CtIP的核酸酶活性及其参与DNA双链断裂修复的机制研究

DNA double-strand breaks (DSB) caused by ionizing radiation (IR) or other reasons are a major source to induce genome instability. Aberrant DSB repair can cause various kinds of chromosomal rearrangements including translocations, which are commonly observed in cancer cells. Thus, clarifying the molecular mechanisms of how DNA DSB are repaired has significant impact on cancer etiology and cancer treatment. CtIP , which associated with Mre11/Rad50/Nbs1 complex and BRCA1, is a new identified critical player in the regulation of homologous recombination (HR)-mediated DSB repair. Both CDK and ATM kinase target CtIP to regulate its repair function although the mechanisms underlying this regulation are not clear. Our recent study found CtIP carries an endonuclease activity similar with its homologous protein Sae2 does in budding yeast. In this project, we will perform biochemical study and a serious repair assay including new established HR:IR-Alu and HR:Flex1 repair assay to study this new identified nuclease activity of CtIP and try to reveal how this nuclease activity affect CtIP's repair function and whether phosphorylation by CDK and ATM can regulate nuclease activity of CtIP.These will help us to understand the exactly molecular mechanism of how CtIP play it's DSB repair function in mammalia cell.

电离辐射等因素造成的DNA双链断裂(DSB)是导致基因组不稳定的最主要原因之一。阐明DSB 修复的分子机制是癌症病因学与癌症治疗的重要研究方向。对DSB修复起关键作用的蛋白CtIP与MRN/BRCA1 协同作用于5'-3'末端的剪切，调节同源重组(HR)介导的DSB 修复。但是，CtIP是否具有核酸酶活性还不清楚。我们最近发现CDK和ATM 激酶对CtIP磷酸化并调节DSB 修复功能，但其确切机制仍有待剖析。我们的前期实验结果显示在体外CtIP具有类似于其在酵母中同源蛋白Sae2 所具有的内切核酸酶活性，并在哺乳动物细胞内建立了研究这种活性的IR-Alu 及HR-Flex1(AT)修复检测系统。本课题将在此基础上研究CtIP 核酸酶活性的酶学性质、CDK/ATM介导的CtIP磷酸化对其酶活性的影响及核酸酶活性对DSB修复的调控机制，进一步阐明CtIP参与DSB修复的分子机制。

CtIP 是一个熟知的DNA修复因子。CtIP与MRN蛋白复合物一道参与DNA双链断裂（DSB）末端修切（end resection），促进同源重组（HR）及微同源末端连接(MMEJ)介导的DSB 修复。本项目试图在前期研究工作的基础上，进一步深入的研究CtIP及其内切核酸酶活性在DSB修复中的功能及调控。经过4年的工作，我们取得了以下的进展：(1) 发现CtIP具有内切核酸酶活性、但不参与经典的末端修切（5’-3’End resection），对于富含AT DNA序列Flex1在DSB末形成的二级结构移除具有重要意义。(2) CtIP在G2/M期被有丝分裂期激酶CDK1/Aurora A/PLK1协同磷酸化，该磷酸化不调控CtIP内切磷酸酶活性，但是却阻碍CtIP介导的长程末端修切，促进微同源末端介导的末端连接修复(MMEJ)。(3) CtIP的内切核酸酶活性对于复制压力及癌基因表达造成的染色体脆性位点（CFS）稳定性的维持具有重要意义。(4) FANCM 移位酶（Translocase）活性在避免在AT富含区生成DSB及避免重组方面发挥重要作用。在FANCM KO细胞中如同时缺失CtIP内切核酸酶活性会明显的影响肿瘤细胞的在体外及裸鼠体内的存活。两种核酸酶活性具有协同致死性。