Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.
Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.
Calsou
Patrick
P
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077, Cedex 4, Toulouse, France. patrick.calsou@ipbs.fr
Delteil
Christine
C
Frit
Philippe
P
Drouet
Jérôme
J
Salles
Bernard
B
D034961:Antigens, Nuclear / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D021122:Protein Subunits; C101467:XRCC4 protein, human; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP