Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway

Mitochondrial DNA (mtDNA) is located close to the respiratory chain, a major source of reactive oxygen species (ROS). This proximity makes mtDNA more vulnerable than nuclear DNA to damage by ROS. Therefore, the efficient repair of oxidative lesions in mtDNA is essential for maintaining the stability of the mitochondrial genome. A series of genetic and biochemical studies has indicated that eukaryotic cells, including the model organism Saccharomyces cerevisiae, use several alternative strategies to prevent mutagenesis induced by endogenous oxidative damage to nuclear DNA. However, apart from base excision repair (BER), no other pathways involved in the repair of oxidative damage in mtDNA have been identified. In this study, we have examined mitochondrial mutagenesis in S. cerevisiae cells which lack the activity of the Ogg1 glycosylase, an enzyme playing a crucial role in the removal of 8-oxoG, the most abundant oxidative lesion of DNA. We show that the overall frequency of the mitochondrial oligomycin-resistant (Olir) mutants is increased in the ogg1 strain by about one order of magnitude compared to that of the wild-type strain. Noteworthy, in the mitochondrial oli1 gene, G:C to T:A transversions are generated approximately 50-fold more frequently in the ogg1 mutant relative to the wild-type strain. We also demonstrate that the increased frequency of Olir mutants in the ogg1 strain is markedly reduced by the presence of plasmids encoding Msh1p, a homologue of the bacterial mismatch protein MutS, which specifically functions in mitochondria. This suppression of the mitochondrial mutator phenotype of the ogg1 strain seems to be specific, since overexpression of the mutant allele msh1-R813W failed to exert this effect. Finally, we also show that the increased frequency of Olir mutants arising in an msh1/MSH1 heterozygote grown in glucose-containing medium is further enhanced if the cells are cultivated in glycerol-containing medium, i.e. under conditions when the respiratory chain is fully active. Taken together, these results strongly suggest that MSH1-dependent repair represents a significant back-up to mtBER in the repair of oxidative damage in mtDNA.