BRCA1对正确和错误性NHEJ亚通路的引导作用

DNA double strand breaks(DSBs)is a kind of DNA damage happened on chromatin of cells. Being repaired correct or incorrect would determine genomic integrity of daughter cells. Concerning about its non-homologous end joining(NHEJ) repair pathway, some papers show that Breast cancer susceptibility gene1(BRCA1) might promote fedelity precise-NHEJ while suppressing mutagenic and error-prone alternative-NHEJ(A-NHEJ), and that is what this project focuses on. Two cell lines carrying chromosomally integrated precise- or A-NHEJ repair report-substrate respectively will be used. Introducing rare endonuclease I-SceI into cells will recongnize its cutting sites located in the substrates, and generate DSBs which suit to be repaired by precise- or A-NHEJ in a large amount of cell individuals. After wild type or mutant BRCA1 expression in cells are changed, several DNA repair indexes（such as the two NHEJ subpathway repair rate, chromatin bound protein expression, DSBs bound protein expression, subpathway-related protein -protein interaction which include with BRCA1, and so on）induced by the two different DSB are determined. This project trys to expose mechanism of BRCA1 modulating the choice of these two opposite NHEJ repair subpathways, and to find possible related repair protein in each and prove them by using their inhibitors. In another word, it trys to explain BRCA1 on genome stability maitenance in a NHEJ subpathway profile, and hopefully to afford an important clinic guidance for illness prevention and treatment of tumors and other genetic diseases.

DNA双链断裂（DSBs）是细胞染色体上的损伤，正确修复与否决定子代基因组的完整性。有文献显示BRCA1可能在其非同源末端连接（NHEJ）修复中促进精确NHEJ而抑制错误性的非经典A-NHEJ，这正是本课题的立题所在。本课题拟用两套分别在染色体上整合了含有I-SceI酶切位点的精确-或A-NHEJ修复报告底物的细胞模型，导入I-SceI酶将分别在其大量的细胞个体内产生适合精确-或A-NHEJ修复的DSBs。改变胞内BRCA1或其突变体表达，检测两种不同DSB引起的亚通路修复概率、染色质结合蛋白表达、DSB结合蛋白表达和各亚通路相关蛋白（包括与BRCA1）相互结合等变化，揭示BRCA1对这两条修复结果相反的NHEJ亚通路的取舍和可能机理，发现可能的亚通路相关蛋白并用蛋白抑制剂证实其作用。试图从DSB修复亚通路角度来诠释BRCA1维护DNA稳定性的机制，对肿瘤等疾病防治具有重大的指导意义。

本课题组已成功完成定量检测A-NHEJ的Ku80缺陷的Xrs5/EJ2GFP细胞模型的构建；定量分析了BRCA1 siRNA对精确NHEJ和A-NHEJ的影响，结果显示：BRCA1表达沉默降低精确NHEJ修复亚通路的效率，同时提高错误倾向的A-NHEJ亚通路的修复效率，提示BRCA1可能在NHEJ亚通路的选择上发挥了一定的作用，而PARP1抑制剂ABT-888的联合作用，可以逆转由BRCA1缺失引起的A-NHEJ修复的升高；染色体结合蛋白表达结果显示：Mre11蛋白质在Ku功能完整或缺陷的不同细胞系中对BRCA1沉默的表现不一致，提示BRCA1与Mre11的相互作用，对精确NHEJ和A-NHEJ的选择可能发挥着一定的作用。