組換え機構の分子的基礎

1993 Fiscal Year Annual Research Report

• Project/Area Number: 04262102
• Research Institution: Nagoya University
• Principal Investigator: 堀田 康雄 名古屋大学, 理学部, 教授 (30190218)
• Co-Investigator(Kenkyū-buntansha): 杉野 明雄 大阪大学, 微生物研究所, 教授 (90231737) ; 城石 俊彦 国立遺伝学研究所, 遺伝実験生物保存研究センター, 助教授 (90171058) ; 小林 一三 東京大学, 医科学研究所, 助教授 (30126057) ; 大坪 栄一 東京大学, 分子細胞生物学研究所, 教授 (10158800) ; 小川 英行 大阪大学, 理学部, 教授 (70028207)
• Keywords: 組換え遺伝子 / 組換え蛋白 / ホリデー構造 / 核骨格 / 相同組換え / 非相同組換え / 組換えホットスポット / 減数分裂

Research Abstract

一連の組換え遺伝子、MRE11,RAD50,RAD51,RAD52,XRS2などの機能解析が進み、これらが複合体を作って2重鎖切断・相同性認識・鎖交換反応を行うことが酵母を主体として示された。欠損株では減数分裂での組換えは起こらないが、体細胞分裂ではDNA修複を欠くにもかかわらず極めて高い組換えを起こすことも明らかに成った。このうちのあるものは酵母からヒトや高等植物に至るまで類似蛋白が存在していることが明らかになった。MRE1やRAD50にはRNA結合領域があり、減数分裂細胞内にsnRNAが存在する事実と併せてRNAがDNA・蛋白と協調して複合体として組換えに作用する可能性を示した。
酵母ではN源を除くと、細胞内cAMPレベルの低下があり、幾つもの遺伝子抑制と発現のカスケードに従って減数分裂の誘導が起こることが知られていたが、isp6遺伝子が単離され、この産物のアミノ酸配列がproteinase βなどセリンプロテアーゼと類似性を示すことが明らかにされ、N_2飢餓とcAMPレベル低下の間に蛋白やRNA分解が起こり、これに携わるisp6がisp5,isp7などと共に減数分裂誘導に不可欠な働きをすることを示した。組換えのホットスポットは遺伝子座、性、発生に関連して調節を受けることがマウスで示され、組換え発生(部位)の制御が初めて手についた。相同染色体の対合はDNAとDNA結合蛋白だけでなく、核の変形や動きによる調節も受けることが核骨格を形成する中間フィラメント蛋白とその遺伝子の解析から明らかになり、DNAポリメラーゼと組換え、ホリデー構造の解離やブランチマイグレーションとruvA,B,CやResolvaseの働き、相同組換えと非相同組換えの構造的関連などについて研究が進展し、組換えの分子レベル解析を主とした国際誌に十数編の論文が発表された。

Research Products

• [Publications] Tabata,S.: "Evidence of meiosis-specific Regulation of Gene Expression." Plant Science. 89. 31-42 (1993)
• [Publications] Kobayashi,T.: "Isolation and characterization of yeast gene which is homo-logous with meiosis-specific cDNA from a plant." Molec.Gen.Genet. 237. 225-232 (1993)
• [Publications] Sato,S.: "Changes in Intracellular cAMP Level and Activities of Adenylcyclase and Phosphodiesterase during Meiosis of Lily Microsporocytes." Cell Struct.and Funct.17. 335-339 (1992)
• [Publications] Olga Bezzubova: "A chicken RAD51 homologue is expressed at high levels in lymphoid and reproductive organs" Nucleic Acids Research. 21. 1577-1580 (1993)
• [Publications] Shinohara,A.: "Cloning of Human,Mouse and Fission Yeast Recombination Genes Homologous to RAD51 and recA." Nature Genetics. 4. 239-243 (1993)
• [Publications] Ajimura,M.: "Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae" Genetics. 133. 51-66 (1993)
• [Publications] Shimizu,K.: "Suppression of Bacteriophage T7 ssb Mutation with Host ssb" J.Mol.Biol.234. 926-931 (1993)
• [Publications] Ogawa,T.: "Similarity of the Yeast Rad51 Filament to the Bacterial RecA Filament." Science. 259. 1896-1899 (1993)
• [Publications] 小川英行: "酵母に観る最新の真核生物像VI.組換え機構 減数分裂期組換え-関与する遺伝子の機能とその普遍性-" 蛋白質 核酸 酵素、「臨時増刊号」. 3(印刷中). (1994)
• [Publications] 小川智子: "真核生物の組換え蛋白質Rad51とRad52の機能と構造" 実験医学「トッピクス」. 12(印刷中). (1994)
• [Publications] Kurumizaka,K.: "A chimera RecA protein exhibits altered double-stranded DNA-binding." J.Biol.Chem.268(in press.). (1994)
• [Publications] Ogawa,T.: "RecA-like Recombination Proteins in Eukaryotes:Functions and Structures of RAD51 Genes." Cold Spring Harbor Symp.Quant.Biol.58(in press.). (1994)
• [Publications] Shinohara,A.: "Cloning of Human,Mouse and Fission Yeast Recombination Genes Homologous to RAD51 and recA." Nature Genetics. 4. 239-243 (1993)
• [Publications] Sekine,Y.: "Translational control in production of the proteins including the transposase encoded by insertion sequence IS3." J.Mol.Biol.234(in press). (1994)
• [Publications] Hirano,H.: "Retroposition of a plant SINE at a single locus in rice evolution." J.Mol.Evol.(in press). (1994)
• [Publications] Ohtsubo,E.: "A simple method to classify rice strains with AA genome and to infer their relationships by identification of transposable elements at various loci." Gamma Field Symposia. 32(in press). (1994)
• [Publications] Mochizuki,K.: "Classification and relationships of rice strains with AA genome by identification of transposable elements at nine loci." Jpn.J.Genet.68. 205-217 (1993)
• [Publications] Maekawa,T.: "DNA-binding domains in Tn3 transposase." Mol.Gen.Genet.236. 267-274 (1993)
• [Publications] Matsutani,S.: "Distribution of the Shigella sonnei insertion elements in Enterobacteriaceae." Gene. 127. 111-115 (1993)
• [Publications] Mochizuki,K.: "Characterization of a plant SINE,p-SINE1,in rice genomes." Jpn.J.Genetics. 67. 155-166 (1992)
• [Publications] Sekine,Y.: "Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS1." Mol.Gen.Genet.235. 317-324 (1992)
• [Publications] Sekine,Y.: "DNA sequences required for translational frameshifting in production of the transposase encoded by IS1." Mol.Gen.Genet.235. 325-332 (1992)
• [Publications] Suzuki,S.: "Effect of SV40 and MMTV promoters on intermolecular homologous recombination in rat cells." Biochem.Biophys.Res.Commun.197. 1194-1202 (1993)
• [Publications] Shimokawa,T.: "Isolstion of multiple activities from mouse FM3A cells which promote homologous pairing of DNA molecules." Biochem.Biophys.Acta.1174. 54-62 (1993)
• [Publications] Fujioka,K.: "Targeted recombination with single-stranded DNA vectors in mammalian cells." Nucleic Acids Res.21. 407-412 (1993)
• [Publications] Kusano,K.: "DNA double-strand break repair:Genetic determinants of flanking crossing-over." Proc.Natl.Acad.Sci.USA. 91(in press). (1994)
• [Publications] 小林一三: "遺伝子間相同相互作用の機構、意味、起源" 蛋白質 核酸 酵素. (刊行中). (1994)
• [Publications] Kusano,K.: "Involvement of recE exonuclaase and recT annealing in double-strand break repair by homologous recombination." Gene. (in press). (1993)
• [Publications] Takahashi,N.K.: "Genetic Analysis of Double-strand Break Repair in Escherichia coil." J.Bacteriol.175. 5176-5185 (1993)
• [Publications] Takahashi,N.K.: "Nonconservative recombination in Escherichia coil." Proc.Natl.Acad.Sci.USA. 89. 5912-5916 (1992)
• [Publications] Aratani,Y.: "End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells." Nucleic Acids Res.20. 4759-4801 (1992)
• [Publications] Mishina,Y.: "Identification of two types of homologous DNA pairing activities in mouse cells." Biochem.Biophys.Res.Commum.185. 987-992 (1992)
• [Publications] Yamamoto,Y.: "Gene conversion in the Escherichia coil RecF pathway:a successive half crossing-over model." Mol.Gen.Genet.234. 1-13 (1992)
• [Publications] Kurumizaka,H.: "A chimeric recA protein exhibita altered double-stranded DNA-binding." J.Biol.Chem.269. in press (1994)
• [Publications] Morishima,N.: "A sequence-specific endonuclease,Endo.SceI,can efficiently induce gene conversion in yeast mitochondria lacking a major exonuclease." Curr.Genet.23. 537-541 (1993)
• [Publications] Mishina,Y.: "Identification of two types of homologous DNA pairing activity in mouse cells." Biochem.Biophys.Res.Commun.185. 987-992 (1992)
• [Publications] Nakagawa,K.: "An endonuclease with multiple cutting sites,Endo.SceI,initiates genetic recombination at its cutting site in yeast mitochondria" EMBO J.11. 2707-2715 (1992)
• [Publications] Arai,N.: "A multi-component protein of a fission yeast which promotes joint molecule formation from homologous DNAs." J.Biol.Chem.267. 3514-3522 (1992)
• [Publications] Ikeda,M.: "Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis" J.Biol.Chem.267. 6291-6296 (1992)
• [Publications] Shiroishi,T.: "Hotspots of meiotic recombination in the mouse major histocompatibility complex." Genetica. 88. 187-196 (1993)
• [Publications] Matsutani,S.: "Distribution of the Shigella sonnei insertion elements in Enterobacteriaceae." Gene. 127. 111-115 (1993)
• [Publications] Mochizuki,K.: "Characterization of a plant SINE,p-SINE1,in rice genomes." Jpn.J.Genetics. 67. 155-166 (1992)
• [Publications] Sekine,Y.: "Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS1." Mol.Gen.Genet.235. 317-324 (1992)
• [Publications] Sekine,Y.: "DNA sequences required for translational frameshifting in production of the transposase encoded by IS1." Mol.Gen.Genet.235. 325-332 (1992)
• [Publications] Suzuki,S.: "Effect of SV40 and MMTV promoters on intermolecular homologous recombination in rat cells." Biochem.Biophys.Res.Commun.197. 1194-1202 (1993)
• [Publications] Shimokawa,T.: "Isolstion of multiple activities from mouse FM3A cells which promote homologous pairing of DNA molecules." Biochem.Biophys.Acta.1174. 54-62 (1993)
• [Publications] Yoshino,M.: "No dosage effect of recombinational hotspots in the mouse MHC." Immunogenetics. (in press). (1993)
• [Publications] 城石俊彦: "マウスMHC領域内組換えと高頻度可突然変異視" 実験医学. 11No.4. 54-60 (1993)
• [Publications] 城石俊彦: "遺伝的組換え研究におけるマウス実験系" 実験医学. 11No.12. 116-121 (1993)
• [Publications] 城石俊彦: "部位特異的なマウス相同染色体間組換え" 実験医学. 11No.20. 147-152 (1993)
• [Publications] Shimizu,K.: "Purification and characterization of a new DNA polymerase from Saccharomyces cerevisiae:A possible homolog of mammalian DNA polymerase β." J.Biol.Chem.268. 27148-27153 (1993)
• [Publications] Morrison,A.: "Progress in Nucleic Acid Research and Molecular Biology DNA polymerase II,the epsilon polymerase of Saccharomyces cerevisial." Academic Press, 37 (1993)
• [Publications] 小川英行: "新生化学実験講座 第2巻" 日本生化学会編:東京化学同人, 23 (1993)