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

描述(申请人提供)：这项研究的长期目标是了解在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)修复的调节机制。我们的目标是了解解除调控的DSB修复对促进癌症发生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胚系突变的患者患乳腺癌和卵巢癌的风险显著增加。虽然许多细胞过程都归因于BRCA1的功能，但为什么BRCA1的缺失会导致基因组的不稳定和癌症的易感性仍然不清楚。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Chk2、Mre11和Ku80等其他DNA损伤信号和修复蛋白的功能相互作用，BRCA1可能通过同源重组(HR)和非同源末端连接(NHEJ)相互竞争的机制来协调DSB的修复。这项研究将集中在确定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。主要的假设是，BRCA1通过促进依赖Ku80的NHEJ来更精确地修复DSB，并通过阻止依赖Mre11的NHEJ来维持基因组的完整性，NHEJ通常不精确地修复DSB，具有高突变潜力。以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出，我们将使用遗传学和siRNA方法：(1)在基因定义良好的人和小鼠细胞系中，研究BRCA1在染色体水平调节两条不同的NHEJ亚通路中的功能；(2)确定BRCA1被DNA损伤反应激酶Chk2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阻止NHEJ突变并通过Ku80促进更精确的NHEJ的分子机制。了解DNA修复缺陷会促进癌症发生并影响肿瘤对放疗和化疗的反应，这可能为携带BRCA1突变的个体预防癌症和为BRCA1缺陷肿瘤患者提供量身定制的治疗提供新的途径。

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.