title: The interaction between polynucleotide kinase phosphatase and the DNA repair protein XRCC1 is critical for repair of DNA alkylation damage and stable association at DNA damage sites.
abstract: XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.
authors.affiliation: Radiation Oncology Research Laboratory, Department of Radiation Oncology and The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Julie
authors.initials: J
authors.lastname: Della-Maria
authors.forename: Muralidhar L
authors.initials: ML
authors.lastname: Hegde
authors.forename: Daniel R
authors.initials: DR
authors.lastname: McNeill
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Miaw-Sheue
authors.initials: MS
authors.lastname: Tsai
authors.forename: Tom
authors.initials: T
authors.lastname: Ellenberger
authors.forename: David M
authors.initials: DM
authors.lastname: Wilson
authors.forename: Sankar
authors.initials: S
authors.lastname: Mitra
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
mesh_terms: D002470:Cell Survival; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017434:Protein Structure, Tertiary; D013912:Threonine / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: C438188:APTX protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D013912:Threonine; C530380:PNKP protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); D011088:DNA Ligases; D045643:DNA Repair Enzymes