(Received for publication, May 19, 1994; and in revised form, November 16, 1994)
From the
Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide
(BPDE), a metabolite of the widespread environmental pollutant
benzo[a]pyrene, is mutagenic in both bacterial and
mammalian systems. Toward understanding the mutagenic effects of
different stereoisomers of BPDE at specific sites in DNA, six
stereochemically defined BPDE adducts were constructed on adenine N at position 2 of the human N-ras 61
codon within an 11-base oligonucleotide fragment. Both the nonadducted
and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted
11-mers into the single-stranded vector were determined by Southern
hybridization and confirmed by electron microscopy. Repair-deficient
AB2480 E. coli cells were transformed with adducted and
nonadducted DNA samples. The resultant plaque-forming abilities were
used to evaluate the replication competence of the various BPDE adducts
with respect to the nonadducted 11-mer. Point mutations due to aberrant
replication at the adducted site were identified by the technique of
differential DNA hybridization. All of the six BPDE adducts examined
were mutagenic in vivo, generating exclusively A
G
mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved
primer extension assays with Klenow fragment. All of the BPDE-modified
templates demonstrated distinct blockage at the adducted site and/or 1
base 3` to the adducted site, allowing essentially no translesion
synthesis to form fully extended polymerization products in
vitro.
Polycyclic aromatic hydrocarbons (PAH) ()are
pervasive in the environment, arising during combustion processes. Some
of these PAH are carcinogens. Benzo[a]pyrene (BP) is
one such PAH that has received intense study in an attempt to define
mechanisms of genotoxicity. This compound is metabolically activated to
bay region 7,8-dihydrodiol 9,10-epoxides that initiate mutagenesis and
carcinogenesis by covalently binding to
DNA(1, 2, 3) . The mutagenic potential of
these diol epoxides is dependent on a variety of interactions including
ones between the carcinogen and the template, the nature of the
polymerase involved in replication past the adduct, and the efficiency
of DNA repair within the cell. DNA lesions caused by such carcinogens
if improperly repaired, may be converted to permanent changes in the
genome during DNA replication. These changes involve base
substitutions, deletions, or frameshift mutations that eventually can
lead to neoplastic transformation(2) . Incubation of BPDE with
DNA containing ras genes can result in
oncogenesis(4, 5) . The mechanism by which these ras genes are activated in tumor cells often involves a single
point mutation, usually resulting in the alteration of amino acid
residue 12 or 61 of the protein encoded by these genes(4) . In vitro mutagenesis experiments showed that activating
mutations could also occur at codons 13, 59, and
63(6, 7) .
There is considerable evidence to
demonstrate that primary nucleotide sequence can modulate the
stereo-selectivity and distribution of BPDE lesions in modified
DNA(8, 9, 10) . Thus, carcinogenic species
may be biased toward or against certain bases because of the
stereoelectronic effects of adjacent bases or the stereochemistry of
the ultimate carcinogen(11) . Furthermore, preferential
site-specific mutation at a particular position could be due to the
relative stability of the adduct or the lack of significant structural
distortions in the nucleotide caused by the carcinogen(12) .
The interaction of these PAH diol epoxides with numerous sites in DNA
involves a nucleophilic attack in every case at the benzylic carbon of
the epoxide resulting in a S
The heterogeneity of base adduction by BPDE is significantly reduced by synthesizing optically pure (+)- or (-) -anti or -syn enantiomers. Variations in biological activity between enantiomers within a given test system are likely to be due to different conformations assumed by these adducts. Studies that differentiate between effects due to adducts produced by cis and trans addition of anti- and/or syn-BPDE to DNA are also gaining importance(16, 17) . Studies examining the metabolism of BP in 3-methylcholanthrene-treated rats have shown four possible BPDE isomers. The ratios of formation for (+)-anti-BPDE/(-)-syn-BPDE/(-)-anti-BPDE/(+)-syn-BPDE were 214:36:24:1, respectively.(18, 19, 20, 21) . However the adduct-forming potentials of these BPDE isomers on adenine have not been firmly established. In spite of the availability of considerable information on both the binding spectra and mutational specificity of BPDE, little is known about the relationship between these two factors within specific sequences. Template-directed mutagenesis employing oligodeoxynucleotides bearing stereo-specific and site-specific lesions offer the possibility of correlating a stereochemically-defined adduct with a particular mutation spectrum. The mutagenicities of these stereoisomers, however, are different in bacterial and mammalian cells (22, 23, 24) . Single-stranded vectors carrying a defined, uniquely located lesion are powerful tools for investigating mutagenic mechanisms in vivo both in prokaryotic and eukaryotic systems(25, 26) .
The objective of this study was
to correlate in vivo and in vitro replication
competence with BPDE adduct chirality. Toward this goal, six
stereochemically-defined BPDE adducts were constructed on adenine
N at position 2 of N-ras codon 61 within an
11-base oligodeoxynucleotide by the postoligomerization
strategy(27, 28) .
BPDE-adducted N-ras 61-containing 11-mers were synthesized by the method of Kim et al. (28) and purified as described by Latham et al.(30) . Both the nonadducted and the BPDE-adducted N-ras 61-containing oligonucleotides were phosphorylated with T4 polynucleotide kinase (New England Biolabs Inc., Beverly, MA). A 100-fold molar excess of the different N-ras 61 11-mers relative to the amount of linearized vector were individually ligated together in the presence of a 2-fold excess of a 51-mer scaffold(25, 30) . Each reaction was incubated overnight at 16 °C with a total of 400 units of T4 DNA ligase (New England Biolabs Inc.).
Ligation efficiencies were further confirmed by electron microscopy subsequent to the removal of the 51-mer scaffold. Samples were prepared using the formamide modification of the basic protein (Kleinschmidt) technique as described by Davis et al.(31) . Grids were rotary shadowed with platinum/palladium in a ratio of 80:20 and examined in either a Philips 300 or 410 electron microscope. Projected images were traced from photographic negatives, and lengths were determined with a map measure.
Figure 1:
Structures of adenine N stereoisomers placed at the second position of N-ras codon 61 within an 11-mer
oligodeoxynucleotide.
The purities of 11-mers containing
the isomeric dA-BPDE adducts were also analyzed on 15% polyacrylamide
sequencing gels subsequent to labeling the 5` terminus using T4
polynucleotide kinase and [-
P]ATP.
Electrophoretic migration of BPDE-modified oligomers was distinctly
slower than that of unmodified 11-mers (Fig. 2). Slight
differences in mobility pattern were also observed among the various
adducted 11-mers. Gels exposed for longer periods revealed no traces of
unmodified 11-mer contaminants in the six dA
-modified
oligodeoxynucleotides.
Figure 2:
Polyacrylamide gel electrophoresis of
11-mers containing a single isomeric dA N-BPDE adduct.
Oligodeoxynucleotides were labeled with
P at the 5`
terminus and subjected to electrophoresis through 15% polyacrylamide
gels (33
44
0.04 cm). The BPDE-adducted oligomers are
shown in lanes2-7. Lane1,
unmodified 11-mer; lane2,
(-)-syn-trans-BPDE 11-mer; lane3,
(+)-syn-trans-BPDE 11-mer; lane4,
(+)-anti-cis-BPDE 11-mer; lane5,
(-)-anti-cis-BPDE 11-mer; lane6,
(+)- anti-trans-BPDE 11-mer; lane7,
(-)-anti-trans-BPDE 11-mer.
Figure 3: Schematic representation for the insertion of N-ras 61 oligodeoxynucleotides in the M13mp7L2 genome and the detection of point mutations.
Figure 4:
Determination of specific point mutations
at position 2 of N-ras codon 61. 11-mers containing the
N-ras 61 codon, differing only by AC or A
G or
A
T at position 2, were inserted into the unique EcoRI
site of M13mp7L2 phage DNA. A shows a mixture of
mutant/wild-type plaques (1:25, approximately) that hybridized
specifically only to their complementary probes. B is a
tabular form of the actual number of wild-type and mutant phage that
give a positive signal, and these values approximately equal the
original number of plaques plated.
To further determine the limits of sensitivity of this differential hybridization, four 51-mer sequences bearing the N-ras 61 codon within them but differing only by a single nucleotide at position 2 were constructed. Dot-blot assays were performed with these DNAs ranging from 1 pg to 5 µg. DNA amounts as low as 1 ng hybridized with their complementary 17-mers with high specificity and were distinctly detected after an overnight exposure. DNAs ranging from 0.5 to 5 µg further exhibited an intense signal with their corresponding probes under exposures as short as 15 min (data not shown).
These control experiments firmly establish the viability of phage containing any type of point mutation and our ability to identify any of these mutations resulting as a consequence of replication past a BPDE-adducted site.
In concert with the results of Southern blot analyses for the determination of ligation efficiencies, independent confirmation was obtained through direct visualization of doubly-ligated molecules by electron microscopy. Using denaturing electron microscopic methodologies, the appearance of circular single-stranded DNA molecules was direct evidence for the presence of doubly ligated 11-mers into the EcoRI restriction site of M13mp7L2 DNA. Linear single-stranded DNA molecules represented either the vector DNA with no insert or those that were singly ligated to an 11-mer. At least 200 molecules were scored for each of the six stereoisomerically-defined BPDE-adducted DNAs and the nonadducted N-ras 61-11-mer template samples. The average full-length circular forms of ligated vector DNA with the insert was 1.76 ± 0.18 µm (Fig. 5). As discussed above, although full-length linear DNA molecules were detected by electron microscopy, it was not possible to distinguish singly-ligated molecules from the linearized vector alone due to the small insert size. This electron microscopic study provides an alternative methodology for verification of the double ligation event.
Figure 5: Ligated circles of BPDE-adducted 11-mer within the single-stranded M13mp7L2 vector as determined by electron microscopy. The BPDE-modified 11-mers were inserted into an unique EcoRI site within M13mp7L2 single-stranded DNA, resulting in covalently closed circular molecules.
Similar to the ligation efficiencies, the plaque-forming abilities of all the six stereochemically defined BPDE-adducted DNAs were distinctly lower than the corresponding values of the unmodified template. Furthermore, a wide spectrum of plaque-forming abilities was observed, ranging from 5.8% for the(-)-anti-trans-BPDE adduct to 22.8% for the (+)-anti-cis-BPDE adduct ( Table 1and Table 3).
Figure 6: Synthesis of site-specific BPDE-adducted 33-mers employed for in vitro studies.
Equimolar ratios of templates (nonadducted and adducted) and P-end-labeled primer were utilized for the polymerization
reactions. A time course study was performed that included primer
extension reactions for 2, 5, 10, and 30 min. Sequence analyses of
extended primers replicated on DNA templates were carried out by
subjecting the reaction mixtures to 15% polyacrylamide gel
electrophoresis. Following electrophoresis, positions of the oligomers
were established by autoradiography as shown in Fig. 7.
Sequences for both the template and primer are depicted above the
results of primer extension (Fig. 7). No qualitative differences
were observed within individual extension reactions for any of the
adducted templates, ranging from 2 to 30 min. The reactions appeared to
be complete within the first 2 min, indicating no further translesion
synthesis over longer incubation times. The
P-end labeled
17-mer primer employed in each of the reactions was completely utilized
as indicated by the absence of a band at the 17-mer position (Fig. 8). The nonadducted 33-mer template exhibited a
full-length product at the end of 2 min of polymerization. In contrast,
none of the six adducted oligodeoxynucleotides accumulated full length
products even after 30 min of synthesis. All six BPDE-modified 33-mers
served as poor templates resulting in blockage of in vitro replication due to synthesis being stopped opposite and/or 1 base
3` to the adducted site. Replication of
(+)-anti-trans-, (+)-syn-trans-, and (-)-anti-cis-BPDE-adducted templates was
completely blocked at 1 base 3` to the adducted site.
With(-)-anti-trans-, (-)-syn-trans-, and
(+)-anti-cis-BPDE-modified 33-mers, a nucleotide was
placed opposite the adducted site in each case but no replication
occurred beyond that point (Fig. 7).
Figure 7: A kinetic analysis of primer extension reactions with templates containing various BPDE stereoisomers. The sequence of the template-primer complex is represented at the top. The adducted site is designated by an asterisk. Chain elongation studies were performed at 2, 5, 10, and 30 min. Lane1, N-ras 61-33-mer; lane2, (+)-anti-trans-BPDE-33-mer; lane3, (-)-anti-trans-BPDE-33-mer; lane4, (-)-syn-trans-BPDE-33-mer; lane5, (+)-syn-trans-BPDE-33-mer; lane6, (+)-anti-cis-BPDE-33-mer; lane7, (-)-anti-cis-BPDE-33-mer.
Figure 8: Differential blockage of replication at the adducted site and 1 base 3` to the site of lesion. Lanes2-7 contain templates with different stereoisomeric BPDE adducts. Lane1, N-ras 61-33-mer; lane2, (+)-anti-trans-BPDE-33-mer; lane3, (-)-anti-trans-BPDE-33-mer; lane4, (-)-syn-trans-BPDE-33-mer; lane5, (+)-syn-trans-BPDE-33-mer; lane6, (+)-anti-cis-BPDE-33-mer; lane7, (-)-anti-cis-BPDE-33-mer. These data were taken from a 30-min primer extension reaction with the Klenow fragment. The 27-mer and 28-mer represent the partially extended products up to 1 base 3` to the adducted site and opposite the site of lesion, respectively. Percentages of fully extended primer and blockage at various positions were analyzed by VISAGE gel electrophoresis analysis system and were tabulated as shown below.
Extended products were quantitated by densitometric analysis of the autoradiographs. A 30-min time point was chosen to measure the amount of fully-extended or truncated products formed with nonadducted and adducted templates (Fig. 8). Based on the autoradiographic signal, 99.3% of the extended product of the nonadducted template was of full length (Fig. 8, lane1). With the adducted templates that inhibited replication beyond 1 base 3` to the site of lesion, approximately 99.7% of the partially extended products were represented by this premature termination (Fig. 8, lanes2, 5, and 7). Two of the lesions ((-)-anti-trans- and (-)-syn-trans-) responsible for significant pause sites 1 base downstream of the adduct revealed polymerized products amounting to two-thirds of all the extended products (Fig. 8, lanes3 and 4). The remaining one-third was represented by those product molecules that terminated opposite the corresponding adduct. The (+)-anti-cis-BPDE enantiomer exhibited roughly a 14:1 ratio of partially extended products 3` to the adducted position to those opposite the lesion (Fig. 8, lane6). In essence, all of the BPDE-adducted templates proved to be poor substrates for in vitro replication with Klenow fragment in contrast to the relatively efficient replication competence of four of these six adducted sequences in repair-deficient E. coli cells after adjusting for ligation efficiencies.
Specific chiral interactions of different diastereomers of BPDE with nucleophilic sites in DNA cause genotoxic lesions that lead to consequences such as mutations, which can initiate a cancerous response in cells, subsequently leading to alterations in gene expression(23, 34, 35) . The focus of this study was to observe the role of six stereochemically-defined BPDE adducts both in vivo and in vitro when anchored at a specific position on DNA. The mutagenic potential of dA-BPDE adducts that were determined by in vivo studies included the relative lethality of these lesions as well as the type and incidence of point mutations arising from replication of the damaged DNA. An additional parameter investigated by in vitro observations involved the role of Klenow fragment in translesion synthesis on encountering the various stereospecific BPDE adducts attached to the DNA template.
To assess the relative abilities of these stereoisomers to adversely influence replication, it is critical that the modified oligonucleotides be of utmost purity(33) . Toward this goal, synthesis of oligodeoxynucleotides bearing adducts at exocyclic amino sites of adenine was carried out by the postoligomerization method, and the integrity of the samples was determined by a variety of analytical techniques(27, 28) . The resultant high purity of the adducted oligonucleotides establishes that within our ability to determine, the mutational frequencies observed were due to the adducts alone and not as a result of contaminants (Fig. 2).
In
vivo studies involved repair-deficient cells that had a recA, uvrA
genotype. This eliminated the effect of inducible responses
attributed to DNA repair. Furthermore, the choice of a single-stranded
vector was advantageous in two ways. First, single-stranded DNA are
poorer substrates than double-stranded genomes for repair, consequently
aiding in a better understanding of template-directed
mutagenesis(36, 37) . Second, the ease of introducing
nonadducted or adducted oligodeoxynucleotides into single-stranded
M13mp7L2 is far greater than inserting oligomers into a gapped
duplex(38, 39, 40, 41) . However it
is not uncommon to obtain poor ligation efficiencies with modified DNA
as observed in this study(40) . This could be attributed to the
interaction of the bulky BPDE adduct with the template, thus causing
structural distortions. The broad range of ligation efficiencies from
8.5 to 31.2% exhibited by the different stereoisomers is likely to be a
result of the chirality of the molecule. The presence of a distinct
population of singly- ligated molecules in the ligation reaction could
be either because of the formation of a secondary structure by the
vector DNA or because the physical presence of the adduct makes it
difficult to form a closed molecule. The recovery of relatively good
yields of the 33-mer constructs for in vitro analyses suggests
that the ligation at least at the 3`-OH of the 11-mer is reasonably
efficient. Therefore, the perturbations inhibiting ligation are more
likely to occur at the 5` end of the 11-mer.
A wide spectrum of
lethality was observed within the various adducted templates examined
even after adjusting for ligation efficiencies. When considering
ligation efficiencies and plaque-forming abilities as contributors to
survival, the data suggest that percentage lethality follows the rank
order of(-)-anti-trans- >
(-)-syn-trans- >
(+)-anti-trans- >
(+)-syn-trans- > (+)-anti-cis-
(-)-anti-cis-BPDE-adducted templates (Table 1-III). It is possible that there is either direct
blockage of polymerase III activity or apparent loss of processivity by
this holoenzyme after bypassing the(-)-anti-
and(-)-syn-trans-BPDE adducts that lead to decreased
survival. Similar diminished levels of enzyme processivity were
observed in replicative bypass of an abasic DNA lesion(42) .
However, the low to nonlethality of the remaining four BPDE isomers
examined could be due to very little or no blockage of DNA replication in vivo(43) . Thus, differences in spatial
configuration influence the template properties of lesions toward DNA
replication and survival of the cell(23) .
In vivo mutagenesis of all of the BPDE adducts studied revealed only
AG transitions. These findings are in contrast to earlier reports
both in prokaryotic and mammalian cells wherein A
T transversions
were prevalent when adenine was the site of lesion for BPDE or other
bulky adducts such as 9,10-dimethy-1,2-benzanthracene and cis-diamminedichloroplatinum
II(4, 5, 11, 44, 45, 46, 47) .
Previous studies using a styrene oxide DNA adduct in the same sequence
context as this work, however, resulted in A
G base
substitutions(30) . These mutations do not follow the
``A-rule'' put forth to explain the mutational behavior of
abasic and bulky, ``noninstructional'' lesions. DNA
polymerases that preferentially insert adenine opposite these sites of
lesions are believed to be subject to an A rule. Therefore, in
contrast, dA
may be directly miscoding or
misinstructional rather than requiring a ``default'' mutation
mode. This misinstructional lesion effect could possibly be influenced
by local sequence context. In addition, it could be a consequence of
the structural distortion of the adducted base that preferentially
allows A
G transitions alone to occur. Results presented in this
study exhibited a frequency of error spanning from 0.26 to 1.20%,
indicating a 5-fold difference. Furthermore, decreasing the molar
concentrations of the adducted 11-mers by 10-50 fold in the
ligation reaction caused no change in the percentage mutations, thus
attributing the mutagenecity to the BPDE adducts rather than to any
contaminants. The small yet significant changes in the mutation
frequency among the BPDE lesions studied, could be a consequence of
adduct conformational polymorphism resulting in varying interactions
with cellular enzyme systems. The adducts could directly be in contact
with the polymerase involved in replication, leading to stabilization
of a mispaired configuration, as proposed for one of the mitomycin
C/DNA lesions(48) . Alternatively, they could cause subtle
structural changes in the polymerase or the template such that optimal
base pairing with the incoming dNTP does not occur. The fact that more
than approximately 98% of the time these bulky adducts were not
mutagenic in this study, implies that DNA polymerases can be flexible
without completely compromising fidelity(17) . These enzymes
may have an additional ``sensor'' to bypass any structural
distortions the adducts make in the major groove, similar to the
observations made with the N
-dG BPDE adducts(49) .
Furthermore, they may also identify bases in spite of improper hydrogen
bonding between base pairs, as observed with the DNA lesions induced by
vinyl chloride(50) . However, the impact of DNA repair enzymes
substantially altering our in vivo results in the present
study was curtailed by allowing replication to occur in a
repair-deficient environment.
In vivo analyses of translesion synthesis of the BPDEadducted templates were complemented by in vitro studies. Previous in vitro primer extension assays with BPDE-adducted templates involved almost exclusively guanine residues (16, 51, 52) . These bulky DNA adducts are known to block DNA replication with certain enzyme systems either at or 1 base prior to the site of the adduct in the template(53) . Recent in vitro studies with oligonucleotides containing stereospecific trans-adducts of anti- and syn-BPDE on adenine indicated that the polymerase (Sequenase) was completely arrested at 1 base 3` to the adduct(54) .
The present study is a further effort to investigate how BPDE-adenine adducts behave upon encountering polymerases in an in vitro system. Although replication in E. coli cells is predominantly performed by DNA polymerase III, the difficulty of assembling all the core proteins involved in the proper functioning of this holoenzyme is a major limiting factor. Therefore, the alternate choice of Klenow fragment was made due to its wide usage with various adducted templates. Kinetic analyses in this investigation revealed that the Klenow fragment had no capacity to perform translesion synthesis even after an incubation period of 30 min. Temporal (from 2-30 min) comparison of the patterns of the partially extended products from each adducted template indicated that the primer extensions were completed as early as 2 min. All of the template and primer were utilized completely as indicated by the fact that no primer was left at the 17-mer position when equimolar substrates were employed in the reactions (Fig. 8).
Quantitative analyses of the truncated products exhibited almost
total termination 3` to the adducted site with
(+)-anti-trans-, (+)-syn-trans-,
and (-)-anti-cis-adducted templates, whereas with
(-)-anti-trans-,(-)-syn-trans- and
(+)-anti-cis-BPDE adducts a stop site was observed 3` to
the adducted site, followed by complete blockage opposite the lesion (Fig. 8). Besides the bulky nature of the adducts that could be
responsible for this resistance to in vitro replication, the
orientation and tilt of the pyrenyl ring relative to the modified
strand could be a causal factor for stalling and abrupt cessation of
primer extension with the Klenow fragment. It is not unlikely that
those adducts impeding the enzyme progress beyond 1 base 3` to the
lesion site (Fig. 8, lanes2, 5, and 7) could be stereo-selectively distinct in angle positioning
from the isomers that allow incorporation of a nucleotide opposite the
adduct (Fig. 8, lanes4 and 6). This
dichotomy is further exacerbated by the fact that isomers categorized
under the first group exhibit a S configuration at the C-10 of
BPDE, whereas the isomers encompassing the second group revealed a R conformation at the same position (Fig. 1). There is
evidence to show that PAH with 10S configuration point in the
opposite direction to those with the 10R configuration (3) . The precedence of exonuclease digestion controlled by
adduct orientation relative to the 5` 3` strand polarity
indicates its influence on enzyme action(55) . Likewise it is
probable that polymerization by a specific enzyme is dictated by the
bulky adduct orientation relative to the site-specifically modified
single-stranded DNA. NMR studies on the duplex structure of adducted
oligonucleotides at N
of dG determined the directionality
of various stereoisomers of
BPDE(49, 56, 57) . Similar observations on
spatial positioning were made with styrene oxide adducts that showed a
pattern of in vitro blockage parallel to those obtained in
this study(30, 58) . In spite of BPDE lesions forming
effective blocks to DNA synthesis by Klenow fragment in vitro,
it is clear that in vivo replication, which is necessary for
the survival of a cell, occurred beyond the adducted site, probably
through polymerase III.