title: The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation.
abstract: DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.
authors.affiliation: Department of Biochemistry & Molecular Biology, University of Calgary, 3300 Hospital Drive, Calgary AB, T2N 4N1, Canada.
authors.forename: Dennis
authors.initials: D
authors.lastname: Merkle
authors.forename: Pauline
authors.initials: P
authors.lastname: Douglas
authors.forename: Greg B G
authors.initials: GB
authors.lastname: Moorhead
authors.forename: Zoya
authors.initials: Z
authors.lastname: Leonenko
authors.forename: Yaping
authors.initials: Y
authors.lastname: Yu
authors.forename: David
authors.initials: D
authors.lastname: Cramb
authors.forename: David P
authors.initials: DP
authors.lastname: Bazett-Jones
authors.forename: Susan P
authors.initials: SP
authors.lastname: Lees-Miller
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D034961:Antigens, Nuclear; D020134:Catalytic Domain; D004249:DNA Damage; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004789:Enzyme Activation; D006801:Humans; D007089:Image Enhancement; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D009690:Nucleic Acid Conformation; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011257:Pregnancy Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D013057:Spectrum Analysis
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011257:Pregnancy Proteins; D000255:Adenosine Triphosphate; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen