Repair Mechanisms of DNA Double Strand Breaks

在DNA复制过程中，DNA双链断裂非常频繁，不仅是由外部遗传毒性物质造成的，也是由正常细胞代谢产生的内部遗传毒性物质造成的。为了阐明DNA双链断裂的修复机制，我们以大肠杆菌和一株嗜热菌为模式原核生物，以酿酒酵母和裂殖酵母为模式真核生物，以热链球菌为模式考古菌。RuvABC蛋白参与原核生物中Holliday连接的处理。为了深入了解RuvABC解析Holliday连接的分子机制，我们将这些蛋白质的突变分析与结晶学研究相结合。大量的RuvABC突变蛋白已经在体内和体外进行了分析。用…揭示了Ruva-Holliday连接复合体的三维结构更多的结晶学分析和Ruva的三个结构域被分配给不同的功能。阐明了嗜热菌单体RuvB蛋白的晶体结构，并根据与最近发现的AAA、ATPase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。我们在S.pombe中发现了4个参与DNA双链断裂修复的新基因。它们还参与DNA复制，从而将DNA重组与复制联系起来。我们研究了MGS(维持基因组稳定性)蛋白的功能，MGS蛋白与RuvB马达蛋白同源，从细菌到人类都高度保守。它具有DNA依赖的ATPase和DNA退火酶活性。我们证明了在DNA复制过程中通过与RecQ解旋酶和拓扑异构酶一起调节DNA的超螺旋来维持基因组的稳定性是必需的。较少

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