The N-terminal Region of the DNA-dependent Protein Kinase Catalytic Subunit Is Required for Its DNA Double-stranded Break-mediated Activation

• Published in: The Journal of biological chemistry 2013-03, Vol.288 (10), p.7037-7046
• Main Authors: Davis, Anthony J., Lee, Kyung-Jong, Chen, David J.
• Format: Article
• Language: English
• Subjects: Animals; Antigens, Nuclear - chemistry; Antigens, Nuclear - genetics; Antigens, Nuclear - metabolism; Blotting, Western; CHO Cells; Cricetinae; Cricetulus; DNA - genetics; DNA - metabolism; DNA and Chromosomes; DNA Breaks, Double-Stranded; DNA Damage; DNA Damage Response; DNA Double-stranded Breaks; DNA Repair; DNA-Activated Protein Kinase - chemistry; DNA-Activated Protein Kinase - genetics; DNA-Activated Protein Kinase - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; DNA-PKcs; DNA-Protein Interaction; Enzyme Activation; HeLa Cells; Humans; Kinase Activity; Ku Autoantigen; Ku70/80; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Molecular Biology; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Binding; Protein Multimerization; Sf9 Cells
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.434498

DNA-dependent protein kinase (DNA-PK) plays an essential role in the repair of DNA double-stranded breaks (DSBs) mediated by the nonhomologous end-joining pathway. DNA-PK is a holoenzyme consisting of a DNA-binding (Ku70/Ku80) and catalytic (DNA-PKcs) subunit. DNA-PKcs is a serine/threonine protein kinase that is recruited to DSBs via Ku70/80 and is activated once the kinase is bound to the DSB ends. In this study, two large, distinct fragments of DNA-PKcs, consisting of the N terminus (amino acids 1–2713), termed N-PKcs, and the C terminus (amino acids 2714–4128), termed C-PKcs, were produced to determine the role of each terminal region in regulating the activity of DNA-PKcs. N-PKcs but not C-PKcs interacts with the Ku-DNA complex and is required for the ability of DNA-PKcs to localize to DSBs. C-PKcs has increased basal kinase activity compared with DNA-PKcs, suggesting that the N-terminal region of DNA-PKcs keeps basal activity low. The kinase activity of C-PKcs is not stimulated by Ku70/80 and DNA, further supporting that the N-terminal region is required for binding to the Ku-DNA complex and full activation of kinase activity. Collectively, the results show the N-terminal region mediates the interaction between DNA-PKcs and the Ku-DNA complex and is required for its DSB-induced enzymatic activity. Background: The Ku70/80-DNA complex recruits DNA-PKcs to DSBs and results in activation of DNA-PKcs kinase activity. Results: Truncation fragments of DNA-PKcs show that different regions of the protein are required for complete functionality of DNA-PKcs. Conclusion: The N-terminal region of DNA-PKcs is required for its ability to interact with the Ku-DNA complex and its full activation. Significance: We provide insights into the biochemical mechanism required for DNA-PKcs activation.