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（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，在基因组稳定性中发挥着关键作用。 RAD51 催化同源重组的核心反应：同源搜索和链入侵。 RAD51-/-突变小鼠在胚胎发生早期死亡，并且RAD51耗尽的细胞在细胞死亡之前积累DSB。由于 RAD51 在同源重组中的核心作用，人们普遍认为 RAD51 的基本功能是在复制过程中自发发生的同源重组介导的 DSB 修复。然而，最近的研究已经确定了 RAD51 和其他同源重组因子（包括乳腺癌易感蛋白 BRCA1 和 BRCA2）在复制叉稳定性中的重组独立作用。此前，我们在芽殖酵母中生成了 RAD51 的功能分离等位基因，该等位基因保留了形成核蛋白丝的能力 位于单链 DNA 上，但在同源性搜索和链入侵方面有缺陷 (rad51-II3A)。令人惊讶的是，这种 Rad51 突变体并没有表现出在 Rad51 缺陷酵母细胞中观察到的生长缺陷，这表明酵母中的生长缺陷不是由于无法修复 DSB 造成的。这一结果与最近的证据相结合，表明 RAD51 在复制中具有与重组无关的作用，使我们得出这样的假设：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 蛋白的人类细胞系衍生物。使用这些细胞系，我们将确定链交换活性丧失对未受干扰的细胞中的细胞活力和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和诱导 DNA 双链断裂的基因毒性剂的反应。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而重新启动的能力，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方面的 HR 独立功能提供有价值的见解。鉴于人力资源在保护方面的重要性 细胞免受基因组不稳定的影响，这项工作将为 HR 破坏如何导致癌症易感性提供重要的机制见解。