The post-replication repair RAD18 and RAD6 genes are involved in the prevention of spontaneous mutations caused by 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae

Abstract

7,8-dihydro-8-oxoguanine (8-oxoG) is an abundant and mutagenic lesion produced in DNA exposed to free radicals and reactive oxygen species. In Saccharomyces cerevisiae, the OGG1 gene encodes the 8-oxoG DNA N-glycosylase/AP lyase (Ogg1), which is the functional homologue of the bacterial Fpg. Ogg1-deficient strains are spontaneous mutators that accumulate GC to TA transversions due to unrepaired 8-oxoG in DNA. In yeast, DNA mismatch repair (MMR) and translesion synthesis (TLS) by DNA polymerase eta also play a role in the prevention of the mutagenic effect of 8-oxoG. In the present study, we show the RAD18 and RAD6 genes that are required to initiate post-replication repair (PRR) are also involved in the prevention of mutations by 8-oxoG. Consistently, a synergistic increase in spontaneous CanR and Lys+ mutation rates is observed in the absence of Rad6 or Rad18 proteins in ogg1 mutant strains. Spectra of CaR mutations in ogg1 rad18 and ogg1 rad6 double mutants show a strong bias in the favor of GC to TA transversions, which are 137- and 189-fold higher than in the wild-type, respectively. The results also show that Poleta (RAD30 gene product) plays a critical role on the prevention of mutations at 8-oxoG, whereas Polzeta (REV3 gene product) does not. Our current model suggests that the Rad6-Rad18 complex targets Poleta at DNA gaps that result from the MMR-mediated excision of adenine mispaired with 8-oxoG, allowing error-free dCMP incorporation opposite to this lesion.

Figure 1

Complementation of the ogg1 rad18 double mutant by OGG1 or RAD18. Plasmids pYCP11 and pYSB210 contain the RAD18 and OGG1 genes placed under the control of constitutively expressed promoters. The mutation rates are determined as described in Table 2.

Figure 2

Role of Rad18 and Rad6 in the prevention of spontaneous mutations by 8-oxoG in S.cerevisiae. Endogenous oxidative stress generates 8-oxoG in DNA yielding (G⁰/C). 8-OxoG mispaired with cytosine (G⁰/C) is removed by the Ogg1 glycosylase in the course of a BER process restoring G/C (WT). If Ogg1 is absent or not available, Polδ replicates past 8-oxoG by inserting an adenine across from the lesion. The resulting 8-oxoG/A (G⁰/A) is recognized and a DNA fragment containing adenine in G⁰/A is excised by MMR. In the absence of MMR, cells accumulate GC to TA transversions (MUTANT). In MMR-proficient cells, the removal of adenine opposite 8-oxoG generates single-stranded DNA gaps that contain the lesions in the template strand nearby the 3′-OH end for DNA repair synthesis. Such DNA structures could be recognized by PCNA (red buoy). Due to its affinity for single-stranded DNA, Rad18 (black rectangle) binds the gap, interacts with PCNA and recruits Rad6 (violet oval). The Rad18–Rad6 complex mono-ubiquinates PCNA (small yellow buoy), allowing the recruitment of Polη (green cube). Then, Polη undergoes error-free TLS across from 8-oxoG, restoring G⁰/C, i.e. the substrate of Ogg1. In the absence of Rad18 or Rad6, unmodified PCNA preferentially recruits Polδ at excision gaps and promotes the mutagenic incorporation of adenine. This model does not exclude minor pathway(s), with recruitment of Polη at unmodified PCNA or that of another DNA polymerase (Polx) at modified PCNA yielding MUTANT and WT.