Separating the function of RAD51 in homologous recombination and replication

分离 RAD51 在同源重组和复制中的功能

• 批准号: 9241382
• 负责人: DOUGLAS K BISHOP
• 金额: $ 7.9万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-09 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9241382
• 关键词: Aftercare; Alleles; Aphidicolin; BRCA1 gene; BRCA2 gene; Binding; Biological Assay; CRISPR/Cas technology; Cell Cycle; Cell Death; Cell Line; Cell Survival; Cells; Chromosomal Stability; DNA; DNA Double Strand Break; DNA Repair; DNA replication fork; Data; Defect; Double Strand Break Repair; Embryo; Embryonic Development; Engineering; Essential Genes; Failure; Family; Fiber; Filament; Generations; Genetic Recombination; Genome Stability; Genomic Instability; Genomics; Growth; Human; Human Cell Line; Ionizing radiation; Knockout Mice; Laboratories; Malignant Neoplasms; Mammalian Cell; Mediating; Meiosis; Meiotic Recombination; Mitotic; Modeling; Mutagens; Mutant Strains Mice; Mutation; Nucleoproteins; Null Lymphocytes; Pathway interactions; Patients; Phenotype; Play; Predisposition; Proteins; Rad51 recombinase; Reaction; Role; Saccharomyces cerevisiae; Saccharomycetales; Single-Stranded DNA; Stress; Testing; Topotecan; Work; Yeasts; experimental study; homologous recombination; hydroxyurea; in vivo; inhibitor/antagonist; insight; kidney cell; malignant breast neoplasm; member; mutant; neoplastic cell; nuclease; osteosarcoma; overexpression; prevent; public health relevance; recombinase; response; tool; tumor progression

 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.

 描述（通过应用程序提供）：通过修复DNA双链断裂（DSB）和拯救停滞的复制叉，同源重组在基因组稳定性中起关键作用。 RAD51催化同源重组的中心反应：同源搜索和链入侵。 RAD51 - / - 突变小鼠在胚胎发生早期死亡，而Rad51耗尽的细胞在细胞死亡之前积累了DSB。由于RAD51在同源重组中的中心作用，这导致了普遍的观点，即Rad51的基本功能是复制过程中发生的DSB的同源重组介导的修复。然而，最近的研究已经确定了RAD51和其他同源重组因子（包括乳腺癌易感性蛋白BRCA1和BRCA2）在复制叉稳定性中的重组角色。以前，我们在发芽酵母中产生了Rad51的功能分离等位基因，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入侵中有缺陷（RAD51-II3A）。令人惊讶的是，该RAD51突变体不存在RAD51缺陷酵母细胞中观察到的生长缺陷，表明酵母中的生长缺陷并不是无法修复DSB的结果。该结果与最近的证据相结合，即RAD51在复制中具有重组独立的作用，这使我们提出了这样的假设：Rad51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，我们将生成从Rad51基因组基因座表达RAD51-II3A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细胞活力和未扰动细胞中染色体稳定性的影响。最后，我们将检查Rad51-II3A表达细胞对复制应激的反应以及诱导DNA双链断裂的遗传学剂的反应。我们将确定RAD51-II3A细胞是否具有保护复制叉并响应复制扰动的能力，但缺乏修复DSB的能力。拟议的工作将为RAD51在细胞活力和复制叉稳定性中的HR无关功能提供宝贵的见解。鉴于人力资源在保护方面的重要性 基因组不稳定性的细胞，这项工作将为人力资源破坏如何导致癌症易感性提供重要的机械洞察力。