分枝性内切酶FEN1蛋白1型SUMO化修饰参与维持基因组稳定性的作用机制

FEN1 is a structure-specific endonuclease which is very important in cellular process and essential in DNA replication and repair. Abnormal expression level or functional deficiency of FEN1can leads to cellular disorder, even threat to life. Previous studies show that FEN1 can be modified after translation in various patterns in response to different growth conditions and environmental stress. Our recent work found that FEN1could be SUMOylated by SUMO1 (type 1 SUMOylation) while the underlying mechanism and the biological meanings were still not clear. To demonstrate how FEN1 type 1 SUMOylation contributes to genome stability maintenance we will carry out several part of works mainly including: 1) detect the FEN1 type 1 SUMOylation level under DNA damage stress, verify the modification sites; 2) analyze the changes of FEN1 activities after type 1 SUMOylation including enzyme activities, protein-protein interactions, cellular localization, et al. In the meanwhile, illustrate the underlying relationship between FEN1 type 1 SUMOlyation with other modifications; 3) analyze how FEN1 type 1 SUMOlyation level influence the cell survivability and genome stability by series cytobiology experiments. All the results will help us to explore the mechanism of how FEN1 type 1 SUMOlyation contributing to cell survival under DNA damage which will improve the integrity of the regulation network of FEN1 post translational modification with its essential role in genome stability maintenance.

分枝性内切酶FEN1是一种结构特异性内切酶，在DNA复制和损伤修复等多种细胞生命活动中发挥着关键作用，其表达水平和活性异常会导致细胞生命活动紊乱，进而危及健康。FEN1蛋白通过其多种翻译后修饰来响应细胞内的不同生理状态或环境胁迫。课题组前期研究发现了FEN1的一种新型修饰类型即1型SUMO化，但该蛋白1型SUMO化修饰的具体生物学意义及其作用机制尚不清楚。本申请项目在前期研究的基础上，计划开展以下研究工作：1）FEN1蛋白1型SUMO化修饰与DNA损伤胁迫的相关性及其修饰位点鉴定；2）1型SUMO化修饰对FEN1蛋白的活性、蛋白互作等的影响以及该修饰与其它类型修饰间的相关性；3）FEN1蛋白1型SUMO化修饰水平变化对基因组稳定性的影响和作用规律。研究结果旨在揭示FEN1蛋白1型SUMO化修饰参与维持基因组稳定性的作用机制，为阐明和完善FEN1的翻译后修饰调控网络提供新依据。

蛋白质翻译后修饰的动态变化过程及其复杂的调控网络一直是研究蛋白生物学功能和作用机制的难点之一。本项目主要针对细胞在受到DNA损伤胁迫时，DNA修复蛋白FEN1是如何被不同翻译后修饰调控，改变其生物学活性，从而参与维持基因组稳定性的过程。通过一系列研究我们发现，当细胞受到DNA损伤剂处理后，细胞中的FEN1蛋白的的1型SUMO化、磷酸化和乙酰化修饰水平同时增加。结合质谱和体内外验证，我们确定了FEN1蛋白的主要1型SUMO化修饰位点。通过免疫荧光实验和Duolink PLA技术都验证了这一现象，即FEN1蛋白与SUMO1蛋白能在体内发生共定位，而SUMO1(GG)点突变细胞株中FEN1与SUMO1的共定位则会消失。当FEN1 蛋白在体外发生1型SUMO化后，其Flap、Nick、GEN的酶切活性都不同程度的提高，表明F EN1的1型SUMO化修饰对DNA损伤修复过程起到了重要的作用。同时，研究SUMO化和其他修饰或蛋白关系时发现，FEN1的磷酸化会促进FEN1的1型SUMO化水平，1型SUMO化会降低FEN1与PCNA的结合，促进其与9-1-1复合物的结合。此外，细胞的凋亡率、周期等细胞表型实验也进一步验证了FEN1的SUMO化修饰对DNA损伤修复的影响以及维持细胞基因组稳定性的重要性。