Repair Mechanisms of DNA Double Strand Breaks

DNA双链断裂的修复机制

• 批准号: 10044206
• 负责人: SHINAGAWA Hideo
• 金额: $ 7.42万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044206/
• 关键词: Double Stand breaks; DNA recombination; Hjc protein; Holliday junction; Escherichia coli; Thermophilic archaea; Thermophilic bacteria; Yeast; rhp57遺伝子; 耐熱細菌; RuvBタンパク質; hjc遺伝子; 古細菌; 相同的組換え; DNA修復; チェックポイント; RecAタンパク質; RadAタンパク質

DNA double strand breaks occur quite frequently during DNA replication not only by external genotoxic agents but also by genotoxic agents internally produced as a result of normal cellular metabolism. To elucidate the repair mechanisms of DNA double strand breaks, we employed Escherichia coli and a thermophilic bacterium as model prokaryotes, Saccharomyces cerevisiaea and Schizosaccharomyces pombe as model eukaryotes, and Pyrococcus furiosus as a model archaea.Holliday junctions are formed as recombination intermediates and during DNA replication when replication fork is blocked. RuvABC proteins are involved in processing the Holliday junctions in prokaryotes. To gain insights into molecular mechanisms of Holliday junction resolution by RuvABC, we combined mutational analysis of these proteins with ctystallographic studies. A large number of RuvABC mutant proteins have been analyzed in vivo and in vitro. The three dimensional structure of RuvA-Holliday junction complex was revealed by … More crystallographic analysis and the three domains of RuvA were assigned to differerest functions. The crystal structure of monomeric RuvB protein from thermophilic bacteria was elucidated and the hexameric model structure of RuvB was proposed based on the similarity with the crystal structures of AAA^+ ATPases recently revealed. Based on mutational studies, two basic residues and a phenylalanine of RuvC critically important for the interactions with Holliday junction were revealed.We identified 4 novel genes involved in repair of DNA double strand breaks in S.pombe. They are also involved in DNA replication and thus link DNA recombination with replication.We studied the function of Mgs (Maintenance of Genome Stability) proteins which are homologous to RuvB motor protein and highly conserved from bacteria to humans. It has DNA-dependent ATPase and DNA annealing activities. We demonstrated that it is required to maintain genome stability by regulating DNA superhelicity during DNA replication together with RecQ helicases and topoisomerases. Less

在DNA复制过程中，DNA双链断裂不仅是由外部遗传毒性物质引起的，而且是由正常细胞代谢过程中内部产生的遗传毒性物质引起的。为了阐明DNA双链断裂的修复机制，我们以大肠杆菌和一种嗜热细菌为模型原核生物，以酿酒酵母和裂糖酵母为模型真核生物，以furiococcus为模型古细菌。假日连接是在DNA复制过程中，当复制叉被阻断时形成的重组中间物。RuvABC蛋白参与处理原核生物的假日连接。为了深入了解RuvABC对Holliday连接分辨率的分子机制，我们将这些蛋白质的突变分析与囊体造影研究结合起来。大量的RuvABC突变蛋白已经在体内和体外进行了分析。进一步的晶体分析揭示了RuvA- holliday结复合物的三维结构，并将RuvA的三个结构域划分为不同的功能域。研究了嗜热细菌中RuvB蛋白单体的晶体结构，并基于其与最近发现的AAA^+ atp酶晶体结构的相似性，提出了RuvB的六聚体模型结构。基于突变研究，揭示了RuvC的两个基本残基和一个苯丙氨酸对与Holliday结的相互作用至关重要。我们发现了4个新的基因参与了S.pombe DNA双链断裂的修复。它们也参与DNA复制，因此将DNA重组与复制联系起来。我们研究了与RuvB运动蛋白同源且从细菌到人类高度保守的Mgs （Maintenance of Genome Stability）蛋白的功能。它具有DNA依赖的atp酶和DNA退火活性。我们证明了在DNA复制过程中，通过调节DNA的超螺旋度以及RecQ解旋酶和拓扑异构酶来维持基因组的稳定性。少

项目成果

Yoshikawa M.,H.Iwasaki & H.Shinagawa: "Evidence that phenylalanine 69 in Escherichia coli RuvC resolvase forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate."J.Biol.Chem.. (in press). (2001)

吉川 M.,H.岩崎

Hishida, T., et al. and Shinaeawa, H.: "Role of Walker motif A of RuvB protein in promoting branch migration of Holliday junctions"J. Biol. Chem.. 274・36. 25335-25342 (1999)

Hishida, T., et al. 和 Shinaeawa, H.：“RuvB 蛋白的 Walker 基序 A 在促进霍利迪连接体分支迁移中的作用”J. Biol. 274・36 (1999)。

Yoshikawa,M.,H.Iwasaki,K.Kinoshita & H.Shinagawa: "Two basic residues, Lys-107 and Lys-118, of RuvC resolvase are involved in critical contacts with the Holliday junction for its resolution."Genes to Cells. 5. 803-813 (2000)

吉川,M.,H.岩崎,K.Kinoshita

Yoshikawa M.,H.Iwasaki & H.Shinagawa: "Evidence that phenylalanine 69 in Escherichia coli RuvC resolvasc forms a stacking interaction during binding and destabilization of a Holliday junction DNA substrate."J.Biol.Chem.. (in press). (2001)

吉川 M.,H.岩崎

Komori, K., Shinagawa, H., et al.: "A Holliday junction resolvase from Pyrococcus furiosus : Functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea"Proc. Natl.

小森 K.、品川 H. 等人：“来自激烈火球菌的霍利迪连接体解析酶：与大肠杆菌 RuvC 的功能相似性为细菌、真核生物和古细菌中同源重组的保守机制提供了证据”Proc.