Interplay between Ku, Artemis, and the DNA-dependent protein kinase catalytic subunit at DNA ends.
Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV.XRCC4.Cernunnos-XLF complex. The Artemis protein exhibits exonuclease and endonuclease activities that are believed to be involved in the processing of a subclass of DSB. Here, we have analyzed the interactions of Artemis and nonhomologous end-joining pathway proteins both in a context of human nuclear cell extracts and in cells. DSB-inducing agents specifically elicit the mobilization of Artemis to damaged chromatin together with DNA-PK and XRCC4/ligase IV proteins. DNA-PKcs is necessary for the loading of Artemis on damaged DNA and is the main kinase that phosphorylates Artemis in cells damaged with highly efficient DSB producers. Under kinase-preventive conditions, both in vitro and in cells, Ku-mediated assembly of DNA-PK on DNA ends is responsible for a dissociation of the DNA-PKcs. Artemis complex. Conversely, DNA-PKcs kinase activity prevents Artemis dissociation from the DNA-PK.DNA complex. Altogether, our data allow us to propose a model in which a DNA-PKcs-mediated phosphorylation is necessary both to activate Artemis endonuclease activity and to maintain its association with the DNA end site. This tight functional coupling between the activation of both DNA-PKcs and Artemis may avoid improper processing of DNA.
Drouet
Jérôme
J
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 Route de Narbonne, 31077 Toulouse, Cedex 4, France.
Frit
Philippe
P
Delteil
Christine
C
de Villartay
Jean-Pierre
JP
Salles
Bernard
B
Calsou
Patrick
P
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear / Q000737:chemistry / Q000502:physiology*; D020134:Catalytic Domain; D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004720:Endonucleases; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D010766:Phosphorylation
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen