组蛋白泛素化修饰与同源重组修复

Ubiquitination is an important form of post-translational modification implicated in a variety of cellular processes. It comes in many forms too, including mono-, di-uniquitination, and poly-ubiquitination via a number of linkages (K6, K11, K27, K48, K63, etc.). It is clear that dynamic ubiquitination and de-ubiquitination play critical roles in transmitting DNA damage signal and in regulating various steps in repair. As a form of epigenetic regulation, histone ubiquitination is now known essential in the recruitment of various factors to DNA damage sites including BRCA1. Our preliminary results indicate that DSB-induced poly-ubiquitin needs to be actively protected if BRCA1 is to be recruited and HR repair to follow, and the protection is provided by the anaphase-promoting complex (APC), an E3 ubiquitin ligase itself. The proposed work will uncover how APC functions at DSBs and generate new insights into DNA damage response (DDR). Further, we will determine if the loss of APC function in tumors is beneficiary to chemotherapy.

泛素化修饰是一种重要的蛋白质翻译后修饰，它通过复杂多样的泛素连接（单泛素、双泛素、各种链接形式的多聚泛素）来调控蛋白的稳定性或功能。近年研究发现，动态的泛素化和去泛素化修饰在决定DNA损伤修复能否正常进行和细胞命运方面发挥着重要作用。这其中，作为表观遗传修饰形式之一的组蛋白泛素化修饰，尤为重要。DNA双链断裂（DSB）后，H2A/H2B上发生的多聚泛素化是招募BRCA1的重要信号。我们的前期研究结果显示这种多聚泛素化不仅其形成受到DNA损伤信号通路的调控，其稳定性也受到调控。本课题组的预实验结果显示，后期促进复合物(anaphase-promoting complex，APC)缺失可引起以K63-连接的多聚泛素化修饰在DSB位点的消失，进而引起BRCA1招募受损和同源重组修复的缺陷。我们将探讨APC是如何保护DSB位点多聚泛素化的，以及其在肿瘤细胞里的功能缺失可否被利用于化疗。

DNA损伤位点的泛素化和去泛素化对于传递DNA损伤信号以及调控下游修复机制具有至关重要的作用。近年来逐步有新的调控分子被发现和证实参与DNA损伤和修复反应。APC是一种多亚基E3泛素连接酶，它通过两个包含WD40重复序列的接头蛋白Cdc20和Cdh1与底物结合。除了参与细胞周期调控，APC/C-Cdh1还被证实可以通过对其底物的泛素化和降解进而影响DNA损伤应答。本研究项目旨在揭示Cdh1在DNA损伤修复中的新功能并验证Cdh1功能缺失是否能促进肿瘤细胞对复制压力的敏感性。课题组利用免疫荧光，HR修复报告基因，RNAi等技术方法探讨APC/C-Cdh1是否对DNA损伤修复方式的选择具有决定性作用及具体分子机制，并通过乳腺癌细胞模型验证Cdh1敲低对PARP抑制剂敏感性的影响。结果发现：1）在G1期，APC/C-Cdh1通过调控RNF8的K11连接的泛素化调节其在DNA损伤区域的聚集情况，进而影响Ku80在DNA损伤位点的水平，从而促进NHEJ修复通路的进行。2）Cdh1缺失能极大提高乳腺癌细胞对PARP抑制剂的敏感性。综上所述，本项目揭示了Cdh1在DNA损伤修复中的又一重要作用，并提示检测Cdh1在肿瘤里的表达、突变对乳腺癌临床用药将具有重要指导意义。