Biomedical subjects

Kazuya Satou

Publications and source records attributed to Kazuya Satou.

5 recordsLinked to original sources

PubMed2005

Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein.

The Escherichia coli Orf135 protein, a MutT-type enzyme, hydrolyzes mutagenic 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP, in addition to dCTP and 5-methyl-dCTP, and its deficiency causes increases in both the spontaneous and H(2)O(2)-induced mutation frequencies. To identify the amino acid residues that interact with these nucleotides, the Glu-33, Arg-72, Arg-77, and Asp-118 residues of Orf135, which are candidates for residues interacting with the base, were substituted, and the enzymatic activities of these mutant proteins were examined. The mutant proteins with a substitution at the 33rd, 72nd, and 118th amino acid residues displayed activities affected to various degrees for each substrate, suggesting the involvement of these residues in substrate binding. On the other hand, the mutant protein with a substitution at the 77th Arg residue had activitiy similar to that of the wild-type protein, excluding the possibility that this Arg side chain is involved in base recognition. In addition, the expression of some Orf135 mutants in orf135(-) E. coli reduced the level of formation of rpoB mutants elicited by H(2)O(2). These results reveal the residues involved in the substrate binding of the E. coli Orf135 protein.

Amino Acid Substitution↗

PubMed2005

Mutagenesis induced by oxidized DNA precursors: roles of Y family DNA polymerases in Escherichia coli.

To reveal the roles of Y family DNA polymerases in the mutagenesis induced by oxidatively damaged DNA precursors, 2-hydroxy-dATP (2-OH-dATP) and 8-hydroxy-dGTP (8-OH-dGTP) were introduced into Escherichia coli strains deficient in the Y family polymerases, DNA polymerase IV (pol IV, encoded by the dinB gene) and DNA polymerase V (pol V, encoded by the umuDC locus). The mutation induced by 2-OH-dATP, but not that induced by 8-OH-dGTP, occurred less frequently in the dinB- strain than in the wild-type (wt) strain, suggesting the involvement of pol IV in the mutagenesis by 2-OH-dATP. Expression of pol IV from plasmid enhanced the mutagenesis by 2-OH-dATP in the dinB- strain. This enhancement depends on the polymerase activity since the expression of a mutant pol IV lacking the polymerase activity did not increase the mutations induced by 2-OH-dATP. In contrast, both 2-OH-dATP and 8-OH-dGTP caused mutations more efficiently in the umuDC- strain than in the wt strain, suggesting that the umuDC gene products suppressed the mutagenesis by these oxidized DNA precursors. The DNA polymerase activity was not required for the suppressive effects because expression of the umuDC gene products lacking the polymerase activity also suppressed the mutagenesis. These results suggest that the E. coli pol IV was involved in mutagenesis by 2-OH-dATP and that the umuDC gene products play suppressive role(s) in the mutagenesis by damaged nucleotides.

Adenosine Triphosphate↗

PubMed2004

Mutagenesis by damaged deoxyribonucleotides and its prevention by MutT-type hydrolyzing enzymes.

Oxidized deoxyribonucleotides, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP) and 8-hydroxydeoxyguanosine 5'-triphosphate (8-OH-dGTP), were introduced into Escherichia coli strains deficient in DNA polymerase IV (a Y-family DNA polymerase encoded in the dinB gene), and the MutT and Orfl35 proteins to examine their in vivo roles in mutagenesis elicited by 2-OH-dATP and 8-OH-dGTP. 2-OH-dATP elicited mutations less efficiently in the dinB- strain than in the wild type strain, suggesting involvement of DNA polymerase IV in 2-OH-dATP-induced mutations. 8-OH-dGTP and 2-OH-dATP elicited mutations more efficiently in mutT- and orfl35- strains, respectively, than those in their isogenic mutT+ and orfl35+ strains. These results indicate that these proteins play important roles in mutagenesis induced by 2-OH-dATP and 8-OH-dGTP in vivo.

Adenosine Triphosphate↗

PubMed2003

Mutagenic effects of 2-hydroxy-dATP on replication in a HeLa extract: induction of substitution and deletion mutations.

The mutagenicity of an oxidized form of dATP, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP induced mutations in a dose-dependent manner and elicited substitution and deletion mutations. Of the substitutions, a G.C-->A.T transition including a tandem (CC-->TT) mutation was mainly observed. This result agrees with our previous observation that mammalian DNA polymerase alpha misincorporates the oxidized nucleotide opposite C, but is in contrast to the finding that 2-OH-dATP elicits G.C-->T.A transversions in Escherichia coli. This type of mutation was also elicited, but to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture, and probably in living cells. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.

Adenosine Triphosphate↗

PubMed2003

Mutations induced by 2-hydroxy-dATP during in vitro replication with a HeLa extract.

The mutagenicity of oxidized dATP, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP), was examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. 2-OH-dATP mainly elicited a G x C to A x T transition, and a G x C to T x A transversion to a lesser extent. Interestingly, the mutagenicity of 2-OH-dATP was enhanced in the presence of 2-hydroxydeoxyadenosine 5'-diphosphate, an inhibitor of the MTH1 protein, suggesting that this protein functions in the hydrolysis of 2-OH-dATP in the replication reaction mixture. These results indicate that 2-OH-dATP is mutagenic and that its mutagenicity is suppressed by the MTH1 protein in mammalian cells.

Adenosine Triphosphate↗