(Received for publication, April 11, 1995; and in revised form, June 25, 1995)
From the
We have previously analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and
frameshift fidelity of SV40-based DNA replication in a human cell
extract (Thomas, D. C., Veaute, X., Kunkel, T. A., and Fuchs, R. P.
P.(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7752-7756).
Here we use two sets of substrates to examine the probability of
replication termination and error-free and error-prone bypass of AAF
adducts. The substrates contained site-specific adducts at one of three
guanines in a NarI sequence (5`-GGCGCC-3`) placed within the lacZ reporter gene and located on the template for either
leading or lagging strand replication. The presence of the adduct at
any position strongly reduces the efficiency of a single round of
replication in a HeLa cell extract. Product analysis reveals
preferential replication of the undamaged strand and termination of
replication of the damaged strand occurring one nucleotide before
incorporation opposite either a leading or lagging strand adduct.
Products resistant to restriction endonuclease cleavage at the adducted
site were generated in amounts consistent with 16-48% lesion
bypass during replication. Most of this bypass was error-free. However,
two-nucleotide deletion errors were detected in the replication
products of DNA containing an AAF adduct in either the leading or
lagging strand, but only when present at the third guanine position.
Collectively, the data suggest that the replication apparatus in a HeLa
cell extract generates a template-primer slippage error at an AAF
adduct once for every 30-100 bypass events.
DNA replication in eukaryotic cells requires multiple proteins
that function in an asymmetric manner to coordinately synthesize the
leading and lagging strands (Kornberg and Baker, 1992). Given the
asymmetry of replication of duplex DNA, unrepaired DNA adducts may have
different potentials for blocking replication or promoting replication
infidelity depending on whether the adduct is located on the leading or
lagging strand. To examine this, we have been using the SV40
origin-dependent replication system (for review, see Stillman(1994),
and references therein) as a model for human chromosomal replication.
With this system, replication of undamaged DNA in mammalian cell
extracts is highly accurate (Roberts and Kunkel, 1988; Hauser et
al., 1988; Thomas et al., 1991). However, replication of
DNA containing randomly placed cyclobutane pyrimidine dimers (CPDs) ()is highly mutagenic (Thomas and Kunkel, 1993; Carty et
al., 1993). The assay system was adapted (Roberts et al.,
1991) to determine whether, as a result of the asymmetry of
replication, CPD-induced errors arise at different rates during leading
and lagging strand synthesis. Overall average error rates were equal
for the leading and lagging strand replication machinery, with
strand-specific differences in fidelity observed at some nucleotide
positions (Thomas et al., 1993).
For several years, we have studied the effects of an encounter between the replication fork and the major adduct of a known carcinogen, N-2-acetylaminofluorene (AAF) located at one template position. Studies in Escherichia coli have revealed that AAF, which binds primarily at the C-8 position of guanine, is a strong mutagen for -1 and -2 frameshift mutations (Fuchs et al., 1981; Koffel-Schwartz et al., 1984; Schaaper et al., 1990), particularly within repetitive sequences that permit slippage to form misaligned intermediates (Burnouf et al., 1989; Lambert et al., 1992). Mutagenesis within these sequences is strongly dependent on adduct location. Template modification at the 3` most base in a run of guanines or at the third guanine in the NarI recognition sequence 5`-GGCGCC-3` is much more mutagenic than at the other guanine residues because stable intermediates can form during replication (Lambert et al., 1992; Garcia et al., 1993; Milhéet al., 1994). More recently, we have extended these studies in human cell extracts using the SV40-based replication system. We examined the efficiency and frameshift fidelity of replication of DNA containing a site-specific AAF adduct present at one of the three guanine positions in the NarI sequence, each on the leading strand template relative to the first fork to encounter the adduct (Thomas et al., 1994). The primary effect of the adduct was to inhibit replication. When replication of the damaged template did occur, evidence was obtained for error-free lesion bypass with all three substrates, and for error-prone lesion bypass with the substrate that carries the AAF adduct at the third guanine.
When a ColE1-derived plasmid is replicated in E. coli, site-specific AAF adducts induced both -1 and -2 frameshift mutations at a 20-fold higher frequency when the adduct is located on the lagging strand as compared to the leading strand (Veaute and Fuchs, 1993). We hypothesized that the asymmetry of leading and lagging strand replication enzymology in human cells might also affect the frequency of mutations induced by AAF adducts. For this study we constructed two sets of four DNA substrates, each containing the SV40 replication origin and either no damage or a single, site-specific AAF adduct at one of the three guanines in a NarI sequence. In both sets of substrates, the adducts are located asymmetrically relative to the origin of replication, such that they will be encountered first by either the leading or lagging strand replication apparatus. After replication in a human cell extract, we performed product analyses to quantitatively compare the probabilities of replication termination, error-free bypass and sequence context-dependent, error-prone bypass.
Figure 1:
Maps of plasmids pMKBNar and pMZBNar
and the location of unique AAF adducts on the leading and lagging
strands. A and B, maps of plasmids containing
site-specific AAF adducts. Vectors contained either no modification or
unique AAF adducts in the lacZ` gene (lacZ` refers to
the modified lacZ sequence containing the NarI
sequence). The precise locations of the adducts are shown in Fig. 5(A and B). For reference, pMKBNar
contains the G3 adduct at nucleotide 419, while pMZBNar contains the G3
adduct at nucleotide 484. Denoted are the locations of restriction
sites used in bypass (BsaHI, AvaII) and uncoupling (PvuII, EcoRI) measurements. Ap is the
resistance gene for ampicillin, -lactamase. C and D, schematic showing location of site-specific AAF adducts on
the leading or lagging strands with respect to the nearest fork
emanating from the SV40 origin. This fork must replicate 520 or 585
nucleotides as the leading or lagging strand, respectively, before
encountering the G3 adduct in pMKBNar or pMZBNar. Thus, 19% (pMKBNar)
or 21% (pMZBNar) of the plasmid is replicated before the fork reaches
the G3 adduct. Only a portion of the entire plasmid is presented in
these linear drawings.
Figure 5: Measurement of strand asymmetry and termination bands among replication products. A and B, map of restriction sites and the strands they generate and the location of unique AAF adducts in the target sequence for leading and lagging strand adducts. Replication products were digested with PvuII and EcoRI to generate two fragments whose individual strands differ in length by four nucleotides. The exact length in nucleotides is noted above and below each strand, and the location of the AAF-modified guanines in the NarI site in the 124-nucleotide strand is indicated for each vector. C, PhosphorImager print of denaturing polyacrylamide gel containing resolved strands of PvuII-EcoRI fragments depicted in A and B. The length of each strand is indicated to the left. The sites of termination for each modified sample are indicated according to their position opposite the bases within the NarI sequence shown on the bottomleft. The exact length of bands representing sites of termination for each substrate can be determined by comparison to a sequence ladder (not shown).
Figure 2: Time course for replication of unmodified or AAF-containing plasmids in a HeLa cell extract. Standard replications reactions were performed containing 10 ng of unmodified (G0) or modified (G3) pNar DNA (Thomas et al., 1994) for varying incubation times as indicated. Samples were processed as described, and aliquots were left untreated or treated with MboI and were resolved on an agarose gel as described under ``Experimental Procedures.'' The positions of RF I, RF II, and the two largest MboI cleavage products (cut) are indicated. To calculate the extent of synthesis beyond one round of replication, the sum of the radioactivity of the MboI cleavage products (extrapolated from the size of the largest fragment) was divided by the total radioactivity in the lane from RF I DNA to the top of the gel, after subtracting background values for a blank lane. The results are expressed as percentage.
The results of a time course for T antigen-dependent replication of undamaged and damaged DNA are shown in Fig. 2. Radiolabeled replication products are detected after a 30-min reaction, and product yield significantly increased in incubations of 1 h or longer. No MboI cleavage products were detected in 30 min. As the incubation time is increased, substantially more MboI cleavage products were observed. In 1-h reactions with G0 DNA, these products comprise approximately 7% of the total. This increases to 29% at 2 h and 38% by 3 h. For G3 DNA, these values were about 5% at 1 h, 29% at 2 h, and 51% at 3 h. Based on these data, we chose to perform subsequent replication reactions for 1 h, because the products at this time point predominantly reflect one round of replication and the yield of replicated DNA was adequate for further analysis.
Figure 3: AAF adduct removal in a HeLa cell extract. Unmodified pMZBNar (G0) or modified pMZBNar (G1 and G3) DNA were incubated in a standard reaction mixture minus T antigen for 1 h. Samples were processed as usual, and equal aliquots were treated or not with restriction endonuclease BsaHI and resolved on an agarose gel as described under ``Experimental Procedures.'' The positions of RF I, II, and III are indicated. Also denoted is the largest BsaHI restriction fragment (uncut, 1,978 bp) resulting from cleavage at the sites indicated in Fig. 1B except the site within lacZ` (which is refractory to incision when the site is modified with AAF or is changed) and the largest cleavage product of this fragment (cut, 1,732 bp). Repair is calculated as described under ``Experimental Procedures.''
Figure 4: Estimation of bypass of AAF adducts by restriction analysis. Following replication of AAF-modified plasmids, samples were extracted and treated or not with BsaHI plus AvaII restriction enzymes, and resolved on an agarose gel as described under ``Experimental Procedures.'' To avoid overexposure, approximately equal amounts of radioactivity, based on RF I products, were loaded in each lane. The positions of RF I, II, and III and the uncut and the cut fragments resulting from BsaHI and AvaII cleavage are indicated. The uncut fragment for the lagging strand vector (1,580 bp) is the largest of six fragments generated by BsaHI and AvaII when the NarI site is modified by AAF (or no longer exists). The smaller fragments are not seen in this figure. Cleavage of the unmodified NarI site yields the one large product indicated (cut, 1,334 bp) and a smaller fragment not shown (246 bp). The uncut fragment for the leading strand vector (1,888 bp) represents the largest of five fragments generated by BsaHI and AvaII when the NarI site is modified by AAF (or no longer exists). Again, the smaller fragments are not seen in this figure. Cleavage of the unmodified NarI site yields the one large product indicated (cut, 1,403 bp) and a smaller fragment not shown (485 bp). Lesion bypass was calculated as described in the legend to Table 1.
The results are shown in Fig. 4, in the alternate lanes marked (+) for BsaHI + AvaII treatment. Digestion of the products of replication of the undamaged G0 substrates with AvaII and BsaHI yielded several bands as predicted from complete digestion and from the location of the known sites ( Fig. 1and legend to Fig. 4). The largest of these bands is shown in the autoradiogram (indicated as cut), while higher mobility bands are not shown. Bands at these same positions were observed with the G1 and G3 substrates in both orientations. However, a new band (indicated as uncut) was observed with the G1 and G3 substrates. For the lagging strand G1 and G3 substrates, the mobility of this band is as predicted by lack of incision at the NarI site containing the AAF adduct. The same is true for the leading strand vector, but here the mobility of the ``uncut'' band is slightly lower, as predicted by the different location of the adducted NarI site relative to its neighbors. The data are consistent with the interpretation that this band represents replicated DNA containing the AAF adduct. To estimate the amount of translesion replication inferred from this result, the uncut band intensities were determined after subtracting background values for minus-T antigen controls (not shown) and the G0 sample. This value was then used to calculate the percentage of BsaHI-resistant product, after correcting for the amount of adduct removed in a 1-h incubation in the extract (Fig. 3, and see legend to Table 1). Estimates ranged from 17 to 24% and were similar for all four substrates (Table 1).
Also observed in Fig. 5C are unique bands in each of the lanes containing products of replication of the modified substrates. The lengths and relative positions of these bands strongly suggest that they are the products of termination of replication of the adducted template strand, as suggested by our previous study (Thomas et al., 1994). The exact lengths of the bands in Fig. 5C were unequivocally determined by mixing an aliquot of each sample with the products of a chain termination sequencing reaction. The results (not shown) suggest that with the G1, G2, and G3 substrates in both orientations, replication terminated after incorporation opposite the template nucleotide immediately preceding the adducted guanine. The intensities of these bands were quantitated and compared to the values for the full-length strand (120 nucleotides) and the complementary strand (124 nucleotides) after adjusting for cytosine content and DNA repair (see above). For the products of replication of all six modified substrates, the sums of the pixel values for the termination bands and the 120-nucleotide product are similar to the values for the complementary 124-nucleotide fragment (Table 2). The relative values are not exactly 100%, perhaps reflecting low levels of second-round synthesis and/or some replication by the fork arriving from the other direction.
Values for the ratio of termination band intensity to the 124-nucleotide fragment intensity ranged from 69 to 95% (Table 2). We suggest that these represent the probability that replication terminates upon encountering an adduct. Correspondingly, the ratio of the 120-nucleotide fragment intensity to the 124-nucleotide fragment intensity may approximate the probability of lesion bypass, which is needed for synthesis of full-length restriction fragment. These probabilities ranged from 16 to 27%.
As noted previously, transfection of
unreplicated G1 or G2 DNAs, or the same DNAs incubated with the extract
in the absence of T antigen, yielded revertant frequencies only
slightly above the background established with undamaged G0 DNA (Thomas et al., 1994). We have noted similar results with the G1 and
G2 substrates in either orientation (Table 3). Control values
obtained for the G3 DNAs were higher than the other DNAs, ranging from
4.0 to 6.6 10
(Table 3). These higher
values are consistent with earlier studies demonstrating that the G3
adduct is mutagenic in E. coli (Burnouf et al., 1989;
Veaute and Fuchs, 1993).
Only the products of replication of the G3
substrates yielded revertant frequencies above background levels (Table 3). For the leading strand vector the frequencies were
about 6-fold above the minus-T antigen control, indicating that most of
the blue colonies reflected errors generated during replication in the
HeLa cell extract. Revertant frequency values for the lagging strand
vector were roughly 3-fold higher than with the leading strand vector
and 20-fold above background reversion frequencies. Sequence
analysis of revertants from the G3 reaction products demonstrated the
deletion of a GC dinucleotide from the NarI site.
The data showing preferential replication of the undamaged strand (Fig. 5C) led us to next examine whether multiple rounds of replication of the undamaged strand lead to an underestimate of AAF-dependent replication infidelity. To test this, we treated the G3 product DNA with MboI to digest second-round replication products. The effect on revertant frequencies was negligible with both G3-modified substrates (Table 3, values in parentheses).
The
appearance of BsaHI-resistant replication products consistent
with lesion bypass (Fig. 4) suggests that AAF adduct blockage of
replication (Fig. 5C) is not absolute. Therefore, we
next examined whether longer incubation times influenced revertant
frequencies. The effect on reversion frequencies for the substrate
containing the lagging strand adduct were negligible (values of 94,
120, and 110 10
, respectively, for
incubations of 60, 90, and 120 min). However, the reversion frequencies
for the substrate containing the leading strand adduct were increased
(values of 33, 51, and 77
10
, respectively,
for incubations of 60, 90, and 120 min). Thus, the
3-fold
difference between leading and lagging strand values for a 1-h
incubation was reduced to only a 1.4-fold difference after 2 h.
The present investigation is an extension of our earlier study (Thomas et al., 1994) of the efficiency and fidelity of replication in human cell extracts of DNA containing unique AAF adducts located at defined template positions. The two main objectives here were to be as quantitative as possible and to compare the effects of adducts on replication by leading and lagging strand replication proteins. Thus, we first established conditions to obtain primarily one round of replication in vitro (1-h incubation, Fig. 2) and then determined the level of adduct repair activity in the extract (14%, Fig. 3) under these conditions. Subsequent analyses of the replication products ( Fig. 4and Fig. 5, Tables II-IV) then permit a quantitative estimate of the probability of replication termination and error-free and error-prone translesion synthesis.
The primary effect of an AAF adduct is termination of
replication. The present study precisely maps the site of termination
as occurring after incorporation opposite the template nucleotide
immediately preceding the adduct, in all six site-specifically modified
substrates examined (Fig. 5C). We did not observe
detectable termination at any upstream template positions or after
incorporation opposite the adduct itself. Thus, any incorporation that
does occur opposite the adduct likely has one of three fates: (i)
correct extension, (ii) misalignment followed by extension (see below),
or (iii) excision by a nuclease. The termination pattern observed
during synthesis by the human replication apparatus is much simpler
than that observed with the replicative DNA polymerase III holoenzyme
of E. coli (Belguise-Valladier et al., 1994). That
multisubunit enzyme complex terminates synthesis at several upstream
locations preceding site-specifically AAF-adducted guanines. The
pattern observed in Fig. 5C is also simpler than that
observed with exonuclease-deficient DNA pol and pol
, which
terminate synthesis both before and opposite AAF adducts (Rabkin and
Strauss(1984), and references therein). Incorporation studies with
AAF-adducted substrates using eukaryotic DNA polymerases having
intrinsic 3`
5` exonuclease activities have not been reported.
The presence of BsaHI-resistant radiolabeled products (Fig. 4) suggests that AAF adducts are present in the replicated
DNA and, thus, are not absolute blocks to replication. Quantitative
analysis shows that approximately 17 to 24% of the products are BsaHI-resistant with all substrates examined. (Note that the
1% mutagenesis for two-base deletions at the NarI site
observed with the G3 vectors (Table 3) should not contribute
significantly to the population of BsaHI-resistant products).
Since these products are derived from replication of both the damaged
and undamaged strand, the efficiency of bypass per encounter with an
AAF adduct in the damaged strand is 2-fold higher than this value, i.e. 34-48%. Lesion bypass efficiencies estimated from
quantitative analysis of full-length restriction fragments versus termination bands (Fig. 5C) are also high, ranging
from 16 to 27% (Table 2). The data suggest that the human
replication apparatus in a HeLa cell extract can bypass one-sixth to
one-half of all AAF adducts encountered. This is remarkable given the
evidence for high fidelity replication with undamaged substrates (Izuta et al.(1995), and references therein). It is also remarkable
from a structural perspective. Although the structures of the major
eukaryotic cellular replicative DNA polymerases are not yet known, some
information does exist for Klenow DNA polymerase (Joyce and Steitz,
1994), human immunodeficiency virus type 1 reverse transcriptase
(Kohlstaedt et al., 1992; Jacobo-Molina et al.,
1993), T7 RNA polymerase (Sousa et al., 1993), and pol
(Pelletier et al., 1994). These studies suggest numerous
contacts between DNA polymerases and template-primers that might be
severely perturbed by the presence of an adduct as bulky as the AAF
adduct studied here. A bulky adduct might be expected to strongly
reduce polymerase binding and/or translocation, as evidenced by the
AAF-induced termination of synthesis observed with numerous DNA
polymerases (e.g. see above). Thus, frequent bypass by the
multiprotein replication complex implies a major role for replication
accessory proteins in assisting lesion bypass. Logical candidates for
modulating lesion bypass during eukaryotic chromosomal replication of
damaged DNA are proteins that influence polymerase
template-primer binding and/or translocation, such as proliferating
cell nuclear antigen, which confers high processivity to pol
(for
review see Stillman(1994), and references therein).
The data in Table 3demonstrate that replication of the G3-modified substrate is mutagenic, whereas replication of the other two modified substrates is not. This is consistent with a model wherein the replication apparatus first incorporates cytosine opposite the modified guanine (Lambert et al., 1992). The template-primer then slips such that the two terminal bases in the primer (3`-CpG-5`) hydrogen-bond with a repeated downstream complementary 5`-GpC-3` dinucleotide in the template, thus forming a misaligned intermediate (Schaaper et al., 1990). Continued synthesis from this intermediate leads to a two-base deletion error. This model predicts that mutagenesis should result from adducts in the G3 but not the G1 or G2 positions, because, given the sequence at the NarI site, only the former allows formation of a misaligned intermediate stabilized by two terminal base pairs. This prediction was realized in E. coli (Burnouf et al., 1989; Veaute and Fuchs, 1993), in our previous study of T antigen-dependent replication in a HeLa cell extract (Thomas et al., 1994) and in this study.
The revertant frequencies in Table 3are for G3 adduct-dependent replication errors among total
replication products generated in a single round of replication and can
therefore be used to estimate quantitatively the probability of
error-free versus error-prone bypass. Given the average
3-fold bias in replication of the undamaged strand (Fig. 5C), a revertant frequency of 1% for the lagging
strand G3-modified substrate (Table 3) suggests that 3% of the
products of replication of the damaged strand are mutagenic with this
substrate in a 1-h incubation. A similar approach suggests that
1%
of bypass events is mutagenic in a 1-h reaction with the leading strand
G3-modified substrate. These estimates are limited to errors that
restore the correct reading frame, which in this and our previous study
are two-base deletions.
Two asymmetries are observed during T
antigen-dependent replication of AAF adduct-modified DNAs in the
extract. These are a 3-fold difference in AAF adduct-induced
replication infidelity with the two opposite-orientation G3-modified
substrates (Table 3) and preferential replication of the
undamaged strand (Fig. 5C). The latter could reflect
uncoupling of the first replication fork to encounter the adduct, such
that replication of the undamaged strand continues while replication of
the damaged strand is completely or transiently blocked. Evidence
exists that uncoupling can occur under some circumstances in E.
coli (Koffel-Schwartz et al., 1987) ()and
during SV40 replication in vivo (Burhans et al.,
1991). Alternatively, the first fork may not replicate either strand
beyond the lesion, with preferential replication of the undamaged
strand accomplished by the fork arriving from the other direction in
the circular substrate. By this same logic, the 3-fold difference in
revertant frequencies can be explained in more than one way. If bypass
is catalyzed by the first fork to encounter the adduct, then the
difference suggests that the leading strand replication apparatus is
3-fold more accurate than the lagging strand apparatus. Alternatively,
error-prone lesion bypass replication may be accomplished only by the
fork arriving from the other direction. For example, it is formally
possible that much or all of the error-prone lesion bypass synthesis
with both G3-modified substrates is catalyzed by the lagging strand
replication apparatus. Consistent with this idea, mutagenesis with the
leading strand G3 vector increases with increasing incubation time,
while that with the lagging strand substrate remains about the same.
Given two replication forks and use of a circular substrate, several
explanations are consistent with the existing data. It is also possible
that the lesion bypass synthesis in the extract is catalyzed by
proteins other than those normally comprising a replication fork. It
may be possible to resolve these issues in the future using reactions
reconstituted from purified proteins or using much larger replicons or
substrates containing a replication termination signal.
Several observations suggest that the misaligned intermediate believed to be responsible for the AAF-dependent deletion errors might be edited by proofreading during replication. First, the evidence presented here for substantial bypass implies that incorporation opposite an AAF adduct is possible, but the bands in Fig. 5C indicate termination of incorporation one base before the adduct but not opposite the adduct itself. This may be interpreted in light of the observation that proofreading-proficient Klenow polymerase generated a termination pattern similar to that seen here, while the exonuclease-deficient polymerase terminated synthesis after incorporation opposite the adduct (Belguise-Valladier et al., 1994). This indicates that a proofreading exonuclease can remove incorporations opposite the adduct. Moreover, the presumed premutational intermediate in this study contains two terminal base pairs and an unpaired modified GpC dinucleotide in the template strand. Several studies suggest that undamaged misaligned intermediates formed during synthesis by proofreading-proficient DNA polymerases (Bebenek et al., 1990; Bebenek and Kunkel, 1990; Thomas et al., 1991; Kunkel et al., 1994) and during T antigen-dependent replication in extracts (Roberts et al., 1993) are subject to editing by exonucleases. In this study, the rate of AAF-induced deletion errors was substantially increased by increasing the dNTP concentration in the reaction (Table 4). This observation is consistent with selective stimulation of polymerization from a misaligned intermediate, allowing less opportunity to proofread the error. The fact that the dNTP-dependent increase was greater with the substrate containing the adduct in the leading strand relative to the first fork to encounter the adduct is consistent with more active proofreading by the leading strand replication apparatus. As discussed above, other explanations are also possible. The second approach to modulate deletion error rates by modulating proofreading, inhibition of exonuclease activity by adding dGMP to the reaction, yielded very small effects (Table 4). Lack of a monophosphate effect on frameshift fidelity during T antigen-dependent replication was also observed with undamaged substrates in an earlier study (Roberts et al., 1993). The reason for this is unclear, especially since addition of dGMP to a replication reaction clearly reduces base substitution fidelity with normal substrates (Roberts et al., 1991; Izuta et al., 1995) and with a dNTP analog (Minnick et al., 1995).
The estimates of termination and error-free and error-prone bypass presented here are for replication in a HeLa cell extract. It will be interesting to obtain similar estimates using extracts of normal human fibroblasts (Boyer et al., 1993) or cell lines having defects in mismatch repair or nucleotide excision repair.