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。我们的目的是了解去调控的DSB修复对细胞致突变过程和促进致癌的基因组不稳定性的影响。携带肿瘤抑制基因BRCA 1生殖系突变的患者患乳腺癌和卵巢癌的风险显著增加。虽然许多细胞过程已归因于BRCA 1的功能，但为什么BRCA 1的缺失会导致基因组不稳定性增加和癌症易感性仍然不清楚。有证据表明BRCA 1可能在细胞对DSB的反应中发挥核心作用。通过与其他DNA损伤信号和修复蛋白（包括Chk 2，Mre 11和Ku 80）的功能相互作用，BRCA 1可能通过同源重组（HR）和非同源末端连接（NHEJ）的竞争机制协调DSB的修复。这项研究将集中在确定BRCA 1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BRCA 1通过促进Ku 80依赖性NHEJ（更精确地修复DSB）和阻止Mre 11依赖性NHEJ（通常不精确地修复DSB，具有高致突变潜力）来维持基因组完整性。利用一系列染色体修复底物作为不同非同源修复亚机制的功能读数，我们将使用遗传和siRNA方法来（1）研究BRCA 1在遗传上明确定义的人和鼠细胞系中在染色体水平上调节两种不同NHEJ亚途径的功能，（2）确定BRCA 1被Chk 2磷酸化的作用，Chk 2是一种DNA损伤反应激酶，蛋白质-蛋白质和蛋白质-DNA相互作用是否是BRCA 1通过Mre 11阻止致突变性NHEJ并通过Ku 80促进更精确的NHEJ的分子机制。了解DNA修复缺陷促进癌发生并影响肿瘤对放疗和化疗的反应，可能为携带BRCA 1突变的个体的癌症预防和BRCA 1缺陷肿瘤患者的定制治疗提供新的途径。

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.