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 stable deleterious products of oxidative DNA damage because it mispairs with adenine during DNA replication. In the fission yeast Schizosaccharomyces pombe, the MutY homolog (SpMYH) is responsible for removing misincorporated adenines from A/8-oxoG or A/G mismatches and thus preventing G:C to T:A mutations. In order to study the functional role of SpMYH, an SpMYH knockout strain was constructed. The SpMYH knockout strain, which does not express SpMYH and has no A/8-oxoG glycosylase activity, displays a 36-fold higher frequency of spontaneous mutations than the wild type strain. Disruption of SpMYH causes increased sensitivity to H2O2 but not to UV-irradiation. Expression of SpMYH in the mutant cells restores the adenine glycosylase activity, reduces the mutation frequency, and elevates the resistance to H2O2. Asp172 of SpMYH is conserved in a helix-hairpin-helix superfamily of glycosylases. The SpMYHA strain expressing D172N SpMYH retained the mutator phenotype. 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 is defective in glycosylase activity exhibits a dominant negative effect. This is the first demonstration that a MutY homolog plays an important role in protecting cells against oxidative DNA damage in eukaryotes.
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Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore 21201, USA.
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D019943:Amino Acid Substitution; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D045647:DNA Glycosylases; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005816:Genetic Complementation Test; D005838:Genotype; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D016296:Mutagenesis; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D010641:Phenotype; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D014466:Ultraviolet Rays
D016877:Oxidants; D006861:Hydrogen Peroxide; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase