組換え機構の分子的基礎

• 研究課題/領域番号: 04262102
• 研究種目: 重点領域研究
• 配分区分: 補助金
• 研究機関: 名古屋大学
• 研究代表者: 堀田 康雄 名古屋大学, 理学部, 教授 (30190218)
• 研究分担者: 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) ; 柴田 武彦 理化学研究所, バイオデザイン研究グループ, 主任研究員 (70087550)
• 研究期間 (年度): 1993
• 研究課題ステータス: 完了 (1993年度)
• 配分額: 137,100千円 (直接経費: 137,100千円); 1993年度: 70,500千円 (直接経費: 70,500千円); 1992年度: 66,600千円 (直接経費: 66,600千円)
• キーワード: 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 / 遺伝子組換え / ホットスポット / RAD51 / RecA

研究概要

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。

研究成果

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• [文献書誌] 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)
• [文献書誌] 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)
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• [文献書誌] 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)
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• [文献書誌] Shimokawa,T.: "Isolstion of multiple activities from mouse FM3A cells which promote homologous pairing of DNA molecules." Biochem.Biophys.Acta.1174. 54-62 (1993)
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• [文献書誌] 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)
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• [文献書誌] Ikeda,M.: "Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis" J.Biol.Chem.267. 6291-6296 (1992)
• [文献書誌] Shiroishi,T.: "Hotspots of meiotic recombination in the mouse major histocompatibility complex." Genetica. 88. 187-196 (1993)
• [文献書誌] Matsutani,S.: "Distribution of the Shigella sonnei insertion elements in Enterobacteriaceae." Gene. 127. 111-115 (1993)
• [文献書誌] Mochizuki,K.: "Characterization of a plant SINE,p-SINE1,in rice genomes." Jpn.J.Genetics. 67. 155-166 (1992)
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• [文献書誌] Sekine,Y.: "DNA sequences required for translational frameshifting in production of the transposase encoded by IS1." Mol.Gen.Genet.235. 325-332 (1992)
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• [文献書誌] Kobayashi,T.: "Isolation and characterization of yeast gene which is homologous with meiosis-specific cDNA from a plant." Molec.Gen.genet.(1992)
• [文献書誌] Shinohara,A.: "Rad51 Protein Involved in Repair and Recombi nation in S.cerevisiae Is aRecA-like Protein." Cell. 69. 457-470 (1992)
• [文献書誌] Leem S H.: "The MRE4 gene encodes a novel protein kinase homologue required for meiotic recombination in Saccharomyces cerevisiae." Nucleic Acids Research. 20. 449-457 (1992)
• [文献書誌] 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)
• [文献書誌] Sekine,Y.: "DNA sequences requied for translational frameshifting in produciton of the trans-posase encoded by IS1." Mol.Gen.Genet.235. 325-332 (1992)
• [文献書誌] Mishina,Y.: "Identification of two types of homologous DNA pairing activity in mouse cells." Biochem.Biophys.Res.Commun.185. 987-992 (1992)
• [文献書誌] Aratani,Y.: "End extension repair of introduced targeting vectors mediated by homologous recomgination in mammalion cells." Nucleic Acid Res.20. 4795-4801 (1992)
• [文献書誌] Kobayashi,I.: "Mechanisms for gene conversion and homologous recombination." Advances in Biophysics. 28. 81-133 (1992)
• [文献書誌] Takahashi,N.: "Nonconservative recombination in Escherichia coli." Proc.Natl.Acad.Sci.89. 5912-5916 (1992)
• [文献書誌] Yamamoto,K.: "Gene conversion in the Escherichia coli RecF pathway." Mol.Gen.Genet.234. 1-13 (1992)
• [文献書誌] Ikeda,M.: "Epitope mapping of anti-recA protein IgGs by region specified polymerase chain reaction mutagenesis." J.Biol.Chem.267. 6291-6296 (1992)
• [文献書誌] Arai,N.: "Phenol-treatment and a homologous pairing-assay." J.Biol.Chem.267. 3679-3684 (1992)
• [文献書誌] Mishina,Y.: "Identification of two types of homologous DNA pairing sctivity in mouse cells." Biochem.Biophys.Res.Commum.185. 987-992 (1992)
• [文献書誌] Ogawa,T.: "Functional Structure of the RecA Protein Found Chimera Analysis." J.Mol.Biol.226. 651-660 (1992)
• [文献書誌] Ogawa,T.: "Similarity of the Yeast Rad51 Filament to the Bacterial RecA Filament." Science. (1993)
• [文献書誌] Koide,T.: "The amplified long genomic sequence(ALGS)located in the centromeric regions of mouse chromosomes." Genomics. 13. 1186-1191 (1992)
• [文献書誌] Shiroishi,T.: "Hotspots of homologous recombination in the mouse major histocompatibility complex." Genetica. (1993)