title: XPF knockout via CRISPR/Cas9 reveals that ERCC1 is retained in the cytoplasm without its heterodimer partner XPF.
abstract: 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.
authors.affiliation: Clinic and Policlinic for Dermatology and Venereology, University Medical Centre Rostock, Strempelstrasse 13, 18057, Rostock, Germany.|Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany.
authors.forename: Janin
authors.initials: J
authors.lastname: Lehmann
authors.affiliation: Clinic and Policlinic for Dermatology and Venereology, University Medical Centre Rostock, Strempelstrasse 13, 18057, Rostock, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Seebode
authors.affiliation: Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Smolorz
authors.affiliation: Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Schubert
authors.affiliation: Clinic and Policlinic for Dermatology and Venereology, University Medical Centre Rostock, Strempelstrasse 13, 18057, Rostock, Germany. steffen.emmert@med.uni-rostock.de.|Department of Dermatology, Venereology and Allergology, University Medical Centre Goettingen, Robert-Koch-Strasse 40, 37075, Goettingen, Germany. steffen.emmert@med.uni-rostock.de.
authors.forename: Steffen
authors.identifier: 0000-0001-5630-4204
authors.initials: S
authors.lastname: Emmert
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D059765:Homologous Recombination / Q000235:genetics; D006801:Humans; D055503:Protein Multimerization / Q000187:drug effects / Q000528:radiation effects; D014118:Toxins, Biological / Q000378:metabolism; D014307:Trioxsalen / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D004268:DNA-Binding Proteins; D014118:Toxins, Biological; C095888:xeroderma pigmentosum group F protein; C071447:ERCC1 protein, human; D004720:Endonucleases; D002945:Cisplatin; D014307:Trioxsalen
keywords: CRISPR/Cas9; Homologous recombination repair; Interstrand crosslink repair; Nucleotide excision repair; Xeroderma pigmentosum group F