RAT 7,8-DIHYDRO-8-OXOGUANINE DNA GLYCOSYLASE - SUBSTRATE-SPECIFICITY,KINETICS AND CLEAVAGE MECHANISM AT AN APURINIC SITE

Reactive oxygen species produce different lesions in DNA, Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative produc ts implicated in mutagenesis, This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isola ted and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1), Expression of the cDNA in the fgp mutY Escherichia coli doub le mutant allowed the purification of the untagged rOGG1 protein. It e xcises 8-oxoG from DNA with a strong preference for duplex DNA contain ing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K-m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' t o the lesion. However, rOGG1 acts on a substrate containing an apurini c site by a beta-delta elimination reaction and proceeds through a Sch iff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppress es its spontaneous mutator phenotype.