title: XRCC1 stimulates polynucleotide kinase by enhancing its damage discrimination and displacement from DNA repair intermediates.
abstract: Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5'-DNA kinase and 3'-phosphatase activities of hPNK can be stimulated by the "scaffold" protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5'-OH termini was only approximately 5-fold tighter than that to identical DNA molecules with 5'-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5'-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5'-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5'-OH termini and those with 5'-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.
authors.affiliation: Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada.
authors.forename: Rajam S
authors.initials: RS
authors.lastname: Mani
authors.forename: Mesfin
authors.initials: M
authors.lastname: Fanta
authors.forename: Feridoun
authors.initials: F
authors.lastname: Karimi-Busheri
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Silver
authors.forename: César A
authors.initials: CA
authors.lastname: Virgen
authors.forename: Keith W
authors.initials: KW
authors.lastname: Caldecott
authors.forename: Carol E
authors.initials: CE
authors.lastname: Cass
authors.forename: Michael
authors.initials: M
authors.lastname: Weinfeld
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D001483:Base Sequence; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; C038518:acrylodan; D015081:2-Naphthylamine; D011116:Polynucleotide 5'-Hydroxyl-Kinase