Table2_PLK1-mediated phosphorylation of PPIL2 regulates HR via CtIP.XLSX

Homologous recombination (HR) is an error-free DNA double-strand break (DSB) repair pathway, which safeguards genome integrity and cell viability. Human C-terminal binding protein (CtBP)—interacting protein (CtIP) is a central regulator of the pathway which initiates the DNA end resection in HR. Ubiquitination modification of CtIP is known in some cases to control DNA resection and promote HR. However, it remains unclear how cells restrain CtIP activity in unstressed cells. We show that the ubiquitin E3 ligase PPIL2 is recruited to DNA damage sites through interactions with an HR-related protein ZNF830, implying PPIL2’s involvement in DNA repair. We found that PPIL2 interacts with and ubiquitinates CtIP at the K426 site, representing a hereunto unknown ubiquitination site. Ubiquitination of CtIP by PPIL2 suppresses HR and DNA resection. This inhibition of PPIL2 is also modulated by phosphorylation at multiple sites by PLK1, which reduces PPIL2 ubiquitination of CtIP. Our findings reveal new regulatory complexity in CtIP ubiquitination in DSB repair. We propose that the PPIL2-dependent CtIP ubiquitination prevents CtIP from interacting with DNA, thereby inhibiting HR.

同源重组（HR）是一种无错误的DNA双链断裂（DSB）修复途径，其作用在于维护基因组完整性与细胞存活能力。人类C端结合蛋白（CtBP）相互作用蛋白（CtIP）是该途径的核心调控因子，负责启动HR过程中的DNA末端切割。在某些情况下，CtIP的泛素化修饰被认为能够调控DNA末端切割并促进HR。然而，细胞如何在未受应激的细胞中抑制CtIP的活性仍是一个未解之谜。本研究发现，泛素E3连接酶PPIL2通过与HR相关蛋白ZNF830的相互作用被招募至DNA损伤位点，暗示PPIL2在DNA修复过程中的参与。我们观察到PPIL2与CtIP在K426位点相互作用并泛素化修饰，这一位点此前尚未发现存在泛素化修饰。PPIL2介导的CtIP泛素化抑制了HR和DNA末端切割。此外，这种PPIL2的抑制作用也受到PLK1在多个位点的磷酸化调控，从而减少PPIL2对CtIP的泛素化。我们的研究揭示了DSB修复过程中CtIP泛素化调控的新复杂性。我们提出，PPIL2依赖性的CtIP泛素化通过阻止CtIP与DNA的结合，从而抑制HR。