Katsuyoshi Fujikawa

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

The possibility that Escherichia coliMutT 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-OH-dATP), oxidized forms of dATP, but not (R)-8,5′-cyclo-dATP, 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-OH-dGTP. 8-OH-dATP was hydrolyzed as efficiently as 8-OH-dGTP. 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.

Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides. New evaluation method for mutagenesis by damaged DNA precursors in vivo

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-OH-dATP) 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 inducedlacI 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-OH-dGTP and 2-OH-dATP into anE. coli suspension induced 12- and 9-fold more substitution mutations than the spontaneous event, respectively. The 8-OH-dGTP induced A·T → C·G transversions, and the 2-OH-dATP elicited G·C → T·A 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 oxidized pyrimidine ribonucleotide, 5-hydroxy-CTP, is hydrolyzed efficiently by the Escherichia coli recombinant Orf135 protein.

The Escherichia coli orf135 gene encodes a 15.4kDa protein with homology to the MutT family of nucleotide hydrolases. The orf135 gene was cloned within a glutathione S-transferase (GST) fusion protein expression vector, which was used to overproduce the GST-Orf135 fusion protein in E. coli. The fusion protein thus obtained was purified by affinity column chromatography and gel filtration chromatography from the crude extract. The recombinant Orf135 protein was obtained by removing the GST tag from the purified fusion protein. Various oxidized nucleotides were tested as substrates for the recombinant Orf135 protein. As a result, we found a novel 5-hydroxy-CTPase activity of Orf135, but the hydrolyzing activities for the other nucleotides, including 5-hydroxy-dCTP, were very low. The activation constant (K(a)) of Mg(2+) for the 5-hydroxy-CTPase activity was 1.2 mM, and the pH optimum was 8.5. The catalytic efficiency (k(cat)/K(m)) for this activity was 630 s(-1) mM(-1) at 30 degrees C, which was 30-fold higher than that for the CTPase activity. This result indicates that 5-hydroxy-CTP is the best substrate of Orf135 among the nucleotides tested.

8‐Chloro‐dGTP, a hypochlorous acid‐modified nucleotide, is hydrolyzed by hMTH1, the human MutT homolog

The human mutT homolog, hMTH1, suppresses spontaneous mutations by degrading the endogeneous mutagen, 8‐hydroxy‐dGTP. We previously reported the broad substrate specificity of hMTH1, which also degrades the oxidatively damaged purine nucleotides, 2‐hydroxy‐dATP, 8‐hydroxy‐dATP, 2‐hydroxy‐ATP, and 8‐hydroxy‐GTP, in addition to 8‐hydroxy‐dGTP. In this paper, we describe the hMTH1 activity for 8‐chloro‐dGTP, which could be formed in inflamed tissue by the reaction of dGTP with hypochlorous acid, a product of myeloperoxidase from activated human neutrophils. The hMTH1 protein was mixed with 1–20 μM of 8‐chloro‐dGTP and 8‐hydroxy‐dGTP, and the reaction products were quantified by anion‐exchange HPLC to measure the pyrophosphatase reaction rate. The kinetic parameters revealed that 8‐chloro‐dGTP was degraded by hMTH1 with 50% efficiency as compared with that of 8‐hydroxy‐dGTP. This result suggests that 8‐chloro‐dGTP is an intrinsic substrate for hMTH1.