Fission yeast (Schizosaccharomyces pombe) cells defective in the MutY-homologous glycosylase activity have a mutator phenotype and are sensitive to hydrogen peroxide

The modified base 7,8-dihydro-8-oxo-guanine (8-oxoG) is one of the most sta ble deleterious products of oxidative DNA damage because it mispairs with a denine during DNA replication. In the fission yeast Schizosaccharomyces pom be, the MutY homolog (SpMYH) is responsible for removing misincorporated ad enines from A/8-oxoG or A/G mismatches and thus preventing G:C to T:A mutat ions. In order to study the functional role of SpMYH, an SpMYH knockout str ain was constructed. The SpMYH knockout strain, which does not express SpMY H and has no A/8-oxoG glycosylase activity, displays a 36-fold higher frequ ency of spontaneous mutations than the wild type strain. Disruption of SpMY H causes increased sensitivity to H2O2 but not to UV-irradiation. Expressio n of SpMYH in the mutant cells restores the adenine glycosylase activity, r educes the mutation frequency, and elevates the resistance to H2O2. Asp172 of SpMYH is conserved in a helix-hairpin-helix superfamily of glycosylases. The SpMYH Delta strain expressing D172N SpMYH retained the mutator phenoty pe. Moreover, when D172N mutant SpMYH was expressed in the wild-type cells, the mutation frequency observed was even higher than that of the parental strains. Thus, a mutant SpMYH that retains substrate-binding activity but i s defective in glycosylase activity exhibits a dominant negative effect. Th is is the first demonstration that a MutY homolog plays an important role i n protecting cells against oxidative DNA damage in eukaryotes.