XPF/ERCC1异二聚复合物基本上参与核苷酸切除修复(NER),链间交联(ICL),和双股断裂修复。XPF的缺陷会导致严重的疾病,例如色素性干皮病(XP)。直到现在,XP-F患者细胞已用于功能分析。由于XPF/ERCC1复合体的多个角色,这些患者细胞保留至少一个全长等位基因和残余修复能力。尽管XPF/ERCC1复合物对人体具有重要的功能,我们在胎儿肺成纤维细胞(MRC5Vi细胞)中使用CRISPR/Cas9技术成功产生了XPF完全缺失的活的永生化人类XPF敲除细胞系.这些细胞对UVC的敏感性明显增加,顺铂,和补骨脂素被UVA激活,以及通过报告基因测定评估的NER和ICL修复的修复能力降低。使用新产生的敲除细胞,我们可以证明,人类XPF明显参与同源重组修复(HRR),但对于非同源末端连接(NHEJ)则不重要。值得注意的是,在XPF敲除细胞的细胞核中检测不到ERCC1,表明功能性XPF/ERCC1异二聚体允许ERCC1进入细胞核的必要性。野生型XPF的过表达可以逆转这种效应以及修复缺陷。
The XPF/ERCC1 heterodimeric complex is essentially involved in nucleotide excision repair (NER), interstrand crosslink (ICL), and double-strand break repair. Defects in XPF lead to severe diseases like xeroderma pigmentosum (XP). Up until now, XP-F patient cells have been utilized for functional analyses. Due to the multiple roles of the XPF/ERCC1 complex, these patient cells retain at least one full-length allele and residual repair capabilities. Despite the essential function of the XPF/ERCC1 complex for the human organism, we successfully generated a viable immortalised human XPF knockout cell line with complete loss of XPF using the CRISPR/Cas9 technique in fetal lung fibroblasts (MRC5Vi cells). These cells showed a markedly increased sensitivity to UVC, cisplatin, and psoralen activated by UVA as well as reduced repair capabilities for NER and ICL repair as assessed by reporter gene assays. Using the newly generated knockout cells, we could show that human XPF is markedly involved in homologous recombination repair (HRR) but dispensable for non-homologous end-joining (NHEJ). Notably, ERCC1 was not detectable in the nucleus of the XPF knockout cells indicating the necessity of a functional XPF/ERCC1 heterodimer to allow ERCC1 to enter the nucleus. Overexpression of wild-type XPF could reverse this effect as well as the repair deficiencies.