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Mutsuo Sekiguchi - One of the best experts on this subject based on the ideXlab platform.
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human mth3 nudt18 protein hydrolyzes oxidized forms of guanosine and deoxyguanosine diphosphates comparison with mth1 and mth2
Journal of Biological Chemistry, 2012Co-Authors: Yasumitsu Takagi, Hiroyuki Kamiya, Yuriko Yamagata, Riyoko Ito, Daiki Setoyama, Mutsuo SekiguchiAbstract:Most of the proteins carrying the 23-residue MutT-related sequence are capable of hydrolyzing compounds with a general structure of nucleoside diphosphate linked to another moiety X and are called the Nudix hydrolases. Among the 22 human Nudix proteins (identified by the sequence signature), some remain uncharacterized as enzymes without a defined substrate. Here, we reveal that the NUDT18 protein, whose substrate was unknown, can degrade 8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2 (NUDT15), we propose that it should be named MTH3. Although these three human proteins resemble each other in their sequences, their substrate specificities differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2 can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme activity of MTH2 is considerably lower than that of MTH1. On the other hand, MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and 8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the finding that MTH1 and MTH2 show a limited activity against the ribonucleotide counterpart, 8-oxo-GTP. These three enzymes may function together to help maintain the high fidelity of DNA replication and transcription under oxidative stress.
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cleavage of oxidized guanine nucleotide and adp sugar by human nudt5 protein
Journal of Biochemistry, 2011Co-Authors: Riyoko Ito, Mutsuo Sekiguchi, Yuriko Yamagata, Daiki Setoyama, Yoshimichi Nakatsu, Hiroshi HayakawaAbstract:MutT-related proteins, including Escherichia coli MutT and the human MTH1 (NUDT1), degrade 8-oxo-7, 8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and thereby prevent mutations caused by the misincorporation of 8-oxoguanine into DNA. The human NUDT5, which has an intrinsic activity to cleave ADP sugars to AMP and sugar phosphate, possesses the ability to degrade 8-oxo-dGDP to the monophosphate. Since 8-oxo-dGDP and 8-oxo-dGTP are interconvertible by cellular enzymes, NUDT5 has the potential to prevent errors during DNA replication. The two activities associated with NUDT5 exhibit different pH dependencies; the optimum for the cleavage of ADP ribose is pH 7-9, while that for 8-oxo-dGDPase is around pH 10. The kinetic parameters for the two types of reactions indicated that ADP ribose is a better substrate for NUDT5 compared with oxidized guanine nucleotides. The 8-oxo-dGDP cleavage was competitively inhibited by ADP ribose and its reaction product, AMP, and in reverse, the cleavage of ADP ribose was inhibited by 8-oxo-dGDP. These results imply that the two types of substrates may share the same binding site for catalysis.
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potential of escherichia coli gtp cyclohydrolase ii for hydrolyzing 8 oxo dGTP a mutagenic substrate for dna synthesis
Journal of Biological Chemistry, 1998Co-Authors: Masahiko Kobayashi, Mutsuo Sekiguchi, Hiroshi Hayakawa, Yuko Oharanemoto, Masaru Kaneko, Kazuo YamamotoAbstract:MutT protein of Escherichia coli prevents the occurrence of A:T → C:G transversion by hydrolyzing an oxidized form of dGTP, 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP), which is produced by active oxygen species. In a search formutT-related genes, we found that the ribAgene, encoding GTP cyclohydrolase II, is able to reduce the increased level of mutation frequency of the mutT strain to almost the normal level, provided that the gene product is overproduced. Purified preparations of Escherichia coli GTP cyclohydrolase II protein as well as the histidine hexamer-tagged recombinant GTP cyclohydrolase II protein efficiently hydrolyze 8-oxo-dGTP and 8-oxo-GTP, producing 8-oxo-dGMP and 8-oxo-GMP, respectively. dGTP was not hydrolyzed by these preparations. GTP cyclohydrolase II catalyzes conversion of GTP to 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)-pyrimidine, which constitutes the first step for riboflavin synthesis. TheK m values for the three types of guanine nucleotides, GTP, 8-oxo-GTP, and 8-oxo-dGTP, were almost the same. In the mutT − background,ribA − cells showed higher spontaneous mutation frequencies as compared with that of ribA +cells. Thus, GTP cyclohydrolase II, the ribA gene product, has a potential to protect genetic material from the untoward effects of endogenous oxygen radicals.
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generation and elimination of 8 oxo 7 8 dihydro 2 deoxyguanosine 5 triphosphate a mutagenic substrate for dna synthesis in human cells
Biochemistry, 1995Co-Authors: Hiroshi Hayakawa, Akinobu Taketomi, Kunihiko Sakumi, Michihiko Kuwano, Mutsuo SekiguchiAbstract:8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a potent mutagenic substrate for DNA synthesis. The present study deals with generation and degradation of 8-oxo-dGTP in the nucleotide pool of human cells. (1) 8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxo-dGDP by nucleoside diphosphate kinase. (2) 8-Oxo-dGTP is rapidly degraded to 8-oxo-dGMP by cellular 8-oxo-dGTPase activity. 8-Oxo-dGMP thus produced cannot be rephosphorylated; guanylate kinase, which phosphorylates both GMP and dGMP to the corresponding nucleoside diphosphates, is totally inactive for 8-oxo-dGMP. (3) 8-Oxo-dGMP is further degraded to 8-oxo-deoxyguanosine by a nucleotidase. The enzyme was partially purified from an extract of human Jurkat cells, and the mode of action was elucidated. 8-Oxo-dGMP is the most preferred substrate of the enzyme, and other nucleoside monophosphates are cleaved at significantly lower rates: Km for 8-oxo-dGMP is 10 times lower than that for dGMP, the second best substrate for the enzyme. The enzyme appears to convert 8-oxo-dGMP, which accumulates in the cellular nucleotide pool, to a form readily excretable to the cell exterior.
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genomic structure and chromosome location of the human mutt homologue gene mth1 encoding 8 oxo dGTPase for prevention of a t to c g transversion
Genomics, 1994Co-Authors: Masato Furuichi, Yusaku Nakabeppu, Michihiro C Yoshida, Tatsurou Tajiri, Teruhisa Tsuzuki, Mutsuo SekiguchiAbstract:8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is produced by active oxygen species in the nucleotide pool of the cell and can be incorporated into cellular DNA. Human cells contain enzyme activity that hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing occurrence of mutations, caused by misincorporation. When the cDNA for human 8-oxo-dGTPase was expressed in Escherichia coli mutT- mutant cells devoid of self 8-oxo-dGTPase activity, the elevated level of spontaneous A:T to C:G mutation frequency reverted to normal. We isolated the genomic sequence encoding the enzyme and named the gene MTH1 (for mutT human homologue). This gene is composed of at least 4 exons, spans approximately 9 kb, and is located on human chromosome 7p22.
Martin Egli - One of the best experts on this subject based on the ideXlab platform.
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kinetics structure and mechanism of 8 oxo 7 8 dihydro 2 deoxyguanosine bypass by human dna polymerase η
Journal of Biological Chemistry, 2014Co-Authors: Amritraj Patra, Leslie D Nagy, Q Zhang, L Muller, F P Guengerich, Martin EgliAbstract:DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.
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structural and functional elucidation of the mechanism promoting error prone synthesis by human dna polymerase kappa opposite the 7 8 dihydro 8 oxo 2 deoxyguanosine adduct
Journal of Biological Chemistry, 2009Co-Authors: Adriana Irimia, Peter F Guengerich, Robert L Eoff, Martin EgliAbstract:Human polymerase kappa (hPol kappa) is one of four eukaryotic Y-class DNA polymerases and may be an important element in the cellular response to polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which can lead to reactive oxygenated metabolite-mediated oxidative stress. Here, we present a detailed analysis of the activity and specificity of hPol kappa bypass opposite the major oxidative adduct 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG). Unlike its archaeal homolog Dpo4, hPol kappa bypasses this lesion in an error-prone fashion by inserting mainly dATP. Analysis of transient-state kinetics shows diminished "bursts" for dATP:8-oxoG and dCTP:8-oxoG incorporation, indicative of non-productive complex formation, but dATP:8-oxoG insertion events that do occur are 2-fold more efficient than dCTP:G insertion events. Crystal structures of ternary hPol kappa complexes with adducted template-primer DNA reveal non-productive (dGTP and dATP) alignments of incoming nucleotide and 8-oxoG. Structural limitations placed upon the hPol kappa by interactions between the N-clasp and finger domains combined with stabilization of the syn-oriented template 8-oxoG through the side chain of Met-135 both appear to contribute to error-prone bypass. Mutating Leu-508 in the little finger domain of hPol kappa to lysine modulates the insertion opposite 8-oxoG toward more accurate bypass, similar to previous findings with Dpo4. Our structural and activity data provide insight into important mechanistic aspects of error-prone bypass of 8-oxoG by hPol kappa compared with accurate and efficient bypass of the lesion by Dpo4 and polymerase eta.
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efficient and high fidelity incorporation of dctp opposite 7 8 dihydro 8 oxodeoxyguanosine by sulfolobus solfataricus dna polymerase dpo4
Journal of Biological Chemistry, 2005Co-Authors: Hong Zang, Adriana Irimia, Jeongyun Choi, Karen C Angel, Martin Egli, Lioudmila V Loukachevitch, Peter F GuengerichAbstract:Abstract DNA polymerases insert dATP opposite the oxidative damage product 7,8-dihydro-8-oxodeoxyguanosine (8-oxoG) instead of dCTP, to the extent of >90% with some polymerases. Steady-state kinetics with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4) showed 90-fold higher incorporation efficiency of dCTP > dATP opposite 8-oxoG and 4-fold higher efficiency of extension beyond an 8-oxoG:C pair than an 8-oxoG:A pair. The catalytic efficiency for these events (with dCTP or C) was similar for G and 8-oxoG templates. Mass spectral analysis of extended DNA primers showed ≥95% incorporation of dCTP > dATP opposite 8-oxoG. Pre-steady-state kinetics showed faster rates of dCTP incorporation opposite 8-oxoG than G. The measured Kd,dCTP was 15-fold lower for an oligonucleotide containing 8-oxoG than with G. Extension beyond an 8-oxoG:C pair was similar to G:C and faster than for an 8-oxoG:A pair, in contrast to other polymerases. The Ea for dCTP insertion opposite 8-oxoG was lower than for opposite G. Crystal structures of Dpo4 complexes with oligonucleotides were solved with C, A, and G nucleoside triphosphates placed opposite 8-oxoG. With ddCTP, dCTP, and dATP the phosphodiester bonds were formed even in the presence of Ca2+. The 8-oxoG:C pair showed classic Watson-Crick geometry; the 8-oxoG:A pair was in the syn:anti configuration, with the A hybridized in a Hoogsteen pair with 8-oxoG. With dGTP placed opposite 8-oxoG, pairing was not to the 8-oxoG but to the 5′ C (and in classic Watson-Crick geometry), consistent with the low frequency of this frameshift event observed in the catalytic assays.
Hiroyuki Kamiya - One of the best experts on this subject based on the ideXlab platform.
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human mth3 nudt18 protein hydrolyzes oxidized forms of guanosine and deoxyguanosine diphosphates comparison with mth1 and mth2
Journal of Biological Chemistry, 2012Co-Authors: Yasumitsu Takagi, Hiroyuki Kamiya, Yuriko Yamagata, Riyoko Ito, Daiki Setoyama, Mutsuo SekiguchiAbstract:Most of the proteins carrying the 23-residue MutT-related sequence are capable of hydrolyzing compounds with a general structure of nucleoside diphosphate linked to another moiety X and are called the Nudix hydrolases. Among the 22 human Nudix proteins (identified by the sequence signature), some remain uncharacterized as enzymes without a defined substrate. Here, we reveal that the NUDT18 protein, whose substrate was unknown, can degrade 8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2 (NUDT15), we propose that it should be named MTH3. Although these three human proteins resemble each other in their sequences, their substrate specificities differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2 can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme activity of MTH2 is considerably lower than that of MTH1. On the other hand, MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and 8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the finding that MTH1 and MTH2 show a limited activity against the ribonucleotide counterpart, 8-oxo-GTP. These three enzymes may function together to help maintain the high fidelity of DNA replication and transcription under oxidative stress.
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base excision repair enzyme endonuclease iii suppresses mutagenesis caused by 8 hydroxy dGTP
DNA Repair, 2008Co-Authors: Tetsuya Suzuki, Hideyoshi Harashima, Kazuo Yamamoto, Hiroyuki KamiyaAbstract:To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) and 2-hydroxy-dATP were introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei) and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. The mutations induced by exogenous 2-hydroxy-dATP were similar in all of the strains tested. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 2-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.
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2 hydroxy 2 deoxyadenosine 5 triphosphate enhances a t c g mutations caused by 8 hydroxy 2 deoxyguanosine 5 triphosphate by suppressing its degradation upon replication in a hela extract
Biochemistry, 2007Co-Authors: Kazuya Satou, Fumio Hanaoka, Hideyoshi Harashima, Chikahide Masutani, Hiroshi Kasai, Hiroyuki KamiyaAbstract:The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.
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Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Teruya Nakamura, Masahiro Shirakawa, Yusaku Nakabeppu, Hiroyuki Kamiya, Yuki Kitaguchi, Masayuki Miyazawa, Sachiko Toma, Shinji Ikemizu, Yuriko YamagataAbstract:Human MutT homologue 1 (hMTH1) hydrolyzes a variety of oxidized purine nucleoside triphosphates, including 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP and 8-oxo-dATP, to their corresponding nucleoside monophosphates, while Esherichia coli MutT possesses prominent substrate specificity for 8-oxoguanine nucleotides. Three types of crystals were obtained corresponding to the following complexes: selenomethionine-labelled hMTH1 with 8-oxo-dGMP (SeMet hMTH1–8-oxo-dGMP), hMTH1 with 8-oxo-dGMP (hMTH1–8-oxo-dGMP) and hMTH1 with 2-oxo-dATP (hMTH1–2-oxo-dATP). Crystals of the SeMet hMTH1–8-oxo-dGMP complex belong to space group P41212, with unit-cell parameters a = b = 45.8, c = 153.6 A, and diffracted to 2.90 A. Crystals of hMTH1–8-oxo-dGMP and hMTH1–2-oxo-dATP belong to space groups P21 and P212121, with unit-cell parameters a = 34.0, b = 59.0, c = 65.9 A, β = 90.7° and a = 59.2, b = 67.3, c = 80.0 A, respectively. Their diffraction data were collected at resolutions of 1.95 and 2.22 A, respectively.
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increased a t c g mutations in the mutt strain upon 8 hydroxy dGTP treatment direct evidence for mutt involvement in the prevention of mutations by oxidized dGTP
Journal of Biochemistry, 2004Co-Authors: Hiroyuki Kamiya, Chieko Ishiguro, Hideyoshi HarashimaAbstract:The Escherichia coli MutT protein hydrolyzes 8-hydroxy-dGTP (8-OH-dGTP) in vitro, and mutT gene deficiencies cause increased spontaneous A:T-->C:G mutations. However, no direct evidence exists for enhanced mutagenicity of 8-OH-dGTP in mutT cells. In this study, 8-OH-dGTP was introduced into wild type and mutT E. coli cells, and mutations of a chromosomal gene were monitored. 8-OH-dGTP induced mutations of the rpoB gene, the degree of the mutation induction in the mutT strain being approximately 6-fold higher than that in the wild type strain. On the other hand, 2-hydroxy-dATP, which is not a substrate of the MutT protein, increased the mutation to similar degrees in the two strains. These results constitute the first evidence that the MutT protein suppresses mutation by 8-OH-dGTP in vivo.
Kazimierz S Kasprzak - One of the best experts on this subject based on the ideXlab platform.
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inhibition of 8 oxo 2 deoxyguanosine 5 triphosphate pyrophosphohydrolase 8 oxo dGTPase activity of the antimutagenic human mth1 protein by nucleoside 5 diphosphates
Free Radical Biology and Medicine, 2003Co-Authors: Karol Bialkowski, Kazimierz S KasprzakAbstract:Abstract The hMTH1 protein, a human homologue of E. coli MutT protein, is an enzyme converting 8-oxo-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP) to 8-oxo-2′-deoxyguanosine 5′-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate. It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As found in our previous investigations, 8-oxo-2′-deoxyguanosine 5′-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1. Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5′-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line). Among them, the strongest inhibitors appeared to be dGDP (Ki = 74 μM), dADP (Ki = 147 μM), and GDP (Ki = 502 μM). Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range. Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 μM and GDP at mean concentration of 30 μM, taken together, can decrease the cellular hMTH1 enzymatic activity vs. 8-oxo-dGTP (expected to remain below 500 pM) by up to 15%. The other five NDPs and dNDPs tested cannot markedly affect this activity.
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a novel assay of 8 oxo 2 deoxyguanosine 5 triphosphate pyrophosphohydrolase 8 oxo dGTPase activity in cultured cells and its use for evaluation of cadmium ii inhibition of this activity
Nucleic Acids Research, 1998Co-Authors: Karol Bialkowski, Kazimierz S KasprzakAbstract:8-Oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a product of oxidative modification of dGTP, thatcan be misincorporated into DNA, causing AT-->CG mutations. Cells are protected against 8-oxo-dGTP by 8-oxo-dGTP 5'-pyrophosphohydrolases (8-oxo-dGTP-ases) that convert it to 8-oxo-dGMP. Thus, inhibition of 8-oxo-dGTPases may lead to cancer. To elucidate the involvement of 8-oxo-dGTPases in carcinogenesis, an assay of the 8-oxo-dGTPase activity is required. This paper presents such an assay developed for Chinese hamster ovary (CHO) cells that can be applied to any biological material. It includes: (i) a convenient method for preparing 8-oxo-2'-deoxyguanosine 5'-phosphates; (ii) an HPLC/UV quantification of 8-oxo-dGTP hydrolysis products and (iii) separation of 8-oxo-dGTPase activity from interfering 8-oxo-dGTP phosphatase(s). The 8-oxo-dGTPase activity of CHO cells depends on magnesium, has a pH optimum of 8.5, Km for 8-oxo-dGTP of 9.3 microM, and is inhibited by 8-oxo-dGDP, the product of interfering 8-oxo-dGTP phosphatases. The latter must be removed from the assayed samples by ultrafiltration through 30 kDa cut-off membranes. The method was used to test the inhibition by cadmium ions of the activity of 8-oxo-dGTPase in CHO cells. The cells cultured with 0.3-3 microM cadmium(II) acetate for up to 24 h had their 8-oxo-dGTPase activity suppressed in a Cd(II) concentration-dependent manner, down to 70% of the control value.
Hiroshi Hayakawa - One of the best experts on this subject based on the ideXlab platform.
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cleavage of oxidized guanine nucleotide and adp sugar by human nudt5 protein
Journal of Biochemistry, 2011Co-Authors: Riyoko Ito, Mutsuo Sekiguchi, Yuriko Yamagata, Daiki Setoyama, Yoshimichi Nakatsu, Hiroshi HayakawaAbstract:MutT-related proteins, including Escherichia coli MutT and the human MTH1 (NUDT1), degrade 8-oxo-7, 8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to 8-oxo-dGMP and thereby prevent mutations caused by the misincorporation of 8-oxoguanine into DNA. The human NUDT5, which has an intrinsic activity to cleave ADP sugars to AMP and sugar phosphate, possesses the ability to degrade 8-oxo-dGDP to the monophosphate. Since 8-oxo-dGDP and 8-oxo-dGTP are interconvertible by cellular enzymes, NUDT5 has the potential to prevent errors during DNA replication. The two activities associated with NUDT5 exhibit different pH dependencies; the optimum for the cleavage of ADP ribose is pH 7-9, while that for 8-oxo-dGDPase is around pH 10. The kinetic parameters for the two types of reactions indicated that ADP ribose is a better substrate for NUDT5 compared with oxidized guanine nucleotides. The 8-oxo-dGDP cleavage was competitively inhibited by ADP ribose and its reaction product, AMP, and in reverse, the cleavage of ADP ribose was inhibited by 8-oxo-dGDP. These results imply that the two types of substrates may share the same binding site for catalysis.
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potential of escherichia coli gtp cyclohydrolase ii for hydrolyzing 8 oxo dGTP a mutagenic substrate for dna synthesis
Journal of Biological Chemistry, 1998Co-Authors: Masahiko Kobayashi, Mutsuo Sekiguchi, Hiroshi Hayakawa, Yuko Oharanemoto, Masaru Kaneko, Kazuo YamamotoAbstract:MutT protein of Escherichia coli prevents the occurrence of A:T → C:G transversion by hydrolyzing an oxidized form of dGTP, 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP), which is produced by active oxygen species. In a search formutT-related genes, we found that the ribAgene, encoding GTP cyclohydrolase II, is able to reduce the increased level of mutation frequency of the mutT strain to almost the normal level, provided that the gene product is overproduced. Purified preparations of Escherichia coli GTP cyclohydrolase II protein as well as the histidine hexamer-tagged recombinant GTP cyclohydrolase II protein efficiently hydrolyze 8-oxo-dGTP and 8-oxo-GTP, producing 8-oxo-dGMP and 8-oxo-GMP, respectively. dGTP was not hydrolyzed by these preparations. GTP cyclohydrolase II catalyzes conversion of GTP to 2,5-diamino-6-hydroxy-4-(5-phosphoribosylamino)-pyrimidine, which constitutes the first step for riboflavin synthesis. TheK m values for the three types of guanine nucleotides, GTP, 8-oxo-GTP, and 8-oxo-dGTP, were almost the same. In the mutT − background,ribA − cells showed higher spontaneous mutation frequencies as compared with that of ribA +cells. Thus, GTP cyclohydrolase II, the ribA gene product, has a potential to protect genetic material from the untoward effects of endogenous oxygen radicals.
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generation and elimination of 8 oxo 7 8 dihydro 2 deoxyguanosine 5 triphosphate a mutagenic substrate for dna synthesis in human cells
Biochemistry, 1995Co-Authors: Hiroshi Hayakawa, Akinobu Taketomi, Kunihiko Sakumi, Michihiko Kuwano, Mutsuo SekiguchiAbstract:8-Oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) is a potent mutagenic substrate for DNA synthesis. The present study deals with generation and degradation of 8-oxo-dGTP in the nucleotide pool of human cells. (1) 8-Oxo-dGTP can be generated not only by direct oxidation of dGTP but also by phosphorylation of 8-oxo-dGDP by nucleoside diphosphate kinase. (2) 8-Oxo-dGTP is rapidly degraded to 8-oxo-dGMP by cellular 8-oxo-dGTPase activity. 8-Oxo-dGMP thus produced cannot be rephosphorylated; guanylate kinase, which phosphorylates both GMP and dGMP to the corresponding nucleoside diphosphates, is totally inactive for 8-oxo-dGMP. (3) 8-Oxo-dGMP is further degraded to 8-oxo-deoxyguanosine by a nucleotidase. The enzyme was partially purified from an extract of human Jurkat cells, and the mode of action was elucidated. 8-Oxo-dGMP is the most preferred substrate of the enzyme, and other nucleoside monophosphates are cleaved at significantly lower rates: Km for 8-oxo-dGMP is 10 times lower than that for dGMP, the second best substrate for the enzyme. The enzyme appears to convert 8-oxo-dGMP, which accumulates in the cellular nucleotide pool, to a form readily excretable to the cell exterior.