RNF4的磷酸化修饰在不同细胞周期中对DNA损伤应答的影响与机制

DNA damage is highly related to tumorigenesis. Different repair pathway is preferred during cell cycle which will facilitate maintaining genomic stability and prevent tumorigenesis. However, the mechanism is still unclear. In our previous study, we found MDC1 will be sumoylated following DNA damage which can be ubiquitinated by RNF4 and degraded. The disruption of this process impairs HR but has no effect on NHEJ. Recently, we found RNF4 could be phosphorylated by CDKs in a cell cycle dependent manner. The phosphorylation of RNF4 may regulate the transition between HR and NHEJ during cell cycle. We will find the CDK protein which phosphorylate RNF4 and find out the phosphorylation sites by using in vitro/in vivo kinas assay, site mutagenesis and mass spectrum. We will study how the phosphorylation affects MDC1 degradation by using double thymidine block, western, and immunofluorescence. Furthermore, we will test the function of the phosphorylation in DNA repair by using NHEJ assay and HR assay. Besides studying the effect on MDC1, we will also check whether the phosphorylation affects other key proteins in DNA damage response pathway by immunofluorescence, including the foci formation and disassembly from the damage sites. On the other hand, we will study the biological function of the phosphorylation in cellular translation and tumorigenesis by using knock-in mice model and MEFs. Taken together, we will further investigate the function of RNF4 phosphorylation in DNA damage response and tumorigenesis in this project.

DNA损伤应答与肿瘤发生密切相关。细胞在不同周期中选择不同的修复方式应对DNA损伤有助于细胞维持基因组稳定性并抑制肿瘤发生，但其调控机制尚不清楚。我们的前期研究发现MDC1在DNA损伤后会被SUMO化修饰从而被RNF4识别并泛素化降解，该通路的异常将导致HR修复异常但对NHEJ修复并无影响。近来申请人发现RNF4能够被CDKs磷酸化，且该修饰受细胞周期调控。申请人推测该修饰很可能调控不同细胞周期中对DNA修复途径的选择。为此，本研究针对RNF4的磷酸化修饰，拟采用体内外激酶实验及质谱技术确定磷酸化RNF4的CDK蛋白及修饰位点，并进一步研究RNF4磷酸化修饰对MDC1及DNA损伤应答通路的影响，利用RNF4磷酸化缺陷型knock-in小鼠模型以及MEFs，分析该修饰在肿瘤发生中的作用。以深入阐明RNF4的磷酸化修饰在不同细胞周期中对DNA损伤应答的影响和机制，并阐述其对肿瘤发生的影响。

细胞在不同的细胞周期选择不同 DNA 损伤修复方式的分子机制一直是本领域的研究热点。在本项目在研究 RNF4-MDC1 调控机制时发现，CDK2- RNF4- MDC1 信号轴介导细胞在 S 期选择 DNA 同源重组修复途径。当细胞在 S 期时， CDK2 被激活，从而磷酸化 RNF4 并降解 MDC1，促进 DNA 损伤同源重组修复因子 Rad51 募集到损伤位点，以保证同源重组修复顺利进行。.这项研究的创新之处在于：.① 阐明细胞周期通过CDK2-RNF4-MDC1-Rad51信号轴调控同源重组修复的分子机制。.② 揭示细胞在 S 期选择同源重组修复方式的新的分子机制。该研究科学意义在于详细解析CDK2- RNF4- MDC1-Rad51 信号通路调控同源重组修复分子机理，进一步完善细胞在不同的细胞周期选择 DNA损失修复方式的作用机理。