Mammalian Ogg1/Mmh gene plays a major role in repair of the 8-hydroxyguanine lesion in DNA

Abstract

8-Hydroxyguanine (7,8-dihydro-8-oxoguanine, abbreviated as 8-OH-G or 8-oxoG) is the site of a frequent mutagenic DNA lesion produced by oxidative damage. MutM of E. coli and OGG1 of Saccharomyces cerevisiae are known to possess 8-OH-G glycosylase activity and apurinic (AP) site lyase activity to repair 8-OH-G lesions. Recently, cDNA clones of four isoforms (types 1a, 1b, 1c, and type 2) of human OGG1 homologs (hMMH) were isolated. However, it is unknown whether expression of endogenous hMMH proteins actually occurs in mammalian cells. We have chosen two approaches to clarify this issue. First, using hMMH type 1a-specific antibody and cells overexpressing tag-fused hMMH type 1a, we found that hMMH type 1a protein is in fact expressed in many types of human cells, showing that endogenous hMMH type 1a protein has 8-OH-G glycosylase/AP lyase activity. Furthermore, we have shown that upon antibody-mediated depletion of hMMH type 1a protein in a whole-cell extract, most of the AP lyase activity is lost, indicating that hMMH type 1a protein is a major enzyme for repair of 8-OH-G lesion in human cells. In our second approach we have generated a mouse line carrying a mutant Mmh allele by targeted gene disruption. Mmh homozygous mutant mice were found to be physically normal in appearance, but to have lost the nicking activity for substrate DNA containing 8-OH-G in liver extracts. In addition, the amount of endogenous 8-OH-G in liver DNA of the homozygous mutant mice at 8 weeks of age was 3-fold higher compared with wild-type or heterozygous mice. A further increase of 8-OH-G up to 7-fold was observed in 14-week-old animals. These results indicate that exposure of DNA to internal oxidative species constantly produces the mutagenic DNA adduct 8-OH-G in mice, and that Mmh plays an essential role in the repair of this type of oxidative DNA damage.