title: Replication protein A confers structure-specific endonuclease activities to the XPF-ERCC1 and XPG subunits of human DNA repair excision nuclease.
abstract: XPF-ERCC1 and XPG proteins are nucleases that are involved in human nucleotide excision repair. In this study, we characterized the structure-specific junction-cutting activities of both nucleases using DNA substrates containing a bubble or loop structure. We found that the junction-cutting activities of XPF-ERCC1 and XPG were greatly stimulated by human replication protein A (RPA), while heterologous single-stranded DNA-binding proteins could not substitute for human RPA. To test for specific interaction between RPA and XPF-ERCC1 as is known to occur between RPA and XPG, we employed a pull-down assay with immobilized "bubble" substrate. We found that the binding of XPF-ERCC1 complex to the bubble substrate was enhanced by RPA, suggesting a possible mechanism for RPA in the excision nuclease system, that is the targeting of the nuclease subunits to their specific sites of action. Furthermore, the RPA-promoted junction cutting by XPF-ERCC1 and XPG nucleases was observed with "loop" substrates as well, raising the possibility that XPF-ERCC1, XPG, and RPA may function in removing loop structures from DNA, independent of the other subunits of the human excinuclease.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
authors.forename: T
authors.initials: T
authors.lastname: Matsunaga
authors.forename: C H
authors.initials: CH
authors.lastname: Park
authors.forename: T
authors.initials: T
authors.lastname: Bessho
authors.forename: D
authors.initials: D
authors.lastname: Mu
authors.forename: A
authors.initials: A
authors.lastname: Sancar
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004260:DNA Repair; D004261:DNA Replication; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D051716:Replication Protein A; D016324:Sequence Tagged Sites; D013379:Substrate Specificity; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5
chemical_list: D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004720:Endonucleases; D008190:Lyases; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D051716:Replication Protein A; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5; C497167:RPA1 protein, human; C071447:ERCC1 protein, human