Separating the function of RAD51 in homologous recombination and replication

 描述（由申请人提供）：同源重组通过修复DNA双链断裂（DSB）和挽救停滞的复制叉在基因组稳定性中起关键作用。RAD 51催化同源重组的中心反应：同源搜索和链侵入。RAD 51-/-突变小鼠在胚胎发生早期死亡，并且RAD 51耗尽的细胞在细胞死亡之前积累DSB。由于RAD 51在同源重组中的核心作用，这导致了普遍的观点，即RAD 51的基本功能是在复制期间自发发生的DSB的同源重组介导的修复。然而，最近的研究已经确定了RAD 51和其他同源重组因子（包括乳腺癌易感蛋白BRCA 1和BRCA 2）在复制叉稳定性中的独立重组作用。以前，我们在芽殖酵母中产生了一个功能分离的RAD 51等位基因，它保留了形成核蛋白丝的能力 在单链DNA上，但在同源性搜索和链侵入方面有缺陷（rad 51-II 3A）。令人惊讶的是，这种Rad 51突变体没有表现出在Rad 51缺陷型酵母细胞中观察到的生长缺陷，表明酵母中的生长缺陷不是不能修复DSB的结果。这一结果结合最近的证据表明，RAD 51在复制中具有不依赖于重组的作用，使我们假设RAD 51在脊椎动物细胞中的基本功能是复制叉稳定性，而不是同源重组。使用Cas9/CRISPR，我们将产生从RAD 51基因组基因座表达RAD 51-II 3A蛋白的人类细胞系的衍生物。使用这些细胞系，我们将确定链交换活性丧失对未扰动细胞中细胞活力和染色体稳定性的影响。最后，我们将研究表达RAD 51-II 3A的细胞对复制应激和诱导DNA双链断裂的遗传毒性剂的反应。我们将确定RAD 51-II 3A细胞是否具有保护复制叉和响应复制扰动而重新启动的能力，但缺乏修复DSB的能力。拟议的工作将提供有价值的洞察力的HR独立功能的RAD 51细胞活力和复制叉的稳定性。鉴于HR在保护 这项工作将提供重要的机制洞察如何破坏HR的结果在易感性癌症。

 DESCRIPTION (provided by applicant): Homologous recombination plays a critical role in genome stability by repairing DNA double strand breaks (DSBs) and rescuing stalled replication forks. RAD51 catalyzes the central reactions of homologous recombination: homology search and strand invasion. RAD51-/- mutant mice die early in embryogenesis and cells depleted of RAD51 accumulate DSBs prior to cell death. Due to the central role of RAD51 in homologous recombination, this led to the prevailing view that the essential function of RAD51 is the homologous recombination-mediated repair of DSBs that occur spontaneously during replication. However, recent studies have identified recombination-independent roles for RAD51 and other homologous recombination factors including the breast cancer susceptibility proteins, BRCA1 and BRCA2, in replication fork stability. Previously, we generated a separation-of-function allele of RAD51 in budding yeast that retains the ability to form a nucleoprotein filament on single-stranded DNA, but is defective in homology search and strand invasion (rad51-II3A). Surprisingly, this Rad51 mutant did not exhibit the growth defect observed in Rad51-deficient yeast cells, indicating the growth defect in yeast is not a result of failure to repair DSBs. This result combined with the recent evidence that RAD51 has recombination-independent roles in replication led us to the hypothesis that the essential function of RAD51 in vertebrate cells is replication fork stability, and not homologous recombination. Using Cas9/CRISPR, we will generate derivatives of human cell lines that express RAD51-II3A protein from the RAD51 genomic locus. Using these cell lines, we will determine the effect of loss of strand exchange activity on cell viability and chromosome stability in unperturbed cells. Finally, we will examine the response of RAD51-II3A expressing cells to replication stress and to genotoxic agents that induce DNA double strand breaks. We will determine if the RAD51-II3A cells have the ability to protect replication forks and restart in response to replication perturbation, but lack the abilityto repair DSBs. The proposed work will provide valuable insight to the HR-independent functions of RAD51 in cell viability and replication fork stability. Given the importance of HR in protecting cells from genome instability, this work will provide important mechanistic insight into how disruption of HR results in predisposition to cancer.