泛素特异性蛋白酶15稳定着色性干皮病F蛋白并促进DNA链间交联损伤修复

着色性干皮病F蛋白 (xeroderma pigmentosum group F, XPF) 和切除修复交叉互补组1蛋白 (excision repair cross complementing group 1, ERCC1) 组成一种结构特异性的核酸内切酶 (XPF-ERCC1)复合物，参与DNA链间交联 (interstrand crosslink, ICL) 损伤修复。其中，XPF蛋白的去泛素化修饰对DNA损伤修复的影响尚未见报道。本工作主要研究泛素特异性蛋白酶15 (ubiquitin-specific protease 15, USP15) 对XPF的稳定性及ICL修复的影响。本研究通过蛋白质质谱和Western印迹法分析发现，XPF蛋白与USP15存在相互作用，进而使XPF蛋白去泛素化修饰；采用CRISPR-Cas9技术构建USP15基因敲除的HeLa细胞株 (USP15 KO) 并进行Western印迹分析，结果显示，敲除组XPF蛋白水平低于对照组 (P<0.001)。克隆形成试验显示，在ICL诱导剂顺铂 (cisplatin，DDP) 和丝裂霉素C (mitomycin, MMC) 的作用下，USP15基因敲除的HeLa细胞增殖能力显著降低 (P<0.01)。本研究表明，去泛素化酶USP15是一种重要的DNA修复调节因子，该酶通过稳定XPF蛋白促进由XPF-ERCC1介导的ICL修复。本研究为改善ICL诱导剂类抗癌药物的耐药性提供了理论依据，并为肿瘤的治疗提供了潜在的新靶点。

Ubiquitin-specific Protease 15 Stabilizes XPF and Promotes Repair of DNA Interstrand Crosslink

Xeroderma pigmentosum group F (XPF) and excision repair cross complementary group 1 protein (excision repair cross complementing group 1, ERCC1) can form a structure-specific endonuclease (XPF-ERCC1) complex, which participates in DNA interstrand crosslink (ICL) repair. However, the effect of deubiquitination of XPF protein on DNA repair has not been reported. This experiment focused on the effect of ubiquitin-specific protease 15 (USP15) on the stability of XPF and ICL repair. In this study, data from the protein mass spectrometry and Western blot showed that XPF protein interacted with USP15, which further deubiquitinated XPF protein. The HeLa cell line with USP15 gene knocked out (USP15 KO) was constructed by CRISPR-Cas9, and Western blot analysis showed that the level of XPF protein in USP15 KO cells was lower than that in WT. Colony formation assay demonstrated that the proliferation of USP15 KO cells was significantly reduced in the presence of ICL inducing agents (P<0.01), such as cisplatin and mitomycin C. In conclusion, this study demonstrated that the USP15 was an important regulator of DNA repair, and it promoted ICL repair mediated by XPF-ERCC1 via stabilizing XPF protein. This study provided a theoretical basis for improving the drug resistance of anticancer drugs such as ICL inducer and finding potential new targets for the treatment of tumors.