Replication Fork Repair

描述（由申请人提供）： 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，修复效率低下与癌症倾向，神经和发育缺陷以及过早衰老有关。这项研究的长期目标是更完整地了解复制缺口的修复机制以及这些过程是如何调节的。目标将是阐明复制缺口修复途径的要素，包括特定的复制体成分，使用遗传和生化实验。由于所有细胞都通过进化相关的途径以基本相似的方式修复DNA，这些使用模式生物大肠杆菌的研究应该揭示适用于人类细胞DNA修复的机制。在本研究中使用叠氮胸苷作为诱导复制间隙的化合物，有可能揭示与其作为抗HIV药物的治疗用途相关的毒性和耐受机制。此外，由于细菌病原体中的DNA损伤反应在毒素产生、抗生素耐药性和感染持续性中起作用，因此这项工作可以为抗生素治疗提供新的靶点。本研究的第一个目的是通过研究YoaA蛋白的遗传和生化特性，阐明YoaA蛋白是如何参与复制间隙修复的，YoaA蛋白是人类疾病综合征中突变的XPD/FANCJ解旋酶家族的成员。这项研究的中心假设是与复制体蛋白质Chi的相互作用将YoaA解旋酶招募到停滞的复制叉，并且其新生链的解旋有利于去除链终止病变。将测试对特定修复途径的影响。该建议的第二个目的是确定不依赖SOS的DNA损伤反应的机制和程度。许多复制和修复基因都有一个共同的特征，即DNA损伤诱导不依赖于SOS反应。这一目标将集中在iraD基因，发现作为一个调节因子所需的生存复制抑制和氧化损伤。DnaA蛋白在这种调节中的作用和通过复制夹的信号传导将通过体内和体外实验进行研究。这项研究将通过提供有关复制缺口如何被感知和修复的新信息来显着推进DNA修复领域。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Replication fork repair is essential for cell survival and stability of genetic material. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. The long-term goal of this study is a more complete mechanistic understanding of the repair of replication gaps and how such processes are regulated. Objectives 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 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 study seeks to clarify how the YoaA protein, a member of the XPD/FANCJ family of helicases mutated in human disease syndromes, participates in replication gap repair by studies of its genetic and biochemical properties. The central hypothesis of this study is that interactions with the replisome protein, Chi, recruits YoaA helicase to a stalled replication fork and that its unwinding of the nascent strand facilitates removal of chain-terminating lesions. Effects on specific repair pathways will be tested. The second aim of this proposal is to identify the mechanism and extent of the SOS-independent DNA damage response. Many replication and repair genes share a common feature of DNA damage inducibility independent of the SOS response. This aim will center on the iraD gene, discovered as a regulatory factor required for survival to replication inhibition and oxidative damage. The role of the DnaA protein in this regulation and signaling via replication clamps will be examined by in vivo and in vitro experiments. This study will significantly advance the field of DNA repair by providing new information regarding how replication gaps are sensed and repaired.