CtIP介导的DNA剪切功能在染色体易位引起的癌症中的作用机制研究

CtIP协同MRN（Mre11-Rad50-Nbs1）复合物作用于DNA末端5’-3’的剪切是DNA双链损伤后进行同源重组修复（HR）和微小同源序列介导的末端连接修复（MMEJ）的重要环节。但CtIP的DNA剪切功能在肿瘤发生发展中的作用尚未明确。CtIP敲除导致小鼠胚胎死亡，限制了对其功能的体内研究。申请者首先利用条件性敲除CtIP基因的小鼠细胞系证明了CtIP对细胞增值生长不可或缺并探讨了其机制；而后通过在细胞水平构建筛选系统，申请者找到了影响DNA剪切功能却不影响细胞增值的磷酸化位点：ATM/ATR介导的CtIP T859位(老鼠为T855)磷酸化。前期实验证明T855A点突变小鼠能够存活且发育正常。申请者将通过进一步分析T855A点突变小鼠并将其与成熟的肿瘤小鼠模型杂交，深入探讨CtIP的DNA剪切功能在染色质易位及肿瘤发生发展中的作用。此研究将为肿瘤的预防和治疗提供指导依据。

CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex to mediate 5’-3’ DNA resection, which is required for both homologous recombination (HR) and Microhomology-mediated end joining (MMEJ). However, the exact role of CtIP in tumorigenesis in not fully explored due to the embryonic lethality caused by CtIP deletion. By conditionally knocking out CtIP in mouse cells, we prove that CtIP is required to mediate endogenous DNA repair and indispensable for the proliferation and growth of cells in physiological conditions. Utilizing the CtIP conditional knock-out cell line, we screened different CtIP mutants and found that T859 (T855 in mouse) phosphorylation by ATM/ATR is required for the function of CtIP in DNA resection, but dispensable for cell viability. T855A mouse is viable and fertile. We will further use this mouse to cross with mouse models that generate chromosome translocations and investigate the function of CtIP in chromosome translocation and tumorigenesis in vivo.