INACTIVATION OF OGG1 INCREASES THE INCIDENCE OF G-CENTER-DOT-C-]T-CENTER-DOT-A TRANSVERSIONS IN SACCHAROMYCES-CEREVISIAE - EVIDENCE FOR ENDOGENOUS OXIDATIVE DAMAGE TO DNA IN EUKARYOTIC CELLS

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase th at excises 7,8-dihydro-8-oxoguanine (8-OxoG) and ,6-diamino-4-hydroxy- 5-N-methylformamidopyrimidine To investigate the biological role of th e OGG1 gene, mutants were constructed by partial deletion of the codin g sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1 ::URA3 mutant strains. The disruption of the OGG1 gene does not compro mise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free ext racts of wild-type and ogg1 strains using a 34mer DNA fragment contain ing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a sub strate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell -free extracts of the Ogg1-deficient strain have no detectable activit y that can cleave the 8-OxoG/C duplex. The biological properties of th e ogg1 mutant have also been investigated. The results show that the o gg1 disruptant is not hypersensitive to DNA-damaging agents such as ul traviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonat e. However, the ogg1 mutant exhibits a mutator phenotype. When compare d to those of a wildtype strain, the frequencies of mutation to canava nine resistance (Can(R)) and reversion to Lys(+) are sevenfold and ten fold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displ ays a 50-fold increase in spontaneously occurring G . C-->T . A transv ersions compared to the wild-type strain. The five other base substitu tion events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cau se DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 pr otein contributes to the maintenance of genetic stability in S. cerevi siae.