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.

UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae

“…It is well established that Pol replicates through CPDs and 8-oxoG with high efficiency and accuracy, whereas Pol is critically important in UVC mutagenesis (27,28,32,33). Cells deficient in Pol (rad30 and rad30 ogg1) or Pol (rev3 and rev3 ogg1) exhibit the same sensitivity to the lethal action of UVA as the WT cells (data not shown), whereas they are sensitive to UVC (28).…”

“…Mutation frequencies were determined from the number of Can R colonies per milliliter divided by the number of viable cells per milliliter and expressed as averages. Spontaneous mutation frequencies were determined as described by using the method of the median (26,27). Can R mutation spectra were determined as described in refs.…”

“…If 8-oxoG is largely responsible for UVA-induced mutations in yeast, the spectrum of Can R mutation should be biased in favor of GC3TA transversions (27,34). To challenge this hypothesis, sequence analysis of Can R mutations was performed in WT, ogg1, or rad30 cells exposed or not to 250 kJ⅐m Ϫ2 of UVA radiation (this UVA dose leads to 50% survival) ( Table 2).…”

Nuclear and mitochondrial genome instability induced by senna (Cassia angustifolia Vahl.) aqueous extract in Saccharomyces cerevisiae strains

“…cerevisiae strains FF18733 (MATa,, CD138 (as FF18733 but ogg1D::TRP1), BPS1031(as FF18733 but msh6D::kANMX6), and BPS1050 (as FF18733 but msh6D::kANMX6, ogg1D::TRP1) were used (Heude and Fabre, 1993;Thomas et al, 1997;Melo et al, 2004;de Pádula et al, 2004). Cells were grown at 30°C in YPD medium (1% yeast extract, 1% bacto-peptone, 2% glucose, with 2% agar for plates) or yeast nitrogen base dextrose (YNBD) medium (0.7% YNBD without amino acids, 2% glucose, with 2% agar for plates) supplemented with appropriate amino acids and bases (de Pádula et al, 2004). Supplemented YNBD medium lacking arginine but containing canavanine (60 mg/L) was used for the selective growth of canavanine-resistant (Can R ) mutants (de Pádula et al, 2004).…”

“…In S. cerevisiae, mismatch repair (MMR) and translesion synthesis cooperate with Ogg1 to prevent the mutagenic effect of 8-oxoG in nuclear DNA (Girard and Boiteux, 1997;Boiteux et al, 2002;de Pádula et al, 2004). Msh2/Msh6 proteins contribute to the prevention of GC to TA tranversions in nuclei, as the Msh6 protein can remove adenine from 8-oxoG:A pairs (Boiteux et al, 2002).…”