The Oxidized Forms of dATP Are Substrates for the Human MutT Homologue, the hMTH1 Protein

Abstract

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...

Mutagenicity of oxidized DNA precursors in living cells: Roles of nucleotide pool sanitization and DNA repair enzymes, and translesion synthesis DNA polymerases

“…Meanwhile, the frequencies of the mutations caused by 2-OH-dATP were similar in the mutT and wt strains, in agreement with the fact that MutT does not act on 2-OH-dATP in vitro [34]. The Orf135 protein catalyzes the hydrolysis of 2-OH-dATP, and that of 8-OH-dGTP somewhat less efficiently, [35].…”

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

“…The MTH1 protein catalyzes the hydrolysis of 2-OH-dATP in addition to 8-OH-dGTP [43]. Since single-base deletions were induced by 2-OH-dATP in in vitro replication reactions using a HeLa extract [45], the accumulation of 2-OH-dATP in the spleen cells of the knock-out mice may partly explain the occurrence of this type of mutation.…”

NUDT5 hydrolyzes oxidized deoxyribonucleoside diphosphates with broad substrate specificity

“…shown to catalyze the hydrolysis of oxidized DNA precursors [7,[16][17][18][19][20][21][22][23]. Among these enzymes, the human NUDT5 protein is unique, since the hydrolysis of…”

“…The expression of the NUDT5 protein in mutT-deficient E. coli mutant cells decreases the spontaneous mutation frequency to the normal level, suggesting its importance in nucleotide pool sanitization [19]. Since the human MTH1, yeast PCD1, and E. coli Orf135 and Orf17 proteins act on multiple substrates containing different damaged nucleobases [17,18,21,23], NUDT5 might also recognize other oxidized deoxyribonucleoside diphosphates besides 8-OH-dGDP.…”