Replication Fork Repair

项目总结 复制分叉修复对于细胞存活和遗传物质的稳定是必不可少的。在人类中，低效的 修复与癌症倾向、神经、免疫、发育缺陷和 过早衰老。 这项研究的长期目标是从机制上更全面地理解复制的修复 差距。这个项目的目标是阐明复制缺口修复途径的要素，包括 利用遗传和生化实验，获得特定的复制体组分。因为所有的细胞都在修复DNA 通过进化相关的途径，这些研究使用了模式生物体， 大肠杆菌，应该揭示适用于人类细胞DNA修复的机制。对.的使用 在本研究中，叠氮胸苷(AZT)作为一种诱导复制缺口的化合物具有潜在的毒性 以及与其作为抗艾滋病毒药物的治疗用途相关的耐受机制。此外，因为DNA 细菌病原体的损伤反应在毒素产生、抗生素耐药性和持久性方面起着作用。 这项工作可能为抗生素治疗提供新的靶点。 这项建议的第一个目的是确定RADA，一种普遍的细菌蛋白，通过其机制与 对于RecA，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 通过生化和遗传学实验推断，以确定RADA和RecA如何相互作用促进 重组。 这项研究的第二个目的是试图阐明复制体夹载复合体和修复蛋白质是如何 互动，以便于维修。YoaA蛋白是XPD/FANCJ解旋酶家族的成员之一，在 人类疾病综合征，将通过遗传和生化方法进行研究，以确定它是否是一种 DNA解旋酶，这一活动是如何专门化的，以及它如何促进复制缺口修复。与 复制体将从遗传和生化角度进行研究。在体内，DNAX变异钳的丰度- 将确定加载器复合体及其调节，以及它们在促进遗传稳定性方面的作用。一个 将开发一种分离与AZT末端GAP相关的蛋白质的新方法，以识别和 检测复制和修复蛋白在复制阻断部位的募集。 这项研究将通过提供关于以下方面的新信息来显著推动DNA修复领域的发展 如何感知和修复复制差距。

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.