AND-1在DNA 双链断裂损伤修复中的作用及分子机制研究

Genomic stability and integrity play an important role in maintaining cell normal physiological function and cell survival, however, every cell in the human body could experience tens of thousands of DNA lesions per day due to the environmental factors and normal metabolic processes inside the cell. DNA double-strand breaks (DSBs) are the most lethal types of DNA damage, and, if they are not repaired or are repaired incorrectly, they can result in gene mutations or deleterious chromosome rearrangements that contribute to tumorigenesis. DSBs can be repaired by homologous recombination (HR) repair and nonhomologous end-join (NHEJ) mechanisms. Our recent study demonstrated that AND-1 interacts with MRN complex (MRE11、RAD50、NBS1) and CTIP, the DNA DSB repair proteins, We also found AND-1 recruitment to CPT and laser-induced DSBs by immunofluorescence microscopy. These data suggest that AND-1 may function in the DNA DSB repair response in concert with MRN complex and CTIP. Based on our own findings and preliminary data, we aim to address the functional role and molecular mechanisms of AND-1 in DNA damage repair by using siRNA, co-Immunoprecipitation, HR reporter assay, Immunofluorescence microscopy, Laser micro-irradiation and molecular biotechnology. The expected findings from this study will provide a new target and basis for cancer therapy.

基因组的稳定性和完整性对于细胞的存活和正常功能至关重要。双链断裂（DSB）是各种DNA损伤中最严重的一种形式，如果不能被正确修复，会导致细胞的死亡或者造成基因的突变，使肿瘤发生机率显著增加。DNA双链断裂主要通过同源重组修复(HRR)和非同源末端连接（NHEJ）两种机制进行修复。申请人近期研究发现，酸性核质DNA结合蛋白1（AND-1）被招募到DNA DSB位点处，同MRN复合物（MRE11、RAD50、NBS1）以及CTIP等DNA双链断裂损伤修复蛋白相互作用，揭示AND-1可能参与DNA DSB修复过程。本项目拟在前期工作基础上，采用siRNA敲除、免疫共沉淀、HR报告及激光微束照射等技术，对AND-1在DNA DSB修复中的作用及分子机制进行深入研究，研究结果可能为肿瘤治疗提供新的分子靶点。

申请人研究发现，酸性核质DNA结合蛋白1（AND-1）同MRN复合物（MRE11、RAD50、NBS1）以及CTIP等DNA双链断裂损伤修复蛋白相互作用，免疫荧光染色显示AND-1被招募到DNA DSB位点处，揭示AND-1可能参与DNA DSB修复过程。本项目对AND-1在DNA DSB修复中的作用及分子机制研究进行研究，我们发现AND-1同双链断裂损伤修复蛋白CTIP相互作用并调控CTIP在DSB位点的聚集，从而影响DSB的同源重组（HR）修复过程；AND-1在DSB位点的聚集依赖于MDC1, BRCA1 以及ATM等损伤修复蛋白；AND-1 表达下调后降低了CTIP在DSB位点的聚集，进而减少了细胞损伤检验点蛋白Chk1的激活以及其他HR修复应答反应过程。本研究揭示了HR修复过程中DNA末端切割的一个重要的调控路径，即MDC1→And-1→CtIP 调控介导的 DNA末端切割及细胞对DNA双链断裂损伤的应答反应。研究结果将为肿瘤治疗提供新的靶点和重要的理论依据。