Replication Fork Repair

项目摘要 复制叉修复对于细胞存活和遗传物质的稳定性至关重要。在人类中， 修复与癌症倾向、神经学、免疫学、发育缺陷和 过早衰老 这项研究的长期目标是对复制修复的机制有一个更完整的了解 差距。本项目的目标是阐明复制缺口修复途径的要素，包括 特定的复制体成分，使用遗传和生物化学实验。因为所有的细胞都会修复DNA， 基本上相似的方式通过进化相关的途径，这些研究使用模式生物， 大肠杆菌，应该揭示适用于修复人类细胞中的DNA的机制。使用 叠氮胸苷（AZT）在本研究中作为一种化合物诱导复制间隙有可能揭示毒性 以及与其作为抗HIV剂的治疗用途相关的耐受机制。此外，由于DNA 细菌病原体的损伤反应在毒素产生、抗生素抗性和持久性中起作用 这项工作可以为抗生素治疗提供新的靶点。 该建议的第一个目的是确定RadA（一种通用的细菌蛋白质）相关的机制。 与RecA结合，协助同源重组。在体外，RadA刺激RecA介导的重组反应 通过加速链交换中间体的分支迁移。这种刺激的机制将是 通过生物化学和遗传实验推断，以确定RadA和RecA如何相互作用， 重组 本研究的第二个目的是试图阐明复制体夹装载复合物和修复蛋白是如何 相互作用以促进修复。YoaA蛋白是XPD/FANCJ解旋酶家族的一员，在大肠杆菌中突变。 人类疾病综合征，将通过遗传和生物化学方法进行研究，以确定它是否是一种 DNA解旋酶，这种活性是如何专门化的，以及它如何促进复制缺口修复。的相互作用 将从遗传学和生物化学的角度对复制体进行研究。在体内，DnaX-变体钳- 将确定装载复合物及其调节，以及它们在促进遗传稳定性中的作用。一 将开发一种分离与AZT终止缺口相关的蛋白质的新方法， 测定复制和修复蛋白到复制阻断位点的募集。 这项研究将通过提供有关DNA修复的新信息， 如何检测和修复复制间隙。

PROJECT SUMMARY Replication fork repair is essential for cell survival and stability of genetic material. In humans, inefficiency of repair is associated with cancer proneness, neurological, immunological, developmental defects and premature aging. The long-term goal of this study is a more complete mechanistic understanding of the repair of replication gaps. The objective of this project will be to elucidate elements of replication gap repair pathways, including specific replisome components, using genetic and biochemical experiments. Because all cells repair DNA in fundamentally similar ways by evolutionarily related pathways, these studies using the model organism, Escherichia coli, should reveal mechanisms applicable to repair of DNA in human cells. The use of azidothymidine (AZT) in this study as a compound to induce replication gaps has the potential to reveal toxicity and tolerance mechanisms relevant to its therapeutical use as an anti-HIV agent. In addition, because the DNA damage response in bacterial pathogens plays a role in toxin production, antibiotic resistance and persistence of infection, this work could provide new targets for antibiotic therapy. The first aim of this proposal is to identify the mechanism by which RadA, a universal bacterial protein related to RecA, assists homologous recombination. In vitro, RadA stimulates RecA-mediated recombination reactions by accelerating branch migration of strand exchange intermediates. The mechanism of this stimulation will be deduced by biochemical and genetic experiments, to determine how RadA and RecA interact to promote recombination. The second aim of this study seeks to clarify how replisome clamp-loader complexes and repair proteins interact to facilitate repair. The YoaA protein, a member of the XPD/FANCJ family of helicases mutated in human disease syndromes, will be studied by genetic and biochemical methods to ascertain whether it is a DNA helicase, how this activity is specialized and how it promotes replication gap repair. Interactions with the replisome will be examined genetically and biochemically. The abundance, in vivo, of DnaX-variant clamp- loader complexes and their regulation will be determined, as well as their role in promoting genetic stability. A new method for isolation of proteins associated with AZT-terminated gaps will be developed to identify and assay recruitment of replication and repair proteins to sites of replication blockage. This study will significantly advance the field of DNA repair by providing new information regarding how replication gaps are sensed and repaired.