XRCC1 Stimulates Polynucleotide Kinase by Enhancing Its Damage Discrimination and Displacement from DNA Repair Intermediates

• Published in: The Journal of biological chemistry Vol. 282; no. 38; pp. 28004 - 28013
• Main Authors: Mani, Rajam S., Fanta, Mesfin, Karimi-Busheri, Feridoun, Silver, Elizabeth, Virgen, César A., Caldecott, Keith W., Cass, Carol E., Weinfeld, Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.09.2007; American Society for Biochemistry and Molecular Biology
• Subjects: 2-Naphthylamine - analogs & derivatives; 2-Naphthylamine - chemistry; Base Sequence; DNA Damage; DNA Repair; DNA-Binding Proteins - physiology; Fluorescence Resonance Energy Transfer; Humans; Kinetics; Models, Biological; Molecular Conformation; Molecular Sequence Data; Polynucleotide 5'-Hydroxyl-Kinase - metabolism; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Substrate Specificity; X-ray Repair Cross Complementing Protein 1
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M704867200

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 ∼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.