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INTRODUCTION |
Various pathways exist in cells to overcome replication blockage
caused by DNA lesions. One such pathway, translesion DNA synthesis,
involves specialized polymerases that, unlike replicative polymerases,
are able to perform DNA synthesis on a damaged DNA template (reviewed
in Refs. 1-3). Translesion DNA synthesis can be error-free or
error-prone, depending on the chemical structure of the lesion and the
polymerase utilized for translesion replication. Among the eukaryotic
DNA polymerases, yeast and human DNA polymerases
perform efficient
and accurate replication past a cis-syn cyclobutane pyrimidine dimer, a predominant DNA lesion formed by ultraviolet irradiation (4-7). In the yeast Saccharomyces cerevisiae,
deletion of RAD30, which encodes pol
,1 confers moderate
sensitivity to UV irradiation and an increase in UV-induced mutagenesis
(8).
Mutations in the human RAD30A gene, the counterpart of the
yeast RAD30, cause the variant form of xeroderma pigmentosum
(9, 10). Xeroderma pigmentosum variant cells are hypermutable in response to UV irradiation, and they exhibit a significantly reduced ability to bypass a T-T dimer (reviewed in Ref. 3). Consequently, xeroderma pigmentosum variant individuals suffer from a high incidence of sunlight-induced skin cancers.
7,8-Dihydro-8-oxoguanine is one of the lesions formed by oxidative
damage to DNA. Yeast and human pol
both efficiently bypass the 7,8-dihydro-8-oxoguanine lesion. Whereas other polymerases insert A
opposite this lesion, pol
preferentially inserts a C (11). Thus,
pol
is unique among DNA polymerases in its ability to bypass a T-T
dimer and a 7,8-dihydro-8-oxoguanine lesion efficiently and accurately.
Here we examined the ability of yeast pol
to carry out translesion
synthesis on DNA substrates containing
N2-guanine adducts of stereoisomeric
1,3-butadiene metabolites. 1,3-Butadiene is a potent carcinogen in mice
and to a lesser extent in rats (12) and has been classified as a
probable human carcinogen. Butadiene-mediated carcinogenesis is
initiated through its reactive metabolites: butadiene monoepoxide,
butadiene diepoxide, and butadiene diolepoxide. Each of these
metabolites is represented by at least two stereoisoforms. The
mutagenicity of butadiene and its reactive metabolites has been
observed in several biological systems, particularly in yeast (13, 14)
and mammalian cells (15). Butadiene epoxides can react at numerous
sites in DNA, forming a multitude of adducts that differ in their
stereochemistry (16, 17). Butadiene epoxides are potent inhibitors of
synthesis by DNA polymerases. Previously, it has been shown that
Escherichia coli DNA polymerases I, II, and III are
incapable of bypassing DNA substrates containing (R)- and
(S)-BDO N2-guanines and
(R,R)- and (S,S)-BDE
N2-guanines (18) as well as
(R,R)- and (S,S)-BDE
N2-guanine-N2-guanine
intrastrand cross-links (19). Here we examine the action of yeast pol
on these two types of the N2-guanine
epoxide-containing DNA substrates.
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MATERIALS AND METHODS |
DNA Substrates with Site-specific Lesions--
The
oligodeoxynucleotides containing butadiene epoxide
N2-guanine adducts were prepared by the
postoligomerization methodology developed by Harris et al.
(20). A detailed description of the synthesis of the 11-mer
oligonucleotides containing the (R)- and (S)-BDO
and (R,R)- and (S,S)-BDE
N2-guanines has been described previously (18).
The 8-mer substrates containing the (R,R)- and
(S,S)-intrastrand BDE
N2-guanine-N2-guanine
cross-links were synthesized as published previously (19).
To construct the templates for polymerase reactions, each adducted
oligonucleotide was ligated by T4 DNA ligase (New England Biolabs Inc.,
Beverly, MA) with two flanking oligonucleotides in the presence of the
complement 45-mer scaffold. The ligation products were purified via
denaturing polyacrylamide gel electrophoresis. The sequences
containing the BDO and BDE N2-guanine lesions
are identical:
5'-AGAATGTGGAAGATACTGTGGGCAGGTGGTGAATGGTCTGGGCAATGTCGTTGACTGGGA-3', where the adducted G is underlined. The sequence containing the BDE
N2-guanine-N2-guanine
cross-link is as follows:
5'-CTAGAATGTGGAAGATACTGTGCATGGTCCAATGGTCTGGGCAATGTCGT-3', where the cross-linked guanines are underlined.
Oligodeoxynucleotides of anion-exchange grade purity were used
as primers in the polymerase reactions and were obtained from the
Midland Certified Reagent Co. (Midland, TX). Their sequences include
5'-ACGACATTGCCCAGACCATT-3', which is complementary to the BDO and
BDE N2-guanine-adducted templates from
positions
6 to
26 relative to the site of lesion. The same primer
was used for the BDE
N2-guanine-N2-guanine
cross-link-containing substrates, being complementary from positions
4 to
24. 5'-ACATTGCCCAGACCATTGGA-3' was used as the
1
primer for the BDE
N2-guanine-N2-guanine
cross-link-containing substrates, and 5'-ATTGCCCAGACCATTCACCA-3' served
as the
1 primer for the DNAs containing the BDO and BDE N2-guanine lesions. 5'-TTGCCCAGACCATTCACCAC-3'
and 5'-GCCCAGACCATTCACCACC-3' served as the 0 and +1 primers,
respectively, overlapping the lesion site in the BDO and BDE
N2-guanine-adducted substrates.
Primer oligodeoxynucleotides were phosphorylated with T4 polynucleotide
kinase (New England Biolabs Inc.) using [
-32P]ATP
(PerkinElmer Life Sciences). The 32P-labeled primers
were mixed with the oligonucleotide substrates in a molar ratio of 1:2
in the presence of 50 mM Tris-HCl (pH 7.0) and 100 mM NaCl, heated at 90 °C for 2 min, and slow cooled to
room temperature. The completeness of the primer annealing was
confirmed by electrophoresis through a native 7.5% polyacrylamide gel.
pol
Purification--
The glutathione
S-transferase-pol
fusion protein was overexpressed and
purified as described previously (4).
DNA Polymerase Reaction--
The pol
polymerase assays were
carried out essentially as described by Johnson et al. (4).
The reaction mixture (10 µl) contained 25 mM potassium
phosphate buffer (pH 7.0), 5 mM MgCl2, 5 mM dithiothreitol, 100 µg/ml bovine serum albumin, 10%
glycerol, 100 µM dNTPs (each of the four dNTPs or one, as
indicated), 5 nM primer annealed to a template, and 2 nM glutathione S-transferase-pol
. After
incubation at room temperature for 20 min, reactions were terminated by
the addition of a 10-fold excess loading buffer consisting of 95%
(v/v) formamide, 20 mM EDTA, 0.02% (w/v) xylene cyanol,
and 0.02% (w/v) bromphenol blue. The pol I (Klenow fragment) polymerase reactions were performed basically under the same conditions as the pol
reactions but in the presence of the buffer provided by
the enzyme supplier (New England Biolabs Inc.). The reaction products
were resolved through a 15% polyacrylamide gel containing 8 M urea. Bands were visualized by autoradiography of the wet gels using Hyperfilm MP x-ray film (Amersham Pharmacia Biotech). Quantitative analyses of the results were performed using a
PhosphorImager screen and ImageQuant 5.0 software (Molecular Dynamics,
Sunnyvale, CA).
Steady State Kinetic Analysis--
Steady state kinetic assays
were carried out under the same conditions as the DNA polymerase assays
except that 1 nM pol
and 10 nM DNA
substrates were used with various concentrations of one of the four
nucleotides, and reactions were quenched after 5 min. DNA band
intensities were quantitated using the PhosphorImager (Molecular
Dynamics) and then used to calculate the rate of nucleotide incorporation as described previously (21). The rate of nucleotide incorporation was graphed as a function of nucleotide concentration, and kcat and Km parameters
were obtained from the best fit of the data to the Michaelis-Menten equation.
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RESULTS |
Translesion DNA Synthesis by pol
on the (R)- and (S)-BDO and
(R,R)- and (S,S)-BDE N2-Guanine-adducted DNA
Substrates--
The structures of the BDO and BDE
N2-guanine stereoisomers, which were examined in
this study, are shown in Fig. 1. Among
the butadiene epoxide guanine species that are formed as a result of
the butadiene exposure, the N2-guanine adducts
are relatively stable (16). In E. coli, replication efficiencies past the BDO and BDE N2-guanines
are significantly reduced in vivo, and the presence of these
lesions in DNA is a complete block to synthesis by E. coli
pol I, II, and III in vitro (18).
Primer extension reactions were carried out to test the ability of
yeast pol
to perform translesion DNA synthesis on the BDO and BDE
N2-guanine-adducted DNA substrates (Fig.
2A). Primers were designed that provided "running start" (
6 primer) and "standing start" (
1 primer) conditions. As shown in Fig. 2A, yeast pol
replicated through all four butadiene lesions, resulting in full-length
products. However, pol
displays a strong stall site one nucleotide
before the DNA lesion (lanes 3-6), suggesting an inhibition
of nucleotide incorporation opposite the lesion. Interestingly, the
bypass efficiency of pol
seems to show stereospecificity. On the
BDO N2-guanine-containing substrates, as
well as on the BDE N2-guanine adducts,
translesion DNA synthesis was more efficient in the case of the
S stereoisomers.

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Fig. 2.
DNA polymerase activity
of S. cerevisiae pol (A) and E. coli pol I (Klenow
fragment) (B) under standing start and running start
conditions on the monoepoxide- and diolepoxide-guanine-adducted
templates. Each of the templates (ND = nondamaged,
(R)-BDO = (R)-BDO
N2-guanine, (S)-BDO = (S)-BDO N2-guanine,
(R,R)-BDE = (R,R)-BDE
N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine)
was annealed to one of two primers. The DNA substrates (5 nM) were incubated for 20 min at 22 °C in the presence
of all four dNTPs and S. cerevisiae pol (2 nM) or E. coli pol I (Klenow fragment) (1 unit,
as defined by New England Biolabs Inc.). Incubation of the nondamaged
substrate under the same conditions but without polymerase was used as
a negative control reaction. The positions of the 20-nt primers and the
51-nt (running start reaction) and 46-nt (standing start reaction)
full-length products are indicated. *, position of the
nondamaged G or the adducted G on the template.
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Primer extension reactions using E. coli pol I (Klenow
fragment) were also carried out on substrates containing the BDO and BDE N2-guanine adducts (Fig. 2B).
These data confirm previous reports that BDO and BDE
N2-guanines block DNA replication by pol I (18).
This polymerase incorporated one nucleotide opposite the lesion but in
contrast to the yeast pol
, failed to extend the primer further on
all four damaged substrates tested. Additionally, heterogeneity in the
mobility of the final products (26-mer in the running start assays and 21-mer in the standing start assays) suggested nucleotide misincorporation in these reactions (lanes 3-6 and
9-12).
Next, the specificity of nucleotide incorporation by pol
opposite
and downstream of these lesions was examined. To identify the
nucleotide that was incorporated by pol
opposite the adducted base,
single-nucleotide incorporation experiments were carried out using the
1 primer (Fig. 3). On a nondamaged
substrate, pol
predominantly incorporated a C opposite G, but some
T was also incorporated. In the case of the BDO and BDE
N2-guanine-containing substrates, a C residue
was the only base that was incorporated opposite the lesions. Taking
into account the lower efficiency of primer extension by pol
on the
adducted templates, the substrate to enzyme ratio in the reaction was
changed from 5:2 to 1:4. Under these conditions, no nonextended primers were left in the reactions on all five substrates tested when dCTP was
in the incubation mixture (data not shown). Again, in the presence of
the dTTP, no primer extension was observed on any of the damaged DNA
substrates, but primer extension occurred on the nondamaged
template.

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Fig. 3.
Single-nucleotide incorporation by S. cerevisiae pol on the monoepoxide-
and diolepoxide-guanine-adducted templates using the 1 primer.
Each of the templates (ND = nondamaged,
(R)-BDO = (R)-BDO
N2-guanine, (S)-BDO = (S)-BDO N2-guanine,
(R,R)-BDE = (R,R)-BDE
N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine)
was annealed to the 1 primer. The DNA substrates (5 nM)
were incubated for 20 min at 22 °C with each of the four dNTPs ( = no nucleotide added, G = dGTP, A = dATP, T = dTTP, C = dCTP) and
S. cerevisiae pol (2 nM). The
positions of the 20-nt primer and 22-nt products are indicated. *,
position of the nondamaged G or the adducted G on the template
strand.
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Although in reactions with the
1 primer no significant level of
misincorporation opposite the lesion was observed, the smearing of
bands was noted one nucleotide beyond the lesion, particularly in the
case of the (R)-BDO
N2-guanine-adducted substrate (Fig.
2A). To determine whether this smearing was attributable to
nucleotide misincorporation past the lesion site, single-nucleotide
incorporation studies were performed on (R)-BDO
N2-guanine-adducted template using a 0 primer,
which contains a C opposite the damaged G. On this template, pol
extended 93% of the 0 primer with dCTP, 23% with dTTP, 6% with dATP,
and 2% with dGTP (Fig. 4). A low level
of primer extension was also observed in the presence of dTTP on the
(S)-BDO N2-guanine-containing
template. On the (R,R)- and
(S,S)-BDE
N2-guanine-containing templates, pol
incorporated only the C residue.

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Fig. 4.
Single-nucleotide incorporation by S. cerevisiae pol on the monoepoxide-
and diolepoxide-guanine-adducted templates using the 0 primer.
Each of the templates (ND = nondamaged,
(R)-BDO = (R)-BDO
N2-guanine, (S)-BDO = (S)-BDO N2-guanine,
(R,R)-BDE = (R,R)-BDE
N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine)
was annealed to the 0 primer. The DNA substrates (5 nM)
were incubated for 20 min at 22 °C with each of the four dNTPs ( = no nucleotide added, G = dGTP, A = dATP, T = dTTP, C = dCTP) and S. cerevisiae pol (2 nM). The positions of the 20-nt
primer and 21-nt products are indicated. *, position of the nondamaged
G or the adducted G on the template strand.
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To test whether any misincorporation occurred beyond one nucleotide
downstream of the lesion site, we performed single-nucleotide incorporation experiments using a +1 primer (Fig.
5). No nucleotide misincorporation was
observed on any of the damaged substrates examined, and pol
synthesized nearly the same amount of DNA on different damaged
substrates when all four dNTPs were added to reactions.

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Fig. 5.
DNA polymerase activity of S. cerevisiae pol under conditions of
postlesion start on the monoepoxide- and diolepoxide-guanine-adducted
templates. Each of the templates (ND = nondamaged,
(R)-BDO = (R)-BDO
N2-guanine, (S)-BDO = (S)-BDO N2-guanine,
(R,R)-BDE = (R,R)-BDE
N2-guanine, (S,S)-BDE = (S,S)-BDE N2-guanine)
was annealed to the +1 primer. The DNA substrates (5 nM)
were incubated for 20 min at 22 °C in the presence of each of the
four dNTPs or all four dNTPs ( = no nucleotide added,
G = dGTP, A = dATP, T = dTTP, C = dCTP, N4 = all four
dNTPs) and S. cerevisiae pol (2 nM). The
positions of the 20-nt primer and the 44-nt full-length products are
indicated. *, position of the nondamaged G or the adducted G on the
template strand.
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To quantitate the efficiency of pol
-catalyzed synthesis past each
of the BDO- and BDE-modified N2-guanines, steady
state kinetic analyses were performed with both
1 and 0 primers. As
shown in Table I, incorporation of dCTP opposite the (S)-BDO and (S,S)-BDE
N2-guanines (
1 primer extension) was
200-300-fold less efficient than incorporation opposite the unmodified
guanine, whereas incorporation opposite the R stereoisomers
was 2000-3000-fold less efficient than incorporation opposite the
unmodified guanine. The reduced efficiency for incorporating dCTP
opposite BDO- and BDE-adducted N2-guanines is
primarily a Km effect, not a
kcat effect. Thus, there is a block to inserting
dCTP opposite these lesions, as is also demonstrated by the pause site
just prior to the adduct in Fig. 2A. The extent of the
blockage depended on the stereochemistry of the adduct, and this result
agrees with the data presented in Figs. 2A and 3.
Interestingly, there is little block to extending from the C residue
paired with the BDO- or BDE-modified N2-guanine
(kinetics of the dCTP incorporation in reactions with 0 primer), as is
also demonstrated by the lack of a pause site at the site of the adduct
(Fig. 2A). In contrast to nucleotide incorporation opposite
the lesion, no differences in efficiencies of elongation from the
resulting base pair were observed. Thus, bypass efficiencies by pol
on the BDO- or BDE-modified N2-guanines are
limited at the step of the nucleotide incorporation opposite the lesion
but not at the extension step.
To further evaluate the accuracy of pol
replication through BDO and
BDE N2-guanine adducts, kinetic analyses of
nucleotide misinsertion were carried out, and frequencies of
misincorporation were calculated as the ratio of
kcat/Km of the incorrect
nucleotide to the correct nucleotide (21). In reactions with the
1
primer, frequencies of misincorporation were below the limit of
detection under conditions used for all four damaged substrates. Thus,
pol
incorporates the correct nucleotide C quite accurately opposite N2-guanine modified with BDO or BDE. In
experiments utilizing the 0 primer, high frequencies of
misincorporation were observed in extension from C base-paired with the
(R)-BDO N2-guanine. On this
substrate, the frequencies of misincorporation were 2.0 × 10
2 for a T misincorporation and 6.2 × 10
4 for an A misincorporation. In all other cases,
nucleotide misincorporation was below the limit of detection, which was
approximately 5 × 10
4. Thus, kinetic data confirmed
the results of the single-nucleotide incorporation experiment (Fig. 4),
indicating that pol
is less accurate in extension from the base
paired with (R)-BDO N2-guanine than
with (S)-BDO N2-guanine.
Lack of Bypass of (R,R)- and (S,S)-BDE
N2-Guanine-N2-Guanine Cross-links by pol
--
Structures of the BDE
N2-guanine-N2-guanine
cross-links are shown in Fig. 6.
Cross-linked adducts are believed to contribute to butadiene-mediated
carcinogenesis (22, 23). Previously, in E. coli, both
(R,R)- and (S,S)-BDE
N2-guanine-N2-guanine
cross-links were shown to be extremely inhibitory to replicative
bypass in vivo, and E. coli DNA pol I, II, and
III were shown to be completely blocked on the templates containing these cross-links in vitro (19). To examine whether yeast
pol
can bypass these lesions, primer extension experiments were performed. As shown in Fig. 7, on the
(R,R)- as well as on the (S,S)-BDE
N2-guanine-N2-guanine
cross-link-containing substrates, DNA synthesis by pol
was
completely blocked just before the lesion, both under standing start and running start conditions.

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Fig. 7.
DNA polymerase activity of S. cerevisiae pol under standing
start and running start conditions on the diolepoxide guanine-guanine
cross-link adducted templates. Each of the templates
(ND = nondamaged, (R,R)-CL = (R,R)-BDE
N2-guanine-N2-guanine
intrastrand cross-link-containing, (S,S)-CL = (S,S)-BDE
N2-guanine-N2-guanine
intrastrand cross-link-containing) was annealed to one of two primers
( 4 or 1 primer). The DNA substrates (5 nM) were
incubated for 20 min at 22 °C in the presence of all four dNTPs and
S. cerevisiae pol (2 nM). Incubation of the
nondamaged substrate under the same conditions but without polymerase
was used as a negative control reaction. The positions of the 20-nt
primers and the 50-nt (running start reaction) and 47-nt (standing
start reaction) full-length products are indicated. *,
position of the nondamaged G or cross-linked G on the template.
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DISCUSSION |
Based on the ability of pol
to bypass a T-T dimer efficiently
and accurately, it has been suggested that its active site is flexible
enough to tolerate the distortion of the Watson-Crick geometry caused
by the T-T dimer (6, 7, 24). However, such a flexibility of the
polymerase active site should decrease its overall fidelity. Indeed,
steady state kinetics assays of nucleotide incorporation have shown
that pol
is a low fidelity enzyme. Both yeast and human pol
misincorporate nucleotides on undamaged DNA with frequencies of
approximately 10
2-10
3 (6, 24, 25).
Interestingly, the accuracy of replication by yeast as well as by the
human pol
opposite a T-T dimer does not differ from that opposite
nondamaged DNA (6, 7). The fact that both yeast (4, 26) and human (25)
pol
do not possess any intrinsic proofreading exonuclease activity
could explain in part the low fidelity of these polymerases. However, pol
has a lower fidelity than the other 3'
5'
exonuclease-deficient DNA polymerases (6, 24, 25), suggesting that its
low fidelity derives from the relaxed requirement of its active site
for correct base-pairing geometry. A flexible active site should enable
pol
to bypass DNA lesions other than the T-T dimer. In agreement with this, both yeast and human pol
also bypass a
7,8-dihydro-8-oxoguanine lesion efficiently, and they do so by
predominantly inserting a C opposite the lesion (11). In addition, both
yeast (26) and human (27) pol
preferentially insert the correct
nucleotide (C) opposite an
N2-acetylaminofluorene-guanine. However, yeast
pol
is unable to further extend DNA synthesis beyond the lesion
(26). Human pol
can incorporate relatively efficiently one more
nucleotide beyond the lesion, but only when the modified guanine is
primed with a C (27).
Here it has been shown that yeast pol
can bypass
(S)-BDO N2-guanine as well as
(S,S)-BDE N2-guanine with
200-300-fold less efficient nucleotide insertion opposite the
lesion relative to the nondamaged guanine. pol
can also bypass the
(R)-BDO N2-guanine and
(R,R)-BDE N2-guanine
adducts, but these lesions pose an approximately 10-fold greater block
to replication by pol
than their S stereoisomers. Thus,
the efficiency of translesion DNA synthesis by yeast pol
is
stereoisomer-specific. Blockage of the pol
-catalyzed replication through the BDO and BDE N2-guanines occurs at
the step of the nucleotide insertion opposite the lesion, not at the
extension step. In its ability to effectively extend synthesis past the
BDO and BDE N2-guanine adducts, yeast pol
differs from E. coli pol I, which fails to continue DNA
synthesis beyond the lesion. Single-nucleotide incorporation
experiments on BDO- and BDE
N2-guanine-containing substrates and steady
state kinetic data indicate that lesion bypass by pol
can be
error-prone at the step of postlesion replication and that the accuracy
of translesion DNA synthesis at this step can also be
stereoisomer-specific. On three out of four substrates tested, namely
on (S)-BDO, (R,R)-BDE, and (S,S)-BDE N2-guanine DNA
adducts, nucleotide insertion opposite the lesion as well as elongation
from the resulting base pair appeared to be quite accurate. On the
(R)-BDO N2-guanine-containing
substrate, pol
inserted the correct nucleotide opposite the lesion,
but it showed a tendency for nucleotide misincorporation in elongation
from the resulting base pair.
Stereoisomeric BDE
N2-guanine-N2-guanine
intrastrand cross-links were also examined in this study. However,
these lesions were a complete block to synthesis by yeast pol
, and
in this case, synthesis terminated one base prior to the first adducted
guanine. Interestingly, it has been recently demonstrated that human
pol
is capable of inserting a C opposite the first G of a
cisplatin-GG intrastrand cross-link, but incorporation of the second C
was highly inefficient, even using higher concentrations of pol
in
the reaction. When the cisplatin cross-link was primed with a CC
opposite the lesion, bypass was achieved (27).
The N2-guanine adducts of stereoisomeric
1,3-butadiene metabolites are a complete block to synthesis by E. coli DNA polymerases I, II, and III. In contrast, yeast pol
can insert nucleotides opposite these lesions and is able to
efficiently extend from the resulting base pair. The ability of yeast
pol
to bypass N2-guanine butadiene adducts
provides further support to the hypothesis (6, 7, 24) that in general,
the pol
active site tolerates geometric distortions within DNA
caused by these and other DNA-damaging agents. However, the inability
of pol
to bypass an
N2-guanine-N2-guanine
intrastrand cross-link suggests that its active site is not flexible
enough to adapt to the rather severe distortion imposed upon DNA by the
cross-link.