The possibility that Escherichia coli MutT and human MTH1 (hMTH1) hydrolyze oxidized DNA precursors other than 8-hydroxy-dGTP (8-OH-dGTP) was investigated. We report here that hMTH1 hydrolyzed 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dATP (8-OHdATP), oxidized forms of dATP, but not (R)-8,5-cyclodATP, 5-hydroxy-dCTP, and 5-formyl-dUTP. The kinetic parameters indicated that 2-OH-dATP was hydrolyzed more efficiently and with higher affinity than 8-OHdGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OHdGTP. The preferential hydrolysis of 2-OH-dATP over 8-OH-dGTP was observed at all of the pH values tested (pH 7.2 to pH 8.8). In particular, a 5-fold difference in the 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. Furthermore, we found that 2-hydroxy-dADP and 8-hydroxy-dGDP competitively inhibited both the 2-OH-dATP hydrolase and 8-OH-dGTP hydrolase activities of hMTH1. The inhibitory effects of 2-hydroxy-dADP were 3-fold stronger than those of 8-hydroxy-dGDP. These results suggest that the three damaged nucleotides share the same recognition site of hMTH1 and that it is a more important sanitization enzyme than expected thus far.Endogenous oxidation of DNA and DNA precursors by reactive oxygen species appears to induce spontaneous mutations, aging, and various diseases, including cancer and neurodegeneration (1, 2). 8-OH-dGTP 1 is an oxidized form of dGTP and induces A:T to C:G transversions because it can pair with adenine as well as cytosine (3-6). It is known that the Escherichia coli MutT protein hydrolyzes 8-OH-dGTP to 8-hydroxydGMP (4). Because the mutation rate in a mutT-deficient strain increases up to 1000-fold as compared with the wild type (7), 8-OH-dGTP is considered to be a major source of spontaneous mutations caused by endogenous reactive oxygen species, and MutT appears to efficiently prevent the spontaneous occurrence of A:T to C:G transversion mutations. In human cells, the hMTH1 protein is considered to be a functional homologue of the E. coli MutT because the hMTH1 protein hydrolyzes 8-OH-dGTP in vitro and suppresses the mutator phenotype of E. coli mutT-deficient cells (8, 9).Recently, we found that 2-hydroxy-dAdo and 2-OH-dATP are produced efficiently by reactive oxygen species treatment of dAdo and dATP, respectively (10, 11). 2-OH-dATP specifically induces G:C to T:A transversion mutations and is more mutagenic than 8-OH-dGTP in vivo (5). Thus, 2-OH-dATP is thought to act as an endogenous mutagen in cells. However, the presence of a hydrolyzing activity for 2-OH-dATP has not been described. We supposed that the MutT and hMTH1 proteins may act on this mutagenic nucleotide, 2-OH-dATP. We report here that the hMTH1 protein, which is known as an 8-OHdGTPase, hydrolyzes 2-OH-dATP more efficiently than 8-OHdGTP. In addition, hMTH1 also hydrolyzed 8-OH-dATP, another oxidized form of dATP, as efficiently as 8-OH-dGTP. On the other han...

We have developed a new strategy for the evaluation of the mutagenicity of a damaged DNA precursor (deoxyribonucleoside 5-triphosphate) in Escherichia coli. 8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and 2-hydroxydeoxyadenosine triphosphate (2-OHdATP) were chosen for this study because they appear to be formed abundantly by reactive oxygen species in cells. We introduced the oxidatively damaged nucleotides into competent E. coli and selected mutants of the chromosomal lacI gene. Both damaged nucleotides induced lacI gene mutations in a dose-dependent manner, whereas unmodified dATP and dGTP did not appear to elicit the mutations. The addition of 50 nmol of 8-OHdGTP and 2-OH-dATP into an E. coli suspension induced 12-and 9-fold more substitution mutations than the spontaneous event, respectively. The 8-OH-dGTP induced A⅐T 3 C⅐G transversions, and the 2-OH-dATP elicited G⅐C 3 T⅐A transversions. These results indicate that the two oxidatively damaged nucleotides are mutagenic in vivo and suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G residues, respectively, in the E. coli DNA. This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.

The human nucleotide pool sanitization enzyme, MTH1, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dATP in addition to 8-hydroxy-dGTP. We report here that human MTH1 is highly specific for 2-hydroxy-ATP, among the cognate ribonucleoside triphosphates. The pyrophosphatase activities for 8-hydroxy-GTP, 2-hydroxy-ATP and 8-hydroxy-ATP were measured by high-performance liquid chromatography. The kinetic parameters thus obtained indicate that the catalytic efficiencies of MTH1 are in the order of 2-hydroxy-dATP > 2-hydroxy-ATP > 8-hydroxy-dGTP > 8-hydroxy-dATP >> dGTP > 8-hydroxy-GTP > 8-hydroxy-ATP. Notably, MTH1 had the highest affinity for 2-hydroxy-ATP among the known substrates. ATP is involved in energy metabolism and signal transduction, and is a precursor in RNA synthesis. We suggest that the 2-hydroxy-ATP hydrolyzing activity of MTH1 might prevent the perturbation of these ATP-related pathways by the oxidized ATP.

The mutational properties of 5-formyl-2'-deoxyuridine 5'-triphosphate (5-CHO-dUTP) and 5-hydroxy-2'-deoxycytidine 5'-triphosphate (5-OH-dCTP), the major oxidatively damaged pyrimidine nucleotides derived from dTTP and dCTP, respectively, were analyzed by an in vivo assay. 5-CHO-dUTP and 5-OH-dCTP were directly incorporated into Escherichia coli , and their mutagenicities were evaluated by the chromosomal lacI forward mutation assay. The mutation frequencies increased, depending on the dose of these damaged nucleotides, indicating that these nucleotides were incorporated into E.coli and acted as mutagens in vivo . The mutagenicities of 5-CHO-dUTP and 5-OH-dCTP were comparable to that of 8-hydroxy-2'-deoxyguanosine 5'-triphosphate, a major form of dGTP oxidative damage. 5-CHO-dUTP induced G.C to A.T, A.T to G.C and G.C to T.A mutations, and 5-OH-dCTP elicited G.C to A.T, A.T to C.G and G.C to T.A mutations.

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