The mutagenic potential of GO lesions and their relevance to cancer and aging have been well documented (11
). Despite decades of study, however, the various mechanisms that act to prevent GO-associated mutagenesis in eukaryotes have yet to be fully described. In addition to Ogg1, which excises GO lesions from GO·C mispairs, both MMR and Polη reduce the mutagenesis that results from the insertion of adenine opposite a template GO lesion during DNA synthesis (18
). Based on epistasis analysis between msh2
, it was proposed that Polη works exclusively in the context of MMR to fill the gaps generated when the MMR system removes the adenine of GO·A mispairs (10
). The data presented here demonstrate that the full relationship between MMR and Polη in limiting GO-associated mutagenesis was obscured in earlier studies because msh2
strains were used for epistasis analysis and because only total mutation rates were considered (18
). Using spectra to focus on GO-associated GC → TA mutations, we were able to clearly observe synergism between msh2
. When msh6
mutants were used instead of msh2
mutants, the synergism with rad30
was evident in total mutation rate measurements and was further exaggerated when GC → TA mutation rates were calculated. Finally, a simple comparison of the ogg1 msh6
and ogg1 msh6 rad30
spectra provided visual confirmation that msh6
is not epistatic to rad30
; if it were, no changes would have been expected upon the additional deletion of RAD30
. Our data thus clearly demonstrate that MMR and Polη can function independently to limit GO-associated mutagenesis. As illustrated in Fig. , we suggest that the MMR-independent role of Polη reflects its direct recruitment for error-free bypass of GO lesions that block the replicative DNA polymerases. Whether such recruitment involves a polymerase switch at the fork and/or occurs through the filling of gaps left behind the fork is not known.
Roles of MMR and Polη activities in preventing GO-associated mutagenesis.
While our data clearly demonstrate that Polη can act independently of the MMR machinery to reduce GO-associated mutagenesis, they do not exclude the possibility that Polη may sometimes be recruited to fill GO-containing gaps created by MMR, as originally proposed (18
). We suggest, however, that Polη would only become involved in MMR if a replicative polymerase is blocked by a lesion during the gap-filling reaction. TLS in this context is likely no different from that triggered by any other replication-blocking lesion, and so it is not unique to the MMR process. It thus requires that the PIP domain of Polη be intact (Table ) (16
) and may involve a specific interaction with the monoubiquitinated form of PCNA (39
). Aside from this specific scenario, however, it is difficult to imagine a more global involvement of Polη in MMR; the relatively low fidelity of this enzyme on undamaged DNA would lead to many nonspecific errors during MMR-associated gap filling (35
). Indeed, the only unequivocal evidence of Polη functioning in MMR is in the specialized somatic hypermutation of B cells (9
One advantage of sequencing a small target is that mutation patterns can be easily discerned. In the case of the SUP4-o allele, there are 45 positions where GO-initiated GC → TA transversions can occur, and transversions at most of these sites were detected (Fig. ). As expected based on overall GC → TA rates, an analysis of mutation rates at individual sites within the hbn1::SUP4-o spectra confirmed that most sites were synergistically affected by the removal of both Polη and Msh6. There were sites, however, where loss of Polη had a greater than average effect and removal of Msh6 had little effect. We suggest that these sites correspond to locations where the replicative DNA polymerase is more efficiently blocked by a GO lesion. In contrast, other sites that were affected only by loss of Msh6 may be locations where the replicative polymerase rarely stalls, resulting in a high incidence of GO·A mismatches that are then subject to MMR. Because of this considerable site-to-site variation, our data underscore the great caution that needs to be exercised when using a single site (e.g., a reversion assay) in mutation analyses.
Even though mutations were much more evenly distributed in the ogg1 rad30 msh6
triple mutant than in other genetic backgrounds, site-to-site differences persisted, indicating significant variability in the susceptibilities of specific guanines to oxidative damage and/or in the propensity of the replicative DNA polymerases to insert A opposite GO lesions. This observed variability is not surprising, as previous studies have shown that mutation and repair rates vary across the genome (20
) and even within small stretches of DNA (4
). Several studies, for example, have shown that GO lesions are less accessible to Ogg1 in areas of DNA that also contain AP sites, single-strand breaks, additional oxidative lesions, and other types of damage (8
). Moreover, efficient nucleotide insertion opposite GO lesions and primer extension from these insertions are also affected by nearby lesions and neighboring bases (22
). Based on previous studies and the work presented here using SUP4-o
, it is clear that multiple factors determine the relative involvements of Ogg1, MMR, and Polη in limiting GO-associated mutagenesis at a given site.
Possible differences in the frequencies or mechanisms of mutagenesis during leading- versus lagging-strand synthesis have been examined using a variety of assays, mutagens, and strain backgrounds (31
). These different studies have inferred a leading-strand bias, a lagging-strand bias, or the complete absence of a bias. A single, unifying explanation for these various findings is unlikely, as mutations result from a wide variety of initiating events and pathways. Our results with the hbn1
alleles demonstrate that, at this specific location, both MMR and Polη-dependent bypass are more efficient on the lagging strand of replication, leading to enhanced mutagenesis on the leading strand (Tables and ). We considered the possibility that the greater efficiency of Polη activity during lagging-strand synthesis at the HBN1
location could simply reflect a role for Polη during MMR and hence the strand bias of MMR. It should be noted, however, that some leading-strand mutation bias persisted in the absence of Msh6 and that this residual bias was completely eliminated upon additional removal of Polη. These results are consistent with earlier work demonstrating that MMR removes errors more efficiently during lagging- than during leading-strand synthesis (40
) and provide the first evidence that the MMR-independent Polη bypass is also more efficient on the lagging strand. Interestingly, all of the leading-strand bias in SUP4-o
mutagenesis at the AGP1
location required the presence of Polη, with the contribution of MMR being relatively minor. This suggests an additional layer of complexity, with effects of the immediate chromosomal environment extending into the mutational target.
It has been suggested that the lagging-strand bias of MMR is due to the increased accumulation either of nicks or of PCNA that accompanies discontinuous DNA synthesis (40
). Just as PCNA would be left behind when the DNA polymerase is recycled to extend the next primer during lagging-strand synthesis, PCNA would presumably be left behind to mark a gap created when a lesion blocks a replicative polymerase. Because lagging-strand DNA replication is an inherently discontinuous process, we speculate that lesion-triggered gaps are more readily formed on the lagging strand than on the leading strand. This would account for the greater efficiency of Polη during lagging-strand synthesis, as well as for the central role of PCNA in orchestrating the bypass reaction.
In addition to demonstrating replication-related strand differences in mutagenesis, the data presented here also indicate that there are more GO lesions present on the nontranscribed strand than on the transcribed strand of SUP4-o
(Tables and ). This could be explained by either more efficient removal of lesions from the transcribed strand or a greater accumulation of lesions on the nontranscribed strand. In the first scenario, GO lesions on the transcribed strand might block RNA polymerases, resulting in the activation of transcription-coupled nucleotide excision repair and a concomitant reduction in potential mutagenesis. In the second scenario, the process of transcription might lead to the formation of a DNA-RNA hybrid between the DNA template and the newly synthesized RNA, leaving the nontranscribed strand transiently single stranded and more chemically reactive (25
). It should be noted that both CG > TA mutations resulting from cytosine deamination and GO-dependent GC → TA transversions have been shown to preferentially accumulate on the nontranscribed strand of a highly transcribed gene in E. coli
ROS are a constant threat to DNA integrity, and defining the mechanisms that limit their mutagenic effects is critical to understanding the regulation of eukaryotic genome stability. As in yeast, both MMR and Polη have been shown to prevent GO-associated mutagenesis in mammalian cells (28
). While purified yeast Polη is both more efficient and more accurate than Polδ in GO lesion bypass, recent data suggest that human Polη has much lower fidelity (34
). Whether this reflects a lack of relevant accessory proteins in vitro or a less prominent role of Polη in the error-free bypass of GO lesions in mammalian cells is unclear. The exact role of MMR in mammals is also unclear, as the MutSβ complex does not bind GO mismatches at all and the MutSα complex does not bind or repair GO mismatches efficiently unless the mismatches are within repeats and are associated with slippage (26
). Consistent with these in vitro data, oxidative lesions have been linked to frameshifts and microsatellite instability in many species (13
). Interestingly, human cells lacking Polη were recently shown to have increased rates of spontaneous fragile-site instability (44
). It is possible that replication is more likely to stall at GO lesions present in fragile sites and, therefore, that Polη is required for bypassing these lesions and maintaining genome stability. Future studies that further elucidate the mechanisms behind MMR-independent Polη bypass will provide additional insight into the role of TLS in the prevention of oxidative mutations and, specifically, the role of human Polη in limiting mutagenesis and preventing human disease.