1996 Fiscal Year Annual Research Report

自然突然変異の起源とその制御機構

Research Project

Project/Area Number	06102006
Research Institution	Fukuoka Dental College
Principal Investigator	関口睦夫福岡歯科大学, 歯学部, 教授 (00037342)
Co-Investigator(Kenkyū-buntansha)	作見邦彦九州大学, 生体防御医学研究所, 助手 (50211933)
Keywords	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
Research Abstract	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。

Research Products

(30 results)

All Other

All Publications (30 results)

[Publications] Ishibashi,T.: "Intracellular localization and function of DNA repair methyltransferase in human cells" Mutation Res.315・3. 199-212 (1994)
[Publications] 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)
[Publications] 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)
[Publications] Ishibashi,T.: "Artificial control of nuclear translocation of DNA repair methyltransferase" J.Biol.Chem.269・10. 7645-7650 (1994)
[Publications] Ito,M.: "Metastatic phenotype in hybrid cells derived from BIG melanoma." Cancer Letters.78・1-3. 33-39 (1994)
[Publications] Ito,T.: "Roles of transcription and repair in alkylation mutagenesis" Mutation Res.314・3. 273-285 (1994)
[Publications] Iyama,A.: "A unique structural feature of rabbit repair methyl-transferase as revealed by cDNA cloning" Carcinogenesis. 15・4. 627-633 (1994)
[Publications] Ohkubo,T.: "Methylation dependent functional switch mechanism newly found in the Escherichia coli Ada protein" J.Am.Chem.Soc.116. 6035-6036 (1994)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] Kawate,H.: "Mouse methyltransferase for repiar of O^6-methylguanine and O^4-methylthymine in DNA" Carcinogenesis.16・7. 1595-1602 (1995)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] Otsuka,M.: "Polymorphism in the human O^6-methylgnanine-DNA methyltransferase gene detected by PCR-SSCP analysis" Pharmacogenetics.6. 361-363 (1996)
[Publications] 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)
[Publications] Iwakuma,T.: "Organization and expression of the mouse gene for DNA repair methyltransferase" DNA Cell Biol.15. 863-872 (1996)
[Publications] Tsuzuki,T.: "Targeted disruption of the methyltransferase gene renders mice extraordinary sensitive to alkylating carcinogens" Carcinogenesis.17. 1215-1220 (1996)
[Publications] 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)
[Publications] Yamagata,Y.: "Three-dimentional structure of a DNA repair enzyme,3-methyladenine DNA glycosylase II,from Escherichia coli" Cell.86・2. 311-319 (1996)
[Publications] Igarashi,H.: "Organization and expression of the mouse MTH1 gene for preventing transversion mutation" J.Biol.Chem.272. 3766-3772 (1997)
[Publications] Iwakuma,T.: "High incidence of nitrosamine-induced tumorigenesis in mice lacking DNA repair methyltransferase" Carcinogenesis. (in press).
[Publications] 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).
[Publications] Cai,J.-P.: "Significance of the conserved amino acid sezuence for human MTH1 protein with antimutator activity" Nucleic Acids Res. (in press).

1996 Fiscal Year Annual Research Report

自然突然変異の起源とその制御機構

Principal Investigator

関口 睦夫 福岡歯科大学, 歯学部, 教授 (00037342)

Research Products

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

[Publications] 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)

[Publications] 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)

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

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

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

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

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

[Publications] 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)

[Publications] 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)

[Publications] 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)

[Publications] 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)

[Publications] 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)

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

[Publications] 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)

[Publications] 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)

[Publications] 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)

[Publications] 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)

[Publications] 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)

[Publications] 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)

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

[Publications] 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)

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

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

[Publications] 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)

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

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

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

[Publications] 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).

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

関口睦夫福岡歯科大学, 歯学部, 教授 (00037342)