在DNA损伤应答中Polo样激酶1（PLK1）活性调控的分子机制

Genomic DNA is constantly attacked by varieties of endogenous and environmental factors, generating millions of DNA damage per cell per day. Fortunately, our body has evolved a highly efficient DNA damage response system to recognize and repair the damaged DNA in order to maintain the genomic stability and prevent cells from diseases. Polo-like kinase1(PLK1) is an essential regulator in the cell cycle progression, however, the mechanism of tightly regulated kinase activity of PLK1 after DNA damage is largely unknown. We found for the first time that PLK1 is rapidly recruited to UV laser-induced DNA damage sties and this recruitment is dependent on poly ADP-ribose polymerase 1 (PARP1). In vitro binding assays revealed that PLK1 binds poly ADP-ribose chain (PAR), resulting in its kinase activity inhibition. In vitro kinase assays demonstrated that CHK1 phosphorylates PLK1 at S137, driving the subsequent phosphorylation of RAD51 by PLK1. This proposal will employ molecular, biochemical, and cellular approaches to determine the molecular mechanisms how the kinase activity of PLK1 is inhibited after DNA damage and reactivated by the checkpoint kinase CHK1 in S/G2 phase and how DNA damage-modulated PLK1 kinase activity has an impact on repair of DNA double strand breaks. These results in sum will reveal function of PLK1 in genome stability maintenance and tumorigenesis.

基因组不间断受到内外因子的攻击而导致大量的DNA损伤，我们机体已经进化出一套高效的DNA损伤应答（DDR）系统来监测并修复受损的DNA，以维持基因组的稳定和防止疾病如癌症的发生。Polo样激酶PLK1是参与细胞周期调控的核心成员，然而DNA损伤发生后其活性调控机制一直不清楚。我们首次发现DNA损伤后，PARP1依赖的PLK1在DNA损伤位点快速募集，体外实验显示PLK1与多聚核糖基化链（PAR）的结合抑制其激酶活性；体外激酶实验显示检验点激酶CHK1对PLK1的S137位点磷酸化驱动其对Rad51磷酸化。本课题拟从分子、生化、细胞等层面剖析DNA损伤后PLK1活性被抑制、在S/G2期CHK1介导的PLK1部分激活的分子机制及其在DNA双链断裂修复中的功能，揭示PLK1在维持基因组稳定性及在肿瘤发生、发展中的作用。

基因组不稳定是癌症的核心特征之一，我们旨在探究DNA损伤后Polo 样激酶 1 (PLK1)活性的调控机制及其在维持基因组稳定性方面的作用。PLK1是一种调节细胞周期进程的关键激酶。DNA 损伤其酶活性如何调控的分子机制尚不清楚。我们的工作表明，PLK1 在几秒钟内迅速募集至DNA双链断裂处，这一过程依赖于 PARP1，并在10分钟内以 PARG 依赖的方式解离。多聚核糖基化链(PAR)在体外与PLK1 直接结合并抑制其激酶活性。蛋白激酶CHK1 介导PLK1的S137和T210位点的磷酸化拮抗其与PAR链结合，并促进PLK1对RAD51 S14的磷酸化。而RAD51 S14位点的磷酸化促进CHK1介导的T309磷酸化，这对于RAD51的完全激活是至关重要的。因此，CHK1-PLK1-RAD51 信号轴促进同源重组 (HR) 介导的修复，并维持基因组的稳定性和细胞放射敏感性。我们的研究诠释了在DNA损伤应答过程中PLK1活性的动态变化及其调控机制，揭示了PLK1的S137位点磷酸化修饰是T210位点的磷酸化修饰和PLK1激活的先决条件，为PARG和 CHK1 抑制剂的联合化疗提供了生物学基础。