Analysis of the Replication Stress Response

项目摘要 在人的一生中，DNA的数十亿个碱基对必须复制数万亿次。更困难的是， 复制受到压力的挑战，包括DNA模板损伤，难以复制序列， 与转录冲突。对复制应激的多重修复和耐受反应作用于完成DNA 合成，同时最大限度地减少错误。在这个项目中，我们试图了解这些机制，重点是 复制叉反转。复制叉逆转被认为是稳定停滞的叉，将DNA损伤放回原位 在双链体DNA的背景下，它可以通过切除修复去除，并提供一个机会， 用于模板切换。还原酶由ATP依赖性DNA移位酶和RAD 51重组酶催化。 它也是高度调节的，这是防止复制和核酸酶不适当减慢所必需的。 复制分叉的相关处理。例如，我们最近发现RADX是一种单链DNA， 通过直接结合RAD 51调节逆转的结合蛋白。在这段时间里，我们试图了解 RADX如何工作，复制如何在挑战后恢复，并严格测试假设， 叉反转是DNA损伤耐受的无错误机制。我们将测试和完善新的概念 利用生物化学、遗传学和细胞生物学方法的组合的模型。我们多年的 积累的知识和试剂以及强大的合作者团队使我们能够完成这些 问题研究鉴于复制应激反应对预防包括癌症在内的疾病的重要性， 研究将产生新的发现，这些发现可以在未来转化为有益于人类健康的成果。

PROJECT SUMMARY Billions of base pairs of DNA must be replicated trillions of times during a human lifetime. Adding to the difficulty, replication is challenged by stresses including DNA template lesions, difficult to replicate sequences, and conflicts with transcription. Multiple repair and tolerance responses to replication stress act to complete DNA synthesis while minimizing errors. In this project we seek to understand these mechanisms with a focus on replication fork reversal. Replication fork reversal is thought to stabilize stalled forks, to place DNA lesions back into the context of duplex DNA where it can be removed through excision repair, and to provide an opportunity for template switching. Reversal is catalyzed by ATP-dependent DNA translocases and the RAD51 recombinase. It is also highly regulated, which is needed to prevent inappropriate slowing of replication and nuclease- dependent processing of replication forks. For example, we recently discovered RADX as a single-stranded DNA binding protein that regulates reversal by directly binding RAD51. In this funding period we seek to understand how RADX functions, how replication resumes following a challenge, and to rigorously test the hypothesis that fork reversal is an error-free mechanism of DNA damage tolerance. We will test and refine new conceptual models utilizing a combination of biochemical, genetic, and cell biological approaches. Our many years of accumulated knowledge and reagents and strong team of collaborators uniquely position us to complete these studies. Given the importance of replication stress responses to preventing diseases including cancer, these studies will generate discoveries that could be translated in the future to benefit human health.