title: The N-terminal region of the DNA-dependent protein kinase catalytic subunit is required for its DNA double-stranded break-mediated activation.
abstract: 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.
authors: Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors: Anthony J
authors: AJ
authors: Davis
authors: Kyung-Jong
authors: KJ
authors: Lee
authors: David J
authors: DJ
authors: Chen
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D061987:Sf9 Cells
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen