The role of BRCA1 in nonhomologous repair of chromosomal double-strand breaks

描述（由申请人提供）：本研究的长期目标是了解调节生理过程中或电离辐射或化疗后出现的染色体双链断裂（DSB）修复的机制。我们的目标是了解解除管制的 DSB 修复对促进致癌的细胞突变过程和基因组不稳定性的影响。携带肿瘤抑制基因 BRCA1 种系突变的患者患乳腺癌和卵巢癌的风险显着增加。尽管许多细胞过程被归因于 BRCA1 的功能，但为什么 BRCA1 的缺失会导致基因组不稳定和癌症易感性增加仍不清楚。有证据表明 BRCA1 可能在细胞对 DSB 的反应中发挥核心作用。通过与其他 DNA 损伤信号传导和修复蛋白（包括 Chk2、Mre11 和 Ku80）的功能性相互作用，BRCA1 可以通过同源重组 (HR) 和非同源末端连接 (NHEJ) 的竞争机制来协调 DSB 的修复。这项研究将重点确定 BRCA1 在控制 NHEJ 中的作用，NHEJ 是哺乳动物细胞中主要的 DSB 修复过程。主要假设是，BRCA1 通过促进 Ku80 依赖的 NHEJ（更精确地修复 DSB）和阻止 Mre11 依赖的 NHEJ（通常不精确地修复具有高诱变潜力的 DSB）来维持基因组完整性。通过一系列染色体修复底物作为不同非同源修复子机制的功能读数，我们将使用遗传和 siRNA 方法来 (1) 研究 BRCA1 在遗传明确的人和鼠细胞系中染色体水平上调节两种不同 NHEJ 子通路的功能，(2) 确定 Chk2（一种 DNA 损伤反应激酶）对 BRCA1 磷酸化在控制 NHEJ 过程中的作用，以及 (3)确定蛋白质-蛋白质和蛋白质-DNA 相互作用是否是 BRCA1 通过 Mre11 预防突变 NHEJ 并通过 Ku80 促进更精确的 NHEJ 的分子机制。了解促进癌变并影响肿瘤对放疗和化疗反应的 DNA 修复缺陷可能为携带 BRCA1 突变的个体的癌症预防和 BRCA1 缺陷肿瘤患者的定制治疗提供新途径。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the mechanisms that regulate the repair of chromosomal double-strand breaks (DSB), arisen during physiological processes or after ionizing radiation or chemotherapy. We aim to learn the impact of deregulated DSB repair on cellular mutagenic processes and genomic instability that promote carcinogenesis. Patients who carry germline mutations in the tumor suppressor gene BRCA1 have significantly increased risk of developing breast and ovarian cancer. Although numerous cellular processes have been ascribed to the function of BRCA1, why loss of BRCA1 leads to increased genomic instability and cancer predisposition is still unclear. Evidence indicates BRCA1 may play a central role in cellular response to DSBs. Through functional interaction with other DNA damage signaling and repair proteins including Chk2, Mre11 and Ku80, BRCA1 may orchestrate the repair of DSBs by competing mechanisms of homologous recombination (HR) and nonhomologous end-joining (NHEJ). This study will focus on determining the role of BRCA1 in controlling NHEJ, the predominant DSB repair process in mammalian cells. The main hypothesis is that BRCA1 maintains genome integrity by promoting the Ku80-dependent NHEJ that repairs DSBs more precisely and by preventing the Mre11-dependent NHEJ that typically repairs DSBs imprecisely with high mutagenic potential. With an array of chromosomal repair substrates as the functional readout for different nonhomologous repair sub-mechanisms, we will use genetic and siRNA approaches to (1) study the function of BRCA1 in regulating the two different NHEJ subpathways at the chromosomal level within genetically well- defined human and murine cell lines, (2) determine the role of BRCA1 phosphorylation by Chk2, a DNA damage response kinase, in controlling NHEJ processes, and (3) identify whether protein-protein and protein-DNA interaction are the molecular mechanisms by which BRCA1 prevents the mutagenic NHEJ through Mre11 and promotes more precise NHEJ through Ku80. An understanding of the defects in DNA repair that promote carcinogenesis and affect tumor response to radiotherapy and chemotherapy may offer novel avenues for both cancer prevention in individuals carrying BRCA1 mutations and tailored therapy in patients with BRCA1-deficient tumors.