期刊
CELLULAR AND MOLECULAR LIFE SCIENCES
卷 74, 期 11, 页码 2081-2094出版社
SPRINGER BASEL AG
DOI: 10.1007/s00018-017-2455-7
关键词
CRISPR/Cas9; Xeroderma pigmentosum group F; Nucleotide excision repair; Interstrand crosslink repair; Homologous recombination repair
资金
- German Cancer Aid (Deutsche Krebshilfe e.V.) [111377]
- Heinz and Heide Duerr Foundation [2014-2.2.1/02]
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.
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