XRCC1 stimulates polynucleotide kinase by enhancing its damage discrimination and displacement from DNA repair intermediates.
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.
Mani
Rajam S
RS
Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada.
Fanta
Mesfin
M
Karimi-Busheri
Feridoun
F
Silver
Elizabeth
E
Virgen
César A
CA
Caldecott
Keith W
KW
Cass
Carol E
CE
Weinfeld
Michael
M
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
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