title: A noncatalytic function of the ligation complex during nonhomologous end joining.
abstract: Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)-DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)-X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4-Cer-XLF complexes on DNA ends.
authors: Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31077, Cedex 4, France.
authors: Jessica
authors: J
authors: Cottarel
authors: Philippe
authors: P
authors: Frit
authors: Oriane
authors: O
authors: Bombarde
authors: Bernard
authors: B
authors: Salles
authors: Aurélie
authors: A
authors: Négrel
authors: Stéphanie
authors: S
authors: Bernard
authors: Penny A
authors: PA
authors: Jeggo
authors: Michael R
authors: MR
authors: Lieber
authors: Mauro
authors: M
authors: Modesti
authors: Patrick
authors: P
authors: Calsou
mesh_terms: D002474:Cell-Free System / Q000378:metabolism; D002478:Cells, Cultured; D004249:DNA Damage; D059766:DNA End-Joining Repair / Q000502:physiology*; D004265:DNA Helicases / Q000378:metabolism / Q000502:physiology; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000378:metabolism / Q000502:physiology; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D042822:Genomic Instability; D020035:Holoenzymes; D006801:Humans; D000072200:Ku Autoantigen; D010766:Phosphorylation
chemical_list: D004268:DNA-Binding Proteins; D020035:Holoenzymes; C000607893:LIG4 protein, human; C508551:NHEJ1 protein, human; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases; D045643:DNA Repair Enzymes; D000072481:DNA Ligase ATP