Interplay between Cernunnos-XLF and nonhomologous end-joining proteins at DNA ends in the cell.
Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends.
Wu
Peï-Yu
PY
Institut de Pharmacologie et de Biologie Structurale, CNRS-Université de Toulouse, UMR 5089, Toulouse, and INSERM, Hôpital Necker-Enfants Malades, U768, Unité Développement Normal et Pathologique du Système Immunitaire, F-75015 Paris, France.
Frit
Philippe
P
Malivert
Laurent
L
Revy
Patrick
P
Biard
Denis
D
Salles
Bernard
B
Calsou
Patrick
P
D018929:Cell Culture Techniques; D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation
D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C508551:NHEJ1 protein, human; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D011088:DNA Ligases; D045643:DNA Repair Enzymes; D000072481:DNA Ligase ATP