1996 年度実績報告書

自然突然変異の起源とその制御機構

研究課題

研究課題/領域番号	06102006
研究機関	福岡歯科大学
研究代表者	関口睦夫福岡歯科大学, 歯学部, 教授 (00037342)
研究分担者	作見邦彦九州大学, 生体防御医学研究所, 助手 (50211933)
キーワード	自然突然変異 / 活性酸素 / 遺伝子破壊マウス / アルキル化剤 / 細胞死 / 発がん / DNA修復 / 酸化
研究概要	生物は自然突然変異率を一定の低い値に保つためのきわめて精緻な機構を持っている。哺乳動物細胞におけるその機構の実体を分子レベルで明らかにし、遺伝子破壊マウスなどを作製してその機構の生物学的意義を明確にするのが本研究の目的である。自然突然変異の原因の1つとして、生体内の代謝過程で生じる活性酸素によるDNAおよびその前駆体ヌクレオチドの酸化が注目されており、その過程に関与するMTH1遺伝子をとりあげた。MTH1のホモローグは細菌からヒトまで存在しており、大腸菌の欠損株(mutT^-)は高い頻度で自然突然変異を生じることが知られている。そこでMTH1のcDNAおよび遺伝子をヒトとマウスから分離し、その構造とタンパク質産物の活性を明らかにした。このタンパクはdGTPの酸化によって生じる8-oxo-dGTPを8-oxo-dGMPに分解する活性をもっており、それによって誤塩基対合によって突然変異が生じるのを防いでいる。遺伝子ターゲティングによってMTH1^<-1->となった細胞株を樹立したが、その株の自然突然変異率は正常株より高く、さらにMTH1^<-1->のマウスでは自然状態での飼育中に各種の臓器に癌が生じるのが観察された。それとは別にアルキル化塩基の修復に関与するMGMT遺伝子についても欠損細胞及び欠損マウスをつくって解析した。MGMT^<-1->の細胞株は正常細胞に比べて明らかにアルキル化剤に対する感受性が高く、高頻度で突然変異を生じた。MGMT^<-1->マウスは比較的低濃度のアルキル化剤の投与によって肝と肺に腫瘍が生じることが認められた。MGMT^<-1->にミスマッチ修復遺伝子欠損を加えることによって、アルキル化剤の致死作用に対しては抵抗性となるが、発癌はよく起こるという現象が見つかり、細胞死と突然変異・発癌の関連について重要な示唆が与えられた。

研究成果
(30件)

すべてその他

すべて文献書誌 (30件)

[文献書誌] Ishibashi,T.: "Intracellular localization and function of DNA repair methyltransferase in human cells" Mutation Res.315・3. 199-212 (1994)
[文献書誌] Furuichi,M.: "Genomic structure and chromosome location of the human must T homologue gene MTH1 encoding 8-oxo-dGTPase for prevention of A : T to C : G transversion." Genomics.24・3. 485-490 (1994)
[文献書誌] Ihara,K.: "Requirement of the Pro-Cys-His-Arg sequence for O^6-methyl-guanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening" Molec.Gen.Genet.243・4. 379-389 (1994)
[文献書誌] Ishibashi,T.: "Artificial control of nuclear translocation of DNA repair methyltransferase" J.Biol.Chem.269・10. 7645-7650 (1994)
[文献書誌] Ito,M.: "Metastatic phenotype in hybrid cells derived from BIG melanoma." Cancer Letters.78・1-3. 33-39 (1994)
[文献書誌] Ito,T.: "Roles of transcription and repair in alkylation mutagenesis" Mutation Res.314・3. 273-285 (1994)
[文献書誌] Iyama,A.: "A unique structural feature of rabbit repair methyl-transferase as revealed by cDNA cloning" Carcinogenesis. 15・4. 627-633 (1994)
[文献書誌] Ohkubo,T.: "Methylation dependent functional switch mechanism newly found in the Escherichia coli Ada protein" J.Am.Chem.Soc.116. 6035-6036 (1994)
[文献書誌] Belguise-Valladier: "Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme. Comparison with other polymerase" J.Mol.Biol.236・1. 151-164 (1994)
[文献書誌] Cai,J.P.: "cDNA and genomic sequences for rat 8-oxo-dGTPase that prevents occurrence of spontaneous mutations due to oxidation of guanine nucleotide" Carcinogenesis.16・10. 2343-2350 (1995)
[文献書誌] Hayakawa,H.: "Generation and elimination of 8-oxo-7,8-dihydro-2'-deoxy-guanosine 5'-triphosphate,a mutagenic substrate for DNA synthesis,in human cells" Biochemistry.34・1. 89-95 (1995)
[文献書誌] Kakuma,T.: "Mouse MTH1 protein with 8-oxo-dGTPase activity that prevents transversion mutation. cDNA cloning and tissue distribution" J.Biol.Chem.270・43. 25942-25948 (1995)
[文献書誌] Kang,D.: "Intracellular localization of 8-oxo-dGTPase in human cells,with special reference to the role of the enzyme in mitochondria" J.Biol.Chem.270・24. 14659-14665 (1995)
[文献書誌] Kawate,H.: "Mouse methyltransferase for repiar of O^6-methylguanine and O^4-methylthymine in DNA" Carcinogenesis.16・7. 1595-1602 (1995)
[文献書誌] Oda,S.: "Stabilization of cyclin E and Cdk2 mRNAs at the Gl-S transtion in rat-lA cells emerged from the Go state" Oncogene.10・7. 1343-1351 (1995)
[文献書誌] Sakashita,H.: "Sequence-specific DNA recognition of the Escherichia coli Ada protein associated with the methylation-dependent functional switch for transcriptional regulation" J.Biochem.118・6. 1184-1191 (1995)
[文献書誌] Tajiri,T.: "Functional cooperation of MutT,MutM and MutY proteins in preventing mutations caused by spont-aneous oxidation of guanine nucelotide in Escherichia coli" Mutation Res.336・3. 257-267 (1995)
[文献書誌] Tanaka,K.: "Cyclic AMP-mediated synthesis of the tissue inhibitors of metalloproteinases supresses the invasive potential of the human fibrosarcoma cell line,HT1080" Cancer Res.55・13. 2927-2935 (1995)
[文献書誌] Wu,C.: "Polymorphisms and probable lack of mutation in a human mutT homolog,hMTH1,in hereditary nonpoliposis colorectal cancer." Biochem.Biophys.Res.Comm.214・3. 1239-1245 (1995)
[文献書誌] Bridges,B.A.: "Effect of mutY and mutM lfpg-1 mutations on starvation-associated mutation in erichia coli : implications for the role of 7,8-dihydro-8-oxoguanine" Molec.Gen.Genet.251・3. 352-357 (1996)
[文献書誌] Otsuka,M.: "Polymorphism in the human O^6-methylgnanine-DNA methyltransferase gene detected by PCR-SSCP analysis" Pharmacogenetics.6. 361-363 (1996)
[文献書誌] Taketomi,A.: "Requirement of two conserved cysteine residues of Ada protein of Escherichia coli for transactivation of ada promoter" Mol.Gen.Genet.250・5. 523-532 (1996)
[文献書誌] Iwakuma,T.: "Organization and expression of the mouse gene for DNA repair methyltransferase" DNA Cell Biol.15. 863-872 (1996)
[文献書誌] Tsuzuki,T.: "Targeted disruption of the methyltransferase gene renders mice extraordinary sensitive to alkylating carcinogens" Carcinogenesis.17. 1215-1220 (1996)
[文献書誌] Tsuzuki,T.: "Targeted disruption of the Rad51 gene leads to lethality in embryonic mice" Proc.Natl.Acad.Sci,U.S.A.93・13. 6236-6240 (1996)
[文献書誌] Yamagata,Y.: "Three-dimentional structure of a DNA repair enzyme,3-methyladenine DNA glycosylase II,from Escherichia coli" Cell.86・2. 311-319 (1996)
[文献書誌] Igarashi,H.: "Organization and expression of the mouse MTH1 gene for preventing transversion mutation" J.Biol.Chem.272. 3766-3772 (1997)
[文献書誌] Iwakuma,T.: "High incidence of nitrosamine-induced tumorigenesis in mice lacking DNA repair methyltransferase" Carcinogenesis. (in press).
[文献書誌] Tominaga,Y.: "Alkylation-induced apoptosis of embryonio stem cells in which the gene for DNA repair methyltransferase had been disrupted by gene targeting" Carcinogenesis. (in press).
[文献書誌] Cai,J.-P.: "Significance of the conserved amino acid sezuence for human MTH1 protein with antimutator activity" Nucleic Acids Res. (in press).

1996 年度 実績報告書

自然突然変異の起源とその制御機構

研究代表者

関口 睦夫 福岡歯科大学, 歯学部, 教授 (00037342)

研究成果

[文献書誌] Ishibashi,T.: "Intracellular localization and function of DNA repair methyltransferase in human cells" Mutation Res.315・3. 199-212 (1994)

[文献書誌] Furuichi,M.: "Genomic structure and chromosome location of the human must T homologue gene MTH1 encoding 8-oxo-dGTPase for prevention of A : T to C : G transversion." Genomics.24・3. 485-490 (1994)

[文献書誌] Ihara,K.: "Requirement of the Pro-Cys-His-Arg sequence for O^6-methyl-guanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening" Molec.Gen.Genet.243・4. 379-389 (1994)

[文献書誌] Ishibashi,T.: "Artificial control of nuclear translocation of DNA repair methyltransferase" J.Biol.Chem.269・10. 7645-7650 (1994)

[文献書誌] Ito,M.: "Metastatic phenotype in hybrid cells derived from BIG melanoma." Cancer Letters.78・1-3. 33-39 (1994)

[文献書誌] Ito,T.: "Roles of transcription and repair in alkylation mutagenesis" Mutation Res.314・3. 273-285 (1994)

[文献書誌] Iyama,A.: "A unique structural feature of rabbit repair methyl-transferase as revealed by cDNA cloning" Carcinogenesis. 15・4. 627-633 (1994)

[文献書誌] Ohkubo,T.: "Methylation dependent functional switch mechanism newly found in the Escherichia coli Ada protein" J.Am.Chem.Soc.116. 6035-6036 (1994)

[文献書誌] Belguise-Valladier: "Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme. Comparison with other polymerase" J.Mol.Biol.236・1. 151-164 (1994)

[文献書誌] Cai,J.P.: "cDNA and genomic sequences for rat 8-oxo-dGTPase that prevents occurrence of spontaneous mutations due to oxidation of guanine nucleotide" Carcinogenesis.16・10. 2343-2350 (1995)

[文献書誌] Hayakawa,H.: "Generation and elimination of 8-oxo-7,8-dihydro-2'-deoxy-guanosine 5'-triphosphate,a mutagenic substrate for DNA synthesis,in human cells" Biochemistry.34・1. 89-95 (1995)

[文献書誌] Kakuma,T.: "Mouse MTH1 protein with 8-oxo-dGTPase activity that prevents transversion mutation. cDNA cloning and tissue distribution" J.Biol.Chem.270・43. 25942-25948 (1995)

[文献書誌] Kang,D.: "Intracellular localization of 8-oxo-dGTPase in human cells,with special reference to the role of the enzyme in mitochondria" J.Biol.Chem.270・24. 14659-14665 (1995)

[文献書誌] Kawate,H.: "Mouse methyltransferase for repiar of O^6-methylguanine and O^4-methylthymine in DNA" Carcinogenesis.16・7. 1595-1602 (1995)

[文献書誌] Oda,S.: "Stabilization of cyclin E and Cdk2 mRNAs at the Gl-S transtion in rat-lA cells emerged from the Go state" Oncogene.10・7. 1343-1351 (1995)

[文献書誌] Sakashita,H.: "Sequence-specific DNA recognition of the Escherichia coli Ada protein associated with the methylation-dependent functional switch for transcriptional regulation" J.Biochem.118・6. 1184-1191 (1995)

[文献書誌] Tajiri,T.: "Functional cooperation of MutT,MutM and MutY proteins in preventing mutations caused by spont-aneous oxidation of guanine nucelotide in Escherichia coli" Mutation Res.336・3. 257-267 (1995)

[文献書誌] Tanaka,K.: "Cyclic AMP-mediated synthesis of the tissue inhibitors of metalloproteinases supresses the invasive potential of the human fibrosarcoma cell line,HT1080" Cancer Res.55・13. 2927-2935 (1995)

[文献書誌] Wu,C.: "Polymorphisms and probable lack of mutation in a human mutT homolog,hMTH1,in hereditary nonpoliposis colorectal cancer." Biochem.Biophys.Res.Comm.214・3. 1239-1245 (1995)

[文献書誌] Bridges,B.A.: "Effect of mutY and mutM lfpg-1 mutations on starvation-associated mutation in erichia coli : implications for the role of 7,8-dihydro-8-oxoguanine" Molec.Gen.Genet.251・3. 352-357 (1996)

[文献書誌] Otsuka,M.: "Polymorphism in the human O^6-methylgnanine-DNA methyltransferase gene detected by PCR-SSCP analysis" Pharmacogenetics.6. 361-363 (1996)

[文献書誌] Taketomi,A.: "Requirement of two conserved cysteine residues of Ada protein of Escherichia coli for transactivation of ada promoter" Mol.Gen.Genet.250・5. 523-532 (1996)

[文献書誌] Iwakuma,T.: "Organization and expression of the mouse gene for DNA repair methyltransferase" DNA Cell Biol.15. 863-872 (1996)

[文献書誌] Tsuzuki,T.: "Targeted disruption of the methyltransferase gene renders mice extraordinary sensitive to alkylating carcinogens" Carcinogenesis.17. 1215-1220 (1996)

[文献書誌] Tsuzuki,T.: "Targeted disruption of the Rad51 gene leads to lethality in embryonic mice" Proc.Natl.Acad.Sci,U.S.A.93・13. 6236-6240 (1996)

[文献書誌] Yamagata,Y.: "Three-dimentional structure of a DNA repair enzyme,3-methyladenine DNA glycosylase II,from Escherichia coli" Cell.86・2. 311-319 (1996)

[文献書誌] Igarashi,H.: "Organization and expression of the mouse MTH1 gene for preventing transversion mutation" J.Biol.Chem.272. 3766-3772 (1997)

[文献書誌] Iwakuma,T.: "High incidence of nitrosamine-induced tumorigenesis in mice lacking DNA repair methyltransferase" Carcinogenesis. (in press).

[文献書誌] Tominaga,Y.: "Alkylation-induced apoptosis of embryonio stem cells in which the gene for DNA repair methyltransferase had been disrupted by gene targeting" Carcinogenesis. (in press).

[文献書誌] Cai,J.-P.: "Significance of the conserved amino acid sezuence for human MTH1 protein with antimutator activity" Nucleic Acids Res. (in press).

1996 年度実績報告書

関口睦夫福岡歯科大学, 歯学部, 教授 (00037342)