Table3_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，它保障了基因组完整性和细胞活力。人C端结合蛋白（CtBP）相互作用蛋白（CtIP）是该途径的核心调节因子，它启动了HR中的DNA末端切除。在某些情况下，CtIP的泛素化修饰被认为可以调控DNA切除并促进HR。然而，细胞如何在不应激状态下抑制CtIP活性尚不清楚。我们的研究表明，泛素E3连接酶PPIL2通过与HR相关蛋白ZNF830的相互作用被募集到DNA损伤位点，这暗示了PPIL2在DNA修复中的参与。我们发现PPIL2与CtIP在K426位点相互作用并泛素化，代表了一个迄今为止未知的泛素化位点。PPIL2介导的CtIP泛素化抑制了HR和DNA切除。这种PPIL2的抑制也受到PLK1在多个位点磷酸化的调节，从而减少PPIL2对CtIP的泛素化。我们的发现揭示了DSB修复中CtIP泛素化的新调控复杂性。我们提出，依赖于PPIL2的CtIP泛素化防止CtIP与DNA相互作用，从而抑制HR。