SPECIFIC RECOGNITION OF A G AND A/7,8-DIHYDRO-8-OXOGUANINE (8-OXOG) MISMATCHES BY ESCHERICHIA-COLI MUTY - REMOVAL OF THE C-TERMINAL DOMAIN PREFERENTIALLY AFFECTS A/8-OXOG RECOGNITION/

Escherichia coli MutY is a 39 kDa adenine DNA glycosylase and 3' apuri nic/apyrimidinic (AP) lyase that is active on DNA substrates containin g A/G, A/C, or A/8-oxoG mismatches. 8-oxoG (7,8-dihydro-8-oxoguanine o r GO) is a major stable product of oxidative damage, and A/GO mismatch es may be particularly important biological substrates for MutY. Prote olytic digestion of MutY using thermolysin was found to produce two re latively stable fragments of 25 and 12 kDa. The 25 kDa fragment begins at the N terminus of MutY and spans the region homologous with E. col i endonuclease III, a DNA glycosylase/AP lyase that repairs oxidativel y damaged pyrimidines. The 12 kDa fragment, which consists of much of the rest of MutY, had no detectable activity. The purified 25 kDa frag ment (M25) had nearly wild-type binding and cleavage activities with A /G-mismatched substrates. Binding to A/GO-mismatched DNA, however, was dramatically reduced in M25 compared to that in intact protein. Boroh ydride-dependent enzyme-DNA cross-linking, which is a hallmark of the reaction of several DNA glycosylases that possess concomitant AP lyase activity, was also substantially reduced when M25 was allowed to reac t with A/GO-mismatched DNA. The significant differences in M25 recogni tion and reactivity with A/G and A/GO mismatches suggest that the C-te rminal region of MutY, a region with no homologous counterpart in E. c oli endonuclease III, plays an important role in the repair of mismatc hed DNA arising from oxidation damage.