Repair Mechanisms of DNA Double Strand Breaks

DNA双链断裂在DNA复制过程中非常频繁地发生，不仅由外部遗传毒性剂引起，而且由正常细胞代谢产生的内部遗传毒性剂引起。为了阐明DNA双链断裂的修复机制，我们以大肠杆菌和嗜热菌为模式原核生物，以裂殖酵母和裂殖酵母为模式真核生物，以激烈火球菌为模式古细菌，研究了Holliday连接作为重组中间体和DNA复制叉被阻断时DNA双链断裂的修复机制。RuvABC蛋白参与原核生物中霍利迪连接的加工。为了深入了解RuvABC的霍利迪连接分辨率的分子机制，我们将这些蛋白质的突变分析与晶体学研究相结合。已经在体内和体外分析了大量RuvABC突变蛋白。RuvA-Holliday连接复合物的三维结构由 ...更多信息 晶体学分析和RuvA的三个结构域被指定为单重功能。阐明了嗜热菌单体RuvB蛋白的晶体结构，并根据其与最近发现的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，我们发现RuvC的两个碱性残基和一个苯丙氨酸对与Holliday连接的相互作用至关重要，并鉴定了4个参与粟酒裂殖酵母DNA双链断裂修复的新基因。我们研究了与RuvB马达蛋白同源的、从细菌到人类高度保守的Mgs（Maintenance of Genome Stability）蛋白的功能。它具有依赖DNA的ATP酶和DNA退火活性。我们证明，它是必要的，以维持基因组的稳定性，通过调节DNA复制过程中的DNA超螺旋与RecQ解旋酶和拓扑异构酶。少

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