The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein

The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investig ated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8 -hydroxy-dATP (8-OH-dATP), oxidized forms of dATP, but not (R)-8,5'-cyclod- dATP, 5-hydroxy-dCTP, and 8-formyl-dUTP. The kinetic parameters indicated t hat 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. The prefe rential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the p H values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in t he hydrolysis efficiencies for 2-OH-dATP over 8-OH-dGTP was found at pH 7.2 . However, E. coli MutT had no hydrolysis activity for either 2-OH-dATP or 8-OH-dATP. Thus, E. coli MutT is an imperfect counterpart for hMTH1. Furthe rmore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibi ted both the 8-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH 1. The inhibitory effects of 2-hydroxy-dADP were S-fold stronger than those of 8-hydroxy-dGDP, These results suggest that the three damaged nucleotide s share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.