Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK.
DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.
Costantini
Silvia
S
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy.
Woodbine
Lisa
L
Andreoli
Lucia
L
Jeggo
Penny A
PA
Vindigni
Alessandro
A
D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D051379:Mice; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D009687:Nuclear Proteins; C101467:XRCC4 protein, human; C511788:XRCC4 protein, mouse; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C497264:Prkdc protein, mouse; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP