DNA-PKcs通过SCF泛素连接酶调控EXO1蛋白稳定性及其DNA同源重组修复中的末端剪切功能机制研究

DNA双链断裂（DSB）是放射诱发的严重损伤，真核生物有同源重组(HR)和非同源末端连接(NHEJ) 两条主要修复通路。DSB经末端剪切形成一段单链DNA，是启动HR修复的关键，也是修复通路选择的调控点。前期发现，DNA-PKcs失活将导致执行DSB末端剪切的核酸酶EXO1的表达和稳定性显著改变。本课题将深入开展DNA-PKcs调控EXO1泛素化降解的上游信号通路和下游修复功能的研究：明确DNA-PKcs对放射损伤及细胞周期（G1 vs G2）EXO1表达和修饰（泛素化、磷酸化等）的影响规律；探讨SCF E3泛素连接酶对EXO1泛素化的调控机制，重点是DNA-PKcs直接或通过与RBX1的相互作用，对SCF催化核心Cullin-1蛋白的类泛素化修饰及作用于EXO1的活性调控机制；通过观测DSB末端剪切，明确DNA-PKcs通过上述作用对EXO1功能的调控机制，加深对放射生物学机制理论认识。

DNA double-strand break (DSB) is a severe damage induced by ionizing radiation. There are two major pathways of DSB repairing in eukaryotic cells: homologous recombination (HR) and non-homologous end joining (NHEJ). The yield 3’ssDNA of DSB through the end resection is a key reaction for initiating HR pathway, also a key regulation point for the DSB repair pathway choice. In our previous study, we found the inaction of DNA-PKcs led to a significant decrease of expression and protein stability of the exonuclease EXO1, the critical molecule of executing DSB end resection of HR pathway. Here, this project will further study the upstream signaling pathway and downstream repair function of EXO1 ubiquitination regulated by DNA-PKcs, including: The effects of DNA-PKcs on the alterations of EXO1 ubiquitination and phosphorylation, and the stability after irradiation and in different cell cycle phases; the regulation mechanism of EXO1 ubiquitination by SCF E3 ubiquitin ligase, especially focus on by the direct action and through interaction with RBX1, DNA-PKcs regulates the neddylation of Cullin-1, a component of SCF E3 ubiquitination ligase catalytic core, and its ubiquitin ligase activity on EXO1. Through the observation of DSB end resection reaction, to further elucidate the regulation mechanism of DNA-PKcs on EXO1 repair function. This project will provide further mechanism theory to advance our knowledge on radiation biology.