OPPOSITE BASE-DEPENDENT REACTIONS OF A HUMAN BASE EXCISION-REPAIR ENZYME ON DNA CONTAINING 7,8-DIHYDRO-8-OXOGUANINE AND ABASIC SITES

The guanine modifications 7,8-dihydro-8-oxoguanine (8-axoG) is a poten t premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), b y molecular cloning and functional analysis;Expression of the human cD NA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels , The hOGH1 enzyme was localized to the nucleus in cells transfected b y constructs of hOGH1 fused to green fluorescent protein, Enzyme purif ication yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA, The enzymatic activities of hOGH1 was analysed o n DNA containing single residues of 8-oxoG or abasic sites opposite ea ch of the four normal bases in DNA, Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimin ation. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without am associated s trand cleavage reaction. Assays with abasic site DIVA showed that stra nd cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue, It thu s appears that strand incisions are made only if repair completion res ults in correct base insertion, whereas excision from mispairs preserv es strand continuity and hence allows for error-free correction by a p ostreplicational repair mechanism.