title: Role of interaction of XPF with RPA in nucleotide excision repair.
abstract: Nucleotide excision repair (NER) is a very important defense system against various types of DNA damage, and it is necessary for maintaining genomic stability. The molecular mechanism of NER has been studied in considerable detail, and it has been shown that proper protein-protein interactions among NER factors are critical for efficient repair. A structure-specific endonuclease, XPF-ERCC1, which makes the 5' incision in NER, was shown to interact with a single-stranded DNA binding protein, RPA. However, the biological significance of this interaction was not studied in detail. We used the yeast two-hybrid assay to determine that XPF interacts with the p70 subunit of RPA. To further examine the role of this XPF-p70 interaction, we isolated a p70-interaction-deficient mutant form of XPF that contains a single amino acid substitution in the N-terminus of XPF by the reverse yeast two-hybrid assay using randomly mutagenized XPF. The biochemical properties of this RPA-interaction-deficient mutant XPF-ERCC1 are very similar to those of wild-type XPF-ERCC1 in vitro. Interestingly, expression of this mutated form of XPF in the XPF-deficient Chinese hamster ovary cell line, UV41, only partially restores NER activity and UV resistance in vivo compared to wild-type XPF. We discovered that the RPA-interaction-deficient XPF is not localized in nuclei and the mislocalization of XPF-ERCC1 prevents the complex from functioning in NER.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Fisher
authors.forename: Mika
authors.initials: M
authors.lastname: Bessho
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Wakasugi
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Tadayoshi
authors.initials: T
authors.lastname: Bessho
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage / Q000235:genetics*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016296:Mutagenesis; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; C095888:xeroderma pigmentosum group F protein