen (N6) of adenine: 1)
8S,9R,10R,11S-10,11-epoxy-8,9,10,11-tetrahydro-BA-8,9-diol; 2)
8R,9S,10S,11R-10,11-epoxy-8,9,10,11-tetrahydro-BA-8,9-diol; 3)
1S,2R,3R,4S-1,2-epoxy-1,2,3,4-tetrahydro-BA-3,4-diol;
and 4) 1R,2S,3S,4R-1,2-epoxy-1,2,3,4-tetrahydro-BA-3,4-diol.
The corresponding adducts will henceforth be referred to as non-bay
region 11R-BA, non-bay region 11S-BA, bay region
1R-BA, and bay region 1S-BA, respectively
(Fig. 1). While we recognize that these
epoxides react mainly at the N2 position of guanine, the
minor adenine lesions examined herein may have a disproportionate
biological significance as has been suggested for adenine adducts
resulting from the diol epoxide derivatives of
dimethylbenz[a]anthracene (16),
benzo[a]pyrene (17), and more recently
dibenzo[a,l]pyrene (18).
[View Larger Version of this Image (25K GIF file)]
EXPERIMENTAL PROCEDURES General Synthetic Procedures
Unless otherwise noted, materials were obtained from commercial
suppliers. Methylene chloride (CH2Cl2) and
pyridine were distilled from calcium hydride after reflux under a
nitrogen atmosphere for 4 h. Benzene and tetrahydrofuran (THF)
were distilled from sodium/benzophenone after reflux under nitrogen
atmosphere for 4 h. All glassware, syringes, needles, and magnetic
stirring bars used in moisture-sensitive reactions were oven dried at
140 °C and stored in desiccators until used. All moisture-sensitive
reactions were conducted under a nitrogen atmosphere. Reactions were
monitored by thin-layer chromatography. TLC plates were purchased from
EM Science (Kieselgel 60 F254, pre-coated, 20 × 20 cm, 0.25-mm
layer thickness). TLC plates were visualized, after development, under ultraviolet (UV) light (254 cm (±)-8 (±)-8 (±)-3 6-Fluoropurine deoxyriboside (0.86 mg, 3.4 µmol) and
(±)-11 6-Fluoropurine deoxyriboside (3.2 mg, 12.6 µmol, 1.1 equiv.) and
(±)-1 Synthesis of Adducted Oligonucleotides
The modified oligonucleotide (2 × 1 µmol), 5 The modified oligonucleotide (3 × 1 µmol
cassette), 5 Characterization of Adducted Oligonucleotides
Capillary gel electrophoresis
was performed on either an Applied Biosystems Model 270, using
gel-filled capillaries (Applied Biosystems) and the manufacturer's
Micro-Gel buffer, or on a Beckman PACE 2000 system with the
manufacturer's e-CAP gel-filled capillaries and Tris borate-urea
buffer. Samples were applied at The oligonucleotides (0.2-0.6 ODs),
lyophilized in 1.5-ml microfuge tubes, were digested in a 2-stage
process. In the first step, buffer (20 µl, 0.01 M
Tris-HCl, 0.01 M MgCl2, pH 7.0) was added,
followed by nuclease P1 (Sigma N-8630, 4 µL). After digesting for
3-6 h at 36 °C, the second digestion step was performed. Tris-HCl buffer (20 µl, 0.1 M, pH 9.0) was added followed by snake
venom phosphodiesterase (Sigma 5785, 0.04 units) and alkaline
phosphatase (Sigma P-4282, 0.4 unit). Digestion was continued at
37 °C for 3-6 h. Before HPLC analysis, H2O (100 µl)
was added to each sample. Following centrifugal filtration, the digests
were analyzed by HPLC on either a C-18 or YMC-ODS-AQ column with a
gradient of 1-10% CH3CN in 0.01 M ammonium
formate, pH 5.9, over 15 min followed by 10-99% CH3CN
over 20 min, at a flow rate of 1.5 ml/min. Under these conditions, the
diastereomeric PAH-adducted nucleosides eluted at 30-32 min. Peak
identification was by comparison with adducted nucleosides synthesized
as described above.
Each of the four BA-adenine lesions described above was constructed
such that it was located in the center of an 11-mer oligonucleotide. The sequence of the 11-mer corresponded to the coding sequence of the
human N-Ras gene from position 3 of codon 59 to position 1 of codon 63 (5 Single-stranded M13mp7L2 DNA was
kindly provided by Dr. Christopher Lawrence (University of Rochester).
This M13 DNA is unique in that the single-stranded form acquires a
hairpin loop structure creating an EcoRI restriction site.
Single-stranded M13mp7L2 DNA was digested with EcoRI
according to the supplier protocol (New England Biolabs), resulting in
the production of linear M13 molecules (cut L2) with only a portion of
the multiple cloning site deleted. The linear DNA was purified using
Microcon-100 microconcentrators according to the manufacturer
instructions (Amicon). The completeness of the digestion was confirmed
by separation of the DNAs via electrophoresis through 1.5% agarose
gels.
A 51-mer scaffold was employed to position each of the adducted
11-mers, or the corresponding non-adducted 11-mer, within the gap
created in the cut L2. A 2.5-fold excess of each 11-mer was annealed to
51-mer scaffold on ice for 30 min. Purified cut L2 was incubated at
90 °C for 2 min, cooled on ice, and added to the annealed
11-mer:51-mer mix. The resulting combination of DNA molecules was
incubated with T4 DNA ligase according to the supplier instructions
(New England Biolabs). The final ratio of cut L2:11-mer:51-mer scaffold
was 1:5:2. Ligation resulted in the production of single-stranded
circular M13 molecules containing either site-specific BA adducts or
the identical non-adducted sequences. To displace the scaffold from the
ligation products, a 5-fold molar excess (relative to the concentration
of the scaffold) of 51-mer whose sequence is complementary to that of
the scaffold was added. The DNAs were heated to 90 °C for 2 min and
then placed on ice for 10 min. Any portion of the ligation mixture that
was not used for transformation immediately was stored at An aliquot of each ligation
mixture was subjected to electrophoresis through a 1.5% agarose gel.
This concentration of agarose efficiently separated ligated, circular
M13 molecules from any remaining linear molecules. To determine the
relative number of circular molecules that contained the 11-mer insert,
the DNAs in these gels were subjected to Southern blot analyses (27) with a radiolabeled 17-mer probe that hybridizes specifically to the
adducted 11-mer and flanking M13 sequences.
Replication of Site-specifically Adducted M13 in Vivo
Repair-deficient E. coli, AB2480 (uvrA6,
recA13) were made competent according to the calcium chloride
method of Sambrook et al. (27) and transformed with aliquots
of the ligation mixtures described above. The resultant plaques were
counted, and the numbers obtained were normalized with respect to the
amount of input circular M13 that contained the 11-mer. The replication
efficiency of each species of adducted M13 was expressed as a
percentage of that of the unmodified M13.
The frequency and spectrum of point
mutations that resulted at the position of the adduct were determined
using a differential hybridization strategy described previously (26,
28).
A saturated culture of AB1157 E. coli was diluted 1:100 with Luria-Bertani medium and grown at
37 °C to an A600 of approximately 0.2. The
cells were pelleted by centrifugation at 3000 × g for 5 min and resuspended in 1/6 volume of 1 × M9 salts (48 mM Na2HPO4, 22 mM
KH2PO4, 8 mM NaCl, 18 mM NH4Cl). They were then transferred to a
sterile 35 × 10 mm tissue culture dish. While stirring, the cells
were irradiated with 254-nm light at a fluence of 40 µW cm Replication of Adducted DNA in Vitro
All proteins used in the in vitro
replication studies (except bovine serum albumin (BSA) and proteinase
K, which were purchased from Sigma and U. S. Biochemical Corp.,
respectively) were provided by Dr. Mike O'Donnell (Cornell University
Medical College).
M13 molecules containing either site-specifically modified adenine
residues, or the corresponding unadducted sequence were produced as
described above. After displacing the 51-mer scaffold, ligation
products were separated from any remaining linear DNAs on a 1.4% low
melting point agarose gel. The gel was stained with 0.5 µg/ml
ethidium bromide, and the DNA was visualized under long wavelength (300 nm) ultraviolet light. The bands containing circular M13 were excised,
and the agarose was digested with Annealing of template to primer was carried out in a 12-µl volume
containing 44 mM Tris-OAc (pH 7.8); 88 mM KOAc;
and 13 mM MgOAc. Templates (200 fmol each) were annealed to
a 5-fold molar excess of a 21-mer primer that was designed to initiate
replication 1.8 kilobases upstream of the adducted adenine. A total of
1.6 µg of single-stranded binding protein (SSB) was added to coat the
remaining single-stranded regions of the circular M13 templates. Samples were incubated at 80 °C for 2 min and slowly cooled to 37 °C. Preinitiation solution (3 µl of 300 µM each
dTTP, dGTP, and dCTP; 110 µM dATP; 110 µM
[ Products of in vitro replication were separated via
electrophoresis through 0.8% alkaline agarose gels according to
Sambrook et al. (27). After neutralization, the gels were
dried under vacuum and placed under a storage phosphor screen for
subsequent quantitation (Molecular Dynamics).
In Vitro Mutagenesis
Both adducted and non-adducted
11-mers were ligated at their 3 Template (6 pmol of 33-mer) was mixed with 1.2 pmol of 17-mer primer in
38.4 µl of a solution containing 50 mM Tris-OAc (pH7.8); 100 mM KOAc; and 16 mM MgOAc. The solution was
incubated at 80 °C for 2 min and then slowly cooled to 37 °C. Of
this 38.4-µl annealing reaction, 6.4 µl was transferred to each of
five tubes containing 1.6 µl of a pre-extension solution comprised of
the following: 20 mM dithiothreitol; 7 mM
ribo-ATP; 0.7 µg/ml BSA; and either 50 µM dATP, 50 µM dTTP, 50 µM dGTP, 50 µM
dCTP, or 50 µM each dATP, dTTP, dGTP, and dCTP. Reactions
were initiated by the addition of 100 ng of Pol III
RESULTS The
usefulness of regio- and stereo-specifically adducted oligonucleotides
for structural and biological studies has been well documented (29,
30). For such studies, evaluation of the purity of the oligonucleotides
is essential. The oligonucleotides used in the studies reported herein
were purified initially by HPLC. The purity at this stage was evaluated
by TLC and capillary gel electrophoresis
(Fig. 2). Mass spectroscopy showed that
the oligonucleotides were of the expected molecular weight. In
addition, the oligonucleotides were enzymatically hydrolyzed to their
component nucleosides, which were present in the predicted ratios.
Inasmuch as the syntheses were carried out with racemic aminotriols, it was important to examine the stereochemical identity and purity of the
PAH adduct in the oligonucleotides. This could be evaluated from the
enzyme hydrolysate by chromatographic comparison with the synthetic
nucleosides. In the case of the non-bay region BA-adducted oligonucleotides, cross-contamination of the 11R and the
11S isomers was seen to the extent of 15-20%; for the well
resolved bay region oligonucleotides, the cross-contamination was less
(~10%).
[View Larger Version of this Image (27K GIF file)]
Further purification of the adducted oligonucleotides was carried out
by polyacrylamide gel electrophoresis, and the purity of the resulting
11-mers was determined by analysis of 32P-end-labeled
products (Fig. 3). All oligonucleotides
were determined to be >99.4% adducted 11-mers.
[View Larger Version of this Image (67K GIF file)]
A scaffolding
strategy was employed to introduce site- and stereo-specifically
modified 11-mers, or their non-adducted counterparts, into the multiple
cloning site of single-stranded M13 (see Ref. 28 and "Experimental
Procedures"). In vivo replication of these DNAs was
carried out in E. coli both with and without prior SOS induction. Plaques that contained site-specific mutations were analyzed
by differential hybridization strategies
(Fig. 4). Neither of the non-bay region
adducts inhibited replication of the M13 (Fig. 4B) or
induced a significant number of point mutations at the site of the
adduct (Fig. 4C). SOS induction of the cells prior to
transformation did not affect the replication of these adducts (Fig.
4B). The M13 molecules that carried the non-bay region
11R-BA lesions were replicated at least as efficiently as
their unadducted counterparts (replication efficiency ranged from 92 to
144% that of the unadducted M13 (Fig. 4B)). Replication of
non-bay region 11S-BA adducted DNA was also uninhibited
within bacteria (replication efficiency range of 94-325% (Fig.
4B)). Of the >18,000 plaques that were generated from these
non-bay region BA adducted M13 molecules (in AB2480, approximately
10,000 and 6000 plaques were screened for the non-bay region
11R-BA and the non-bay region 11S-BA adducted
DNAs respectively, and approximately 2000 (11R-BA) and 1200 (11S-BA) plaques were screened under SOS+ conditions in
AB1157 cells), only four were positive for point mutations at the site
of the lesion (see Fig. 4C). Therefore, we conclude that
these lesions are non-mutagenic when processed within E. coli.
[View Larger Version of this Image (68K GIF file)]
In contrast, the bay region BA adducts did inhibit M13
replication as evidenced by decreased plaque forming abilities (Fig. 4B). They also induced a relatively large proportion of
point mutations at the site of the adduct (Fig. 4C).
Specifically, when allowed to propagate within repair-deficient AB2480,
those M13 DNAs bearing bay region 1R-BA and bay region
1S-BA adducts produced 17 and 11%, respectively, of the
number of plaques as the same quantity of unadducted M13. Under these
conditions, the template DNAs carrying bay region 1R-BA
lesions produced 2.6% A All data described above were the result of multiple experiments in
which each species of adducted DNA was introduced into each type of
E. coli, under a specified set of conditions, in a minimum
of three separate experiments. In the case of the bay region BA
adducted M13, as many as 12 different transformations were carried out
in an attempt to generate large numbers of plaques. The decrease in the
plaque-forming efficiencies of these bay region adducted M13 molecules
made it impossible to screen an equal number of plaques, but the
concomitant increase in mutagenesis opposite these lesions was so
dramatic that it was quite obvious that these lesions were indeed more
mutagenic than their non-bay region complements. Further, during the
course of the investigations described, multiple batches of each
species of adducted oligonucleotide had to be prepared, and in no case
did the results across these experiments vary greatly.
Purified circular M13
molecules, either adducted or unmodified, were used as templates for
polymerization by isolated E. coli Pol III holoenzyme (Pol
III) in vitro. Replication was initiated 1.8 kilobases
upstream of the adducted adenine, and polymerization was carried out in
the presence of [
[View Larger Version of this Image (24K GIF file)]
Initially, we had hypothesized that those lesions which inhibited
replication and were mutagenic in vivo (the bay region
lesions) would block Pol III in vitro, whereas the
non-mutagenic, non-blocking adducts (non-bay region) would be readily
bypassed. This, however, proved not to be the case as replication of
each of the adducted templates resulted in essentially complete
blockage of Pol III at or near the site of the lesion (Fig. 5).
Due
to the lack of correlation between adduct-induced blockage in
vitro and in vivo, we chose to examine the propensity
of the purified polymerase complex to incorporate each of the common nucleotides opposite each of the adducted adenine residues. The strategy employed for these studies also afforded an increased degree
of precision over the system that utilized adducted M13 molecules as
templates since the products of synthesis could be resolved to the
level of a single nucleotide as opposed to an approximate size in
kilobases (as was the case in the experiments utilizing adducted M13
templates). Synthetic oligonucleotide template-primer complexes in
which the 3
[View Larger Version of this Image (84K GIF file)]
DISCUSSION Since BA is known to be metabolized to both bay region and non-bay
region diol epoxides, and there is a clear distinction between the
mutagenicity of these two classes of metabolites (4, 31-33), we
consider site-specific benz[a]anthracene-DNA adducts to
represent ideal models to study the mechanism by which PAHs induce
mutation. This study, therefore, was designed to examine the processing
of two bay region BA-DNA adducts and two non-bay region BA-DNA adducts
by E. coli DNA polymerases both in vivo and
in vitro. M13 molecules carrying these lesions were allowed to replicate within E. coli either with or without prior
induction of the cellular SOS response. The same lesions were also
incorporated into templates that could be replicated in
vitro.
Mutagenesis of BA-DNA Lesions in Vivo
The results of these experiments supported our hypothesis that the
cellular replication of templates carrying bay region lesions would
result in a higher frequency of point mutations at the site of the
adduct than would replication of phage that contained non-bay region
adducts. To our knowledge, this is the first study which has
demonstrated that even in the same sequence context, site-specific bay
region lesions are mutagenic, and the corresponding non-bay region
lesions are not. These data suggest that indeed, there is something
inherently different about the structure of the different adducted DNA
molecules that determines their mutagenicity. While it is not our
intent to overemphasize the quantitative aspects of these data, clearly
some generalizations can be drawn. First, M13 molecules carrying bay
region BA lesions produce fewer plaques than either those carrying
non-bay region BA adducts or the corresponding non-adducted M13.
Second, the bay region BA adducts induce point mutations at the site of
the adduct, whereas the non-bay region lesions are essentially
non-mutagenic. Third, the bay region 1R-BA lesion is the
most mutagenic of all the adducts examined in this study. Fourth, there
is a distinct difference in the number of mutations that were induced
by the bay region lesions when they were propagated in different types
of E. coli. And fifth, the primary type of substitution
introduced is a transition mutation (A Regarding differential mutagenesis in different cell types, since
AB2480 E. coli are deficient in recA and the
binding of RecA protein to single-stranded DNA is thought to be the
event that initiates the SOS response, AB1157 cells were chosen to
examine the mutagenesis of BA lesions when SOS conditions were induced. AB1157 and AB2480 are essentially isogenic strains of E. coli that vary in their repair capacity. AB1157 cells are wild
type with respect to repair, whereas AB2480 cells are deficient in both
RecA and UvrA activities. As we had hypothesized, the bay region BA
lesions induced the highest frequency of point mutations when
replicated in AB1157 cells that had been exposed to UV irradiation prior to transformation. Somewhat surprising, however, was the dramatic
difference in mutation frequency that was solely dependent on the cell
type in which the adducted DNAs were processed. We propose that the
presence of RecA, along with basal levels of UmuC and UmuD, in the
AB1157 cells can explain this differential processing of BA lesions in
different cell types.
Normally, SOS functions are activated in E. coli in response
to massive amounts of DNA damage. Cellular repair activities produce
regions of single-stranded DNA to which RecA protein binds, and this is
thought to initiate the SOS response (35-37). Under normal conditions,
the extensive nature of DNA damage requires that the proteins involved
in translesion synthesis (RecA, UmuC, and the activated form of UmuD)
be expressed in high quantity. In the experiments described above,
however, there is a single DNA lesion per M13 molecule being introduced
into the cell. Further, this lesion is contained on a single-stranded
DNA template. It seems reasonable, therefore, that the normal
complement of RecA protein (estimated to be 7200 copies per cell (38))
would be sufficient to bind to the single-stranded DNA template and
activate some of the constitutive UmuD protein of the cell to its
active form (UmuD In Vitro Replication
We interpreted the inhibition of cellular
replication of M13 containing bay region BA adducts as being
potentially due to physical blockage of cellular polymerase molecules
at or near the site of the adduct. In an attempt to reproduce this
blockage in vitro, we used site-specifically adducted,
covalently closed circular M13 molecules as templates for replication
by purified E. coli DNA polymerase III holoenzyme. Synthesis
was initiated 1.8 kilobases upstream of the adduct site. Thus, if Pol
III were blocked at the site of the lesion, one would expect a
1.8-kilobase product, whereas bypass synthesis would result in a
product DNA of approximately 7.2 kilobases (the size of M13).
In these assays, all four adducts served as efficient blocks to
replication (see Fig. 5). The observation that the non-bay region
lesions were preferentially bypassed in vivo, suggests that
the adducts are processed differently in the intracellular environment.
Of multiple possible explanations, our favored interpretation is that
the polymerase machinery that encounters the adducts in vitro is somehow fundamentally different from the polymerase that allows translesion synthesis in vivo. It may be that a
different subassembly of polymerase III is responsible for the
replication of adducted M13 molecules in vivo, and this
subassembly is more likely to bypass the lesion than the holoenzyme.
This idea is supported by Kornberg (1) who argues that the ~10 Pol
III holoenzyme molecules present in a rapidly dividing cell (our cells
are in log phase when they are transformed) are likely to be involved in replication of cellular DNA and are not available for replication of
exogenous M13 molecules. Maki et al. (41) offer further
support for this hypothesis in a study demonstrating that Pol I may
substitute for the activity of a defective Pol III core. Bonner
et al. (42) also show that Pol II can become processive when
in the presence of Pol III accessory factors. Perhaps the most
convincing argument for a different polymerase, however, can be made in
light of a study that was published from the laboratory of Dr. Myron
Goodman (43) subsequent to the submission of this manuscript. In this study, they demonstrate that Pol II is indeed involved in the replication of episomal DNAs in E. coli. It seems probable,
therefore, that something other than the replicative form of Pol III is
responsible for the replication of the exogenous DNAs and that this
polymerase complex more efficiently bypasses non-bay region BA adducts.
Indeed, we have observed a marked difference in the ability of various polymerases to conduct translesion synthesis on oligonucleotides carrying these same non-bay region BA lesions (data not shown).
When replicated within E. coli, there
is a clear distinction in the mutagenic frequency of the various BA
adducts examined in this study. By far, the most mutagenic lesion
examined is the bay region 1R-BA adduct, and the most
abundant mutation is a transition (A Whatever the reason, we conclude that the in vitro assays
described herein are not predictive of the processing of BA adducted DNAs intracellularly. These findings emphasize the importance of
exercising extreme caution when attempting to make generalizations about the cellular processing of DNA lesions based on data obtained in vitro.
FOOTNOTES ACKNOWLEDGEMENTS We are grateful to Drs. P. Chary, M. Dodson,
and G. Latham for insights and helpful discussions during the course of
this study. Also, we appreciate the work of Dr. H. Jajoo, Dr. A. Chaudhary, and B. Nobes at the Mass Spectrometry Center in the
Department of Pharmacology, Vanderbilt University for help in obtaining
the mass spectra reported in this paper.
REFERENCES
Lack of Correlation between in Vitro and in
Vivo Replication of Precisely Defined
Benz[a]anthracene Adducted DNAs*
,
, and**
Department of Human Biological Chemistry and
Genetics and Sealy Center for Molecular Science, The University of
Texas Medical Branch, Galveston, Texas 77555, the
§ Microbiology Department, Hearst Research Foundation, and
Howard Hughes Medical Institute, Cornell University Medical College,
New York, New York 10021, the ¶ Departments of Chemistry and
Biochemistry and the 
Center in Molecular Toxicology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37235
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
5 proof-reading, and postreplicative mismatch correction.
Factors that compromise the fidelity of any of these steps can lead to
increased mutation rates and potentially to cell death or
transformation/cancer in higher eukaryotes. Polycyclic aromatic
hydrocarbons (PAHs)1 are
ubiquitous carcinogens that are thought to exert their effects by
decreasing polymerase fidelity.
Fig. 1.
Synthesis scheme for the production of site-
and stereo-specifically adducted oligonucleotides. The structures
of the parent hydrocarbon (benz[a]anthracene) and the
adducts employed in this study are shown. Each adducted adenine was
engineered into the center of an 11-mer oligonucleotide. The sequence
of the 11-mer corresponded to the coding sequence of the human
N-Ras gene from position 3 of codon 59 to
position 1 of codon 63 (5-C GGA CAA GAA G-3).
1) followed by staining
with anisaldehyde-sulfuric acid. TLC of oligonucleotides was carried
out using the SureCheckTM system (U. S. Biochemical
Corp.). Column chromatography was conducted using silica gel 60 (70-230 mesh) from EM Science.
,9
-Dihydroxy-10,11-epoxy-8,9,10,11-tetrahydrobenz[a]anthracene
and
(±)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene were purchased from the NCI Chemical Carcinogen Repository. Diol epoxide was converted to aminotriol by trans opening of the
epoxide with liquid ammonia by a previously reported procedure
(15).
-Amino-8,9,10,11-tetrahydrobenz[a]anthracene-8,9,10triol
,9-Dihydroxy-10,11-epoxy-8,9,10,11-tetrahydrobenz[a]anthracene
(10 mg dissolved in anhydrous THF (1 ml)) was converted to
aminotriol by the published procedure (reaction carried out at
100 °C for 24 h) (15). Aminotriol, as a yellow powder, was obtained in essentially quantitative yield. 1H NMR
(Me2SO-d6/D2O) 
8.90 (s, 1H, Ar), 8.79 (d, 1H, Ar), 7.94~7.58 (m, 6H, Ar), 4.88 (d, 1H,
H11), 4.03 (d, 1H, H8), 3.96 (dd, 1H, H10), 3.81 (dd, 1H, H9): LRMS
(electrospray) MH+ 296; N-acetylated derivative:
1H NMR (acetone-d6/D2O)
8.73 (d, 1H, H1), 8.69 (s, 1H, H12), 8.03 (s, 1H, H7), 7.92 (dd,
1H, H4), 7.75 (dd, 2H, H5, H6), 7.58~7.68 (m, 2H, H1, H3), 5.43 (d,
1H, H11), 4.96 (d, 1H, H8), 4.22 (dd, 1H, H10), 4.08 (dd, 1H, H9). Mass
spectrum (electrospray) MH+ 338.
-Amino-1,2,3,4-tetrahydrobenz[a]anthracene-2,3,4-triol
,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene
(15 mg in anhydrous THF (1 ml)) was converted to aminotriol by
reaction with liquid ammonia for 72 h at 75 °C (15). The ammonia and THF were evaporated, and the residue was suspended in a
small volume of MeOH:CH2Cl2 (1:4). Filtration
yielded the aminotriol (15.4 mg, 96%) as a yellow-brown powder.
1H NMR (400 MHz, Me2SO + D2O, ppm)
8.84 (s, 1H, H12), 8.50 (s, 1H, H7), 8.12-8.07 (m, 2H, H8, H11),
7.96 (d, 1H, H6, J = 8.9 Hz), 7.65 (d, 1H, H5,
J = 8.9 Hz), 7.55-7.48 (m, 2H, H9, H10), 4.68 (d, 2H,
H1, H4, J = 8.0 Hz), 3.99 (d, 2H, H2, H3,
J = 8.0 Hz). LRMS (FAB+,
glycerol/Me2SO/TFA) 296.1208 (M+H+), HRMS
(FAB+, glycerol/TFA/PEG) calculated for
C18H17N1O3: 296.1 (M+H+); found: 296.1255 (M+H+). The aminotriol
was converted to the N-acetylated derivative by stirring
with excess acetic anhydride in methanol. 1H NMR (400 MHz,
MeOD, ppm) 8.45 (s, 1H, H12), 8.38 (s, 1H, H7), 8.08-7.95 (m, 3H,
H11, H8, H6), 7.73 (d, 1H, H5, J = 8.9 Hz), 7.48-7.45
(m, 2H, H9, H10). LRMS (FAB+,
glycerol/Me2SO/TFA) 338.1 (M+H+), HRMS
(FAB+, glycerol/TFA/PEG) calculated for
C20H19N1O4: 338.1314 (M+H+): found: 338.1354 (M+H+).
,9,10,11)-2-Deoxy-N6-(8,9,10,11-tetrahydro-8,9,10-trihydroxy-benz[a]anthracene-11-yl)-adenosine
(non-bay BadA)
-amino-8,9,10,11-tetrahydrobenz[a]anthracene-8,9,10-triol
(1.9 mg, 6.4 µmol) were weighed into a conical vial. Triethylamine (2.4 µl, 17.2 µmol) and N,N-dimethylacetamide
(50 µl) were added, and the reaction was stirred at 55 °C for 2 days. The reaction was monitored by HPLC (C-18 column, 4.6 × 250 mm) using a linear gradient of 40-90% MeOH in H2O over 30 min at a flow rate of 1 ml/min. Diastereomeric products were eluted at
21.1 and 21.8 min. The diastereomers were separated by preparative HPLC
(C-18 column) using the same gradient as for the analytical column
chromatography over 40 min at a flow rate of 2.0 ml/min (yield 82%).
The CD spectra of the first eluted diastereomer displayed a negative
Cotton effect at 328 nm and a positive Cotton effect at 282 nm, and the
second eluted diastereomer displayed a positive Cotton effect at 328 nm
and a negative Cotton effect at 282 nm. The absolute configurations of
the adducts were assigned by comparison with the CD spectra of other
PAH deoxyadenosine adducts (19-22). The isomer with a strong negative
band at longer wavelength and strong positive band at shorter
wavelength was assigned the S configuration at the
N-substituted benzylic carbon of the tetrahydroaromatic
substituent. First eluted (11S isomer): 1H NMR
(400 MHz, MeOD-d4,) 8.70 (s, 1H, H12), 8.44 (m, 1H, H1), 8.37 (s, 1H, H-8), 8.30 (s, 1H, H-2), 8.08 (s, 1H, H7),
7.86 (m, 1H, H4), 7.77 (d, 1H, H6), 7.72 (d, 1H, H5), 7.53 (m, 1H, H2), 7.53 (m, 1H, H3), 6.47 (dd, 1H, H1), 5.99 (s, br, 1H, H11), 5.00 (d,
1H, H8), 4.60 (m, 1H, H3), 4.44 (d, 1H, H10), 4.22 (dd, 1H, H9), 4.09 (m, 1H, H4), 3.86 -3.76 (m, 2H, H5, H5"), 2.87 (m, 1H, H2), 2.44 (m,
1H, H2"). Second eluted (11R isomer): 1H NMR (400 MHz,
MeOD-d4) 8.70 (s, 1H, H12), 8.45 (m, 1H,
H1), 8.37 (s, 1H, H-8), 8.30 (s, 1H, H-2), 8.08 (s, 1H, H7), 7.86 (m, 1H, H4), 7.77 (d, 1H, H6), 7.72 (d, 1H, H5), 7.54 (m, 1H, H2), 7.54 (m,
1H, H3), 6.47 (dd, 1H, H1), 5.99 (s, br, 1H, H11), 5.00 (d, 1H, H8),
4.56 (m, 1H, H3), 4.44 (d, 1H, H10), 4.22 (dd, 1H, H9), 4.10 (m, 1H,
H4), 3.88-3.76 (m, 2H, H5, H5"), 2.86 (m, 1H, H2), 2.44 (m, 1H,
H2"). Mass spectra were obtained on (±) material. LRMS
(FAB+, glycerol/Me2SO/TFA) 530.2 (M+H+), HRMS (FAB+, glycerol/TFA/PEG)
calculated for
C28H28N5O6: 530.2039 (M+H+); found: 530.2026 (M+H+).
,2,3,4)-2-Deoxy-N6-(1,2,3,4-tetrahydro-2,3,4-trihydroxybenz-[a]anthracene-1-yl)-adenosine
(Bay BadA)
-amino-1,2,3,4-tetrahydrobenz-[a]anthracene-2,3,4-triol (3.2 mg, 11.0 µmol) were weighed into a conical vial. Dry
N,N-diisopropylethylamine (11 µl, 63 µmol, 5 equiv.) and dimethylacetamide (100 µl) were added, and the reaction
mixture was stirred at 45 °C for 92 h. The reaction mixture was
monitored by HPLC on a C-18 reverse phase column (4.6 × 250 mm)
using a linear gradient of 45-65% MeOH in H2O over 20 min
at a flow rate of 1.0 ml/min. Diastereomeric products were eluted at
8-10 min. The diastereomers were separated by preparative HPLC (C-18
reverse phase column, 10 × 250 mm) with the same gradient as for
the analytical column at a flow rate of 2.0 ml/min. The CD spectra of
the first eluted diastereomer showed a negative Cotton effect at 263 and 283 nm and a positive Cotton effect at 252 nm. The second eluted
diastereomer displayed a positive Cotton effect at 263 and 283 nm and a
negative Cotton effect at 252 nm. Stereochemistry of the adducts was
assigned in accordance with literature values (19). First eluted
(1S isomer): 1H NMR (400 MHz, MeOD, ppm) 8.58 (br, 1H, H2), 8.43 (d, 1H, H12, J = 14 Hz), 8.18 (br, 1H, H8), 8.07 (d, 1H, H5, J = 8.9 Hz), 7.97 (d,
1H, H8, J = 8.3 Hz), 7.80 (d, 1H, H5, J = 9.0 Hz), 7.65 (d, 1H, H11, J = 8.7 Hz), 7.42-7.35
(m, 2H, H9 and H10), 6.44 (t, 1H, H1), 6.35 (br, 1H, H1), 4.95 (d, 1H,
H4, J = 8.9 Hz), 4.58 (m, 1H, H3), 4.47 (m, 1H, H2),
4.17 (d, 1H, H3, J = 8.8 Hz), 4.08 (br, 1H, H4),
3.88-3.74 (m, 2H, H5, H5"), 2.82 (m, 1H, H2"), 2.40 (m, 1H, H2).
LRMS (FAB+, glycerol/Me2SO/TFA) 530.2 (M+H+), HRMS (FAB+, glycerol/TFA/PEG)
calculated for
C28H28N5O6: 530.2039 (M+H+); found: 530.2048 (M+H+). Second eluted
(1R isomer): 1H NMR (400 MHz, MeOD) 8.58 (br, 1H, H2), 8.43 (d, 1H, H12, J = 14 Hz), 8.18 (br,
1H, H8), 8.07 (d, 1H, H5, J = 8.9 Hz), 7.97 (d, 1H, H8,
J = 8.3 Hz), 7.80 (d, 1H, H5, J = 9.0 Hz), 7.65 (d, 1H, H11, J = 8.7 Hz), 7.42-7.35 (m, 2H,
H9, H10), 6.44 (t, 1H, H1), 6.35 (br, 1H, H1), 4.95 (d, 1H, H4,
J = 8.9 Hz), 4.58 (m, 1H, H3), 4.47 (m, 1H, H2), 4.17 (d, 1H, H3, J = 8.8 Hz), 4.08 (br, 1H, H4), 3.88-3.74
(m, 2H, H5, H5"), 2.82 (m, 1H, H2"), 2.40 (m, 1 H, H2). LRMS
(FAB+, glycerol/Me2SO/TFA) 530.2 (M+H+), HRMS (FAB+, glycerol/TFA/PEG)
calculated for
C28H28N5O6: 530.2039 (M+H+); found: 530.2048 (M+H+).
-CGGA-CA*A-GAAG-3 (Ras codon 61), where A* is
the 6-fluoropurine nucleoside, was prepared using automated solid-phase
synthesis (Applied Biosystems Model 391 synthesizer) with
6-fluoro-5-DMTr-3-phosphoramidite (14, 15). The cassettes were
emptied into a 1-dram conical vial to which was added non-bay region
(±)-anti-trans-benz[a]anthracene aminotriol
(8.3 mg, 28 µmol), dry Me2SO (600 µl), and
N,N-diisopropylethylamine (10 µl). The mixture
was heated for 3 days at 55 °C under N2. After cooling
to room temperature, the supernatant was removed, and the residual
beads were washed with CH3OH (3 × 1 ml) and diethyl ether (1 ×) and air-dried. The beads were transferred to a 3-dram vial, and concentrated NH4OH (3 ml) was added. The tightly
closed vial was heated for 8 h at 60 °C to effect cleavage from
the solid support and deblocking. After cooling, the vial was allowed
to stand until the excess NH4OH had evaporated. The
supernatant was transferred to a 50-ml centrifuge tube. The beads were
washed with water (2 × 1 ml), and the washings were added to the
original supernatant. After lyophilization, the dry materials were
dissolved in H2O (1 ml), filtered through a 0.45-mm syringe
filter, and passed through a 1.5 × 44 cm column of Sephadex G-15
with elution by H2O. The major UV-absorbing peak was
collected, concentrated, and purified on a reverse-phase column (PRP-1,
7 × 305 mm, Hamilton) at 45 °C using a linear gradient
of 10 mM ethylenediamine acetate, pH 7.45, containing
6-12% CH3CN over 40 min, flow rate 3.0 ml/min. The two
diastereomers were eluted between 22 and 24 min. A second separation on
the same column was performed at 25 °C using 0.1 M
ammonium formate, pH 6.39, containing from 27 to 30% MeOH over 20 min.
After removal of solvent by lyophilization, the fractions were desalted
on oligonucleotide purification cartridges (Applied Biosystems). Peak 1 (5.3 ODs, 11S isomer): enzyme digestion: dC (2.0), dG (4.1),
dA (3.7), dAN6 (11S)-non-brBA (1.3);
theory: dC (2.0), dG (4), dA (4.0),
dAN6 (11S)-non-brBA (1.0). LRMS
(electrospray) calculated Mr = 3677.62; observed ions 1837.93 (M-2H)/2z, 1224.58 (M-3H)/3z, representing a
measured mass = 3677.30. Peak 2 (5.2 ODs, 11R isomer):
enzyme digestion: dC (2.0), dG (4.1), dA (3.9), dAN6
(11R)-non-brBA (1.0); theory: dC (2.0), dG (4), dA
(4.0), dAN6 (11R)-non-brBA (1.0). LRMS
(electrospray) calculated Mr = 3677.62; observed ions 1837.97 (M-2H)/2z, 1224.57 (M-3H)/3z, representing a measured mass = 3677.32.
-CGGA-CA*A-GAAG-3 (Ras codon 61), where A* is
the 6-fluoropurine nucleoside, was prepared by the same method as for
the non-bay benz[a]anthracene-adducted oligonucleotide.
Bay region (±)-anti-trans-benz[a]anthracene
aminotriol (15.4 mg, 52 µmol) was placed in a conical vial containing
the modified oligonucleotide in Me2SO (700 µl) and
N,N-diisopropylethylamine (15 µl). The mixture
was heated for 5 days at 60 °C under N2. After cooling
to room temperature, the beads were treated with the procedure
described above. Following passage through Sephadex G-15 and
lyophilization, the adducted oligonucleotide was dissolved in
H2O (1 ml), and purified on a reversed-phase column
(YMC-ODS-AQ (4.6 × 250 mm)) using a linear gradient of 100 mM ammonium formate, pH 6.5, containing 5-30%
CH3OH over 30 min, 30% for 5 min, and 30-100% over 10 min, flow rate 1.25 ml/min. The collected peaks were lyophilized,
redissolved, and desalted on OPC cartridges according to the
manufacturer directions (Applied Biosystems, Foster City, CA). Peak 1 (7.0 ODs, 1R isomer): enzyme digestion: dC (2.0), dG (3.8),
dA (4.1), dAN6 (1R)-brBA (0.9); theory: dC
(2.0), dG (4), dA (4.0), dAN6 (1R)-brBA
(1.0). LRMS (electrospray) calculated Mr = 3677.62; observed ions 1224.87 (M-3H)/3z, representing a measured
mass = 3677.61. Peak 2 (6.5 ODs, 1S isomer): enzyme digestion: dC
(2.0), dG (3.8), dA (4.1), dAN6 (1S)-brBA
(0.9); theory: dC (2.0), dG (4), dA (4.0), dAN6
(1S)-brBA (1.0). LRMS (electrospray) calculated
Mr = 3677.62; observed ions 1837.53 (M-2H)/2z,
1224.72 (M-3H)/3z, 918.20 (M-4H)/4z, representing a measured
mass = 3677.01.
5 kV and run at 15 kV at
30 °C.
-C GGA CAA GAA G-3). Thus, the adducted adenine residue was located at position 2 of codon 61, a biologically relevant site since point mutations in this sequence have been detected
in numerous tumor types (23-25).
20 °C
(see Latham et al. (26) for a diagrammatic representation of
these procedures).
2 for 50 s. A mock control was not exposed to
254-nm light. Two volumes of 2 × YT medium were then added, and
the cells were grown in the dark at 37 °C for 45 min (final
A600 was approximately 0.7). Finally, cells were
made competent and transformed as described above. To allow
bacteriophage propagation, transformed cells were plated on
Luria-Bertani medium in the presence of a 5-fold excess of UT481
E. coli (met thy 
(lac-pro) hsdR
BamHI hsdM+ supDTn10/F traD36 proAB
lacIqZ M15). The resultant
plaques were treated as those described above.
-agarase according to the supplier
instructions (New England Biolabs). Purification of the circular M13
molecules was again achieved using Microcon-100 microconcentrators. An
aliquot of each sample was subjected to electrophoresis through a 1.5%
agarose gel, and DNA concentrations were estimated via ethidium bromide
staining. All templates were diluted to approximately 40 fmol/µl.
32P]dATP; 0.2 µg/µl beta sliding clamp; 6 mM dithiothreitol; 3 mM ATP; 6 µg/µl BSA;
33 mM Tris-OAc (pH 7.8); 66 mM KOAc; and 10 mM MgOAc) was then added. Finally, 0.1 µg of purified
E. coli DNA polymerase III* (Pol III*) was added to initiate
replication. Pol III* is the complete replication complex except for
the sliding clamp. The final reaction volume was 17 µl. After 3 and 10 min, 8 µl of each reaction were removed and added to 1.7 µl of 6 × stop buffer containing 3% SDS and 120 mM
EDTA. Proteinase K (1 µl of a solution containing 2 µg in 10 mM CaCl2) was then added, and incubation at
37 °C was continued for 30 min. Alkaline agarose gel electrophoresis
loading buffer (1.5 µl of a 7 × solution consisting of 21%
Ficol 400 (Sigma); 350 mM NaOH; 7 mM EDTA;
0.2% bromcresol green; and 0.3% xylene cyanol) was added, and the
samples were stored at 70 °C until analyzed.
ends to 22-mers as described in Latham
et al. (26) and Chary et al. (28). The 33-mer
templates that resulted had the following sequence: 5-C GGA
CAA GAA GAA TTC GTC GTG ACT GGG AAA AC-3. The adducted
adenine was thus located at position 28 (counting from the 3 end). A
17-mer oligonucleotide primer with the sequence 5-TCA CGA CGA ATT CTT
CT-3 was utilized to determine the relative propensity of
exonuclease-deficient E. coli DNA polymerase III (Pol III
) to insert each of
the four common nucleosides opposite the adducted adenine residue
in vitro.
. This brought the
reaction volume to 10 µl. Extension was carried out at 37 °C, and
reactions were terminated by the addition of 2 µl of 6 × stop
buffer (3% SDS, 120 mM EDTA). Reaction products were
separated by electrophoresis through 15% polyacrylamide sequencing gels. Visualization and quantitation of reaction products was via a
Molecular Dynamics PhosphorImager.
Fig. 2.
Capillary gel electrophoresis of adducted
oligonucleotides following HPLC purification. A, non-bay
region 11S-BA; B, non-bay region
11R-BA; C, bay region 1R-BA;
D, bay region 1S-BA.
Fig. 3.
End-labeled 11-mer oligonucleotides (adducted
and non-adducted) after purification by polyacrylamide gel
electrophoresis. 1, non-adducted; 2, non-bay
region 11R-BA adducted; 3, non-bay region
11S-BA adducted; 4, bay region 1R-BA
adducted; 5, bay region 1S-BA adducted. The
purity of the oligonucleotides was determined via PhosphorImager
analysis and is indicated as a percentage of signal beneath each
lane.
Fig. 4.
Site-specifically modified, single-stranded
M13 molecules were allowed to replicate within E. coli
either with or without prior induction of SOS functions. DNAs from
the resultant plaques were probed by differential hybridization to
detect point mutations at the site of the lesion (A).
Replication efficiency of each species of adducted M13 expressed as a
percentage of that of the unmodified M13 (B). Shown are the
frequency and spectrum of point mutations produced opposite the
adducted adenine under different conditions (C).
G transition mutations, whereas the bay
region 1S-BA adducted M13 molecules produced 0.3%
transitions at the lesion site. In total, replication of these DNAs
within AB2480 cells produced 665 (1R-BA adducted) and 345 (1S-BA adducted) plaques. In wild-type AB1157 cells, in the
absence of SOS induction, bay region 1R-BA lesions decreased replication efficiency to 7% of that of unadducted M13 and produced 0.4% A T transversions and 7.3% A G transitions. In the
same cell line, bay region 1S-BA adducted DNAs were
replicated 16% as efficiently as unadducted and induced 4.2%
A G transitions. Under these conditions, bay region
1R-BA adducted M13 generated 275 plaques, and 570 plaques
were generated from the bay region 1S-BA adducted DNAs. When
SOS conditions were induced in this bacterial strain, bay region
1R-BA containing DNAs were replicated 21% as efficiently as
unadducted and induced A T transversions (1.4%) and A G
transitions (10.7%). Those M13 molecules carrying bay region
1S-BA decreased replication efficiency to 19% of that of
unadducted M13 when SOS functions were induced and produced 0.14%
A T transversions and 2.1% A G transitions (Fig. 4, B and C). In total, ~800 plaques were produced
from bay region 1R-BA adducted M13, and ~700 plaques were produced
from the replication of bay region 1S-BA adducted DNAs when
SOS functions were induced.
32P]dATP. Reaction products were
separated by electrophoresis through alkaline agarose gels and analyzed
via autoradiography. As indicated in Fig.
5, when M13 molecules which were linearized in the vicinity of the
adduct site (Cut-L2 Control) were utilized as templates, 92-95% of the products accumulated in a single band corresponding to
1.8 kilobases in length. When a non-adducted 11-mer was substituted for
the adducted 11-mer during ligation, those templates supported a
10-fold increase in the amount of primer that was extended to lengths
greater than 1.8 kilobases. A circular M13 molecule, which had not been
subjected to manipulation (linearization, ligation, and isolation)
prior to reaction (Circular-L2 Control) coded for the
production of more than 73% of primers that were extended beyond the
1.8-kilobase length (Fig. 5).
Fig. 5.
Blockage of Pol III in
vitro. Circular M13 molecules containing either
site-specifically modified adenine residues or the corresponding
unadducted sequences were utilized as templates for polymerization by
E. coli DNA polymerase III holoenzyme in vitro.
The percent of products in which synthesis was blocked in the vicinity
of the lesion is given.
terminus of the primer was one nucleoside upstream of the
adducted adenine were used to determine the efficiency with which
purified Pol III
would insert a given dNTP opposite the site of the lesion
(Fig. 6A). The sequence
context in which the adducted adenine was located was kept the same as
in the other assays described. The most prevalent mutation that was
seen when these lesions were processed intracellularly, A G
transition, was not recapitulated in vitro. Even after 120 min, there was essentially no incorporation of dCTP opposite any of the
adducted adenine residues (Fig. 6B). It is interesting to
note, however, that after 120 min, the adduct that induced the most
mutations in vivo, bay region 1R-BA, was the lesion opposite which Pol III most
efficiently incorporated dT. This differential is not seen at the
10-min time point (see Fig. 6).
Fig. 6.
Single nucleotide incorporation opposite
adducted adenine. Site-specifically modified 33-mer
oligonucleotides were hybridized to 17-mer primers whose 3 termini
were positioned 1 base upstream of the adducted adenine (the sequences
of these DNAs are depicted in the illustration) (A). The
exonuclease-deficient form of E. coli DNA polymerase III
(Pol III ) was
examined for its propensity to incorporate each of the four common
nucleosides opposite the site of the lesion. Percent incorporation is
given (B).
G), consistent with the
mutations induced by other site-specific adenine N6 adducts
in the same sequence context (26, 28).
), which in conjunction with UmuC would promote error prone bypass of the BA lesion even in the absence of a full SOS response. Estimates of the cellular complement of UmuD and UmuC are 180 and 15 copies, respectively, per uninduced cell (39). This hypothesis
is supported by observations that the SOS response is activated simply
by the presence of single-stranded DNA when E. coli are
infected with mutant M13 that replicate much more slowly than wild-type
M13 (40).
G). This would correspond to
the incorporation of dC opposite the adducted adenine residue. In
vitro, there was essentially no difference in the amount of dC
that was incorporated opposite the bay region 1R-BA adducted
adenine and any of the other lesions. Again, these data suggest that
there is a fundamental difference in either the polymerase machinery
that replicates the adducted DNAs in vitro and in
vivo or in the structure that the adduct assumes in the different
assay systems. Work from the Grollman laboratory (34) has demonstrated
a marked difference in the propensity of various polymerases to insert
a given nucleotide opposite an 8-oxoguanine residue. This further
supports the idea that there is a different enzyme system acting on
these DNAs in vivo and in vitro.
**
To whom correspondence should be addressed. Tel.: 409-772-2179;
Fax: 409-772-1790; E-mail: rslloyd{at}scms.utmb.edu.
1
The abbreviations used are: PAH, polycyclic
aromatic hydrocarbon; BA, benz[a]anthracene; non-bay
region 11R-BA, adduct resulting from the trans
opening of
8S,9R,10R,11S-10,11-epoxy-8,9,10,11-tetrahydro-BA-8,9-diol by the exocyclic nitrogen (N6) of adenine; non-bay region
11S-BA, adduct resulting from the trans opening
of
8R,9S,10S,11R-10,11-epoxy-8,9,10,11-tetrahydro-BA-8,9-diol by the exocyclic nitrogen (N6) of adenine; bay region
1R-BA, adduct resulting from the trans opening of
1S,2R,3R,4S-1,2-epoxy-1,2,3,4-tetrahydro-BA-3,4-diol by the exocyclic nitrogen (N6) of adenine; bay region
1S-BA, adduct resulting from the trans opening of
1R,2S,3S,4R-1,2-epoxy-1,2,3,4-tetrahydro-BA-3,4-diol by the exocyclic nitrogen (N6) of adenine; cut L2, linear
M13mp7L2 resulting from the cleavage of the single-stranded form by
EcoRI; BSA, bovine serum albumin; Pol III*, E. coli DNA polymerase III*; Pol III
,
exonuclease-deficient E. coli DNA polymerase III; Pol III,
E. coli Pol III holoenzyme; THF, tetrahydrofuran; TLC,
thin-layer chromatography; TFA, trifluoroacetic acid; PEG, polyethylene
glycol; HPLC, high performance liquid chromatography; LRMS, low
resolution mass spectrum; HRMS, high resolution mass spectrum; FAB,
fast atom bombardment.
Volume 272, Number 52,
Issue of December 26, 1997
pp. 33211-33219
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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  J. C. Delaney, P. T. Henderson, S. A. Helquist, J. C. Morales, J. M. Essigmann, and E. T. Kool High-fidelity in vivo replication of DNA base shape mimics without Watson-Crick hydrogen bonds PNAS, April 15, 2003; 100(8): 4469 - 4473. [Abstract] [Full Text] [PDF]
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 M. Kanuri, I. G. Minko, L. V. Nechev, T. M. Harris, C. M. Harris, and R. S. Lloyd Error Prone Translesion Synthesis Past gamma -Hydroxypropano Deoxyguanosine, the Primary Acrolein-derived Adduct in Mammalian Cells J. Biol. Chem., May 17, 2002; 277(21): 18257 - 18265. [Abstract] [Full Text] [PDF]
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 I. Ponten, L. S. Waters, J. M. Sayer, A. S. Pilcher, A. Dipple, and D. M. Jerina Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2 Mutagenesis, January 1, 2001; 16(1): 65 - 69. [Abstract] [Full Text] [PDF]
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J. R. Carmical, A. Kowalczyk, Y. Zou, B. Van Houten, L. V. Nechev, C. M. Harris, T. M. Harris, and R. S. Lloyd Butadiene-induced Intrastrand DNA Cross-links: A Possible Role in Deletion Mutagenesis J. Biol. Chem., June 23, 2000; 275(26): 19482 - 19489. [Abstract] [Full Text] [PDF]
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