Distinct effects of DNA-PKcs and Artemis inactivation on signal joint formation in vivo.
The assembly of functional immune receptor genes via V(D)J recombination in developing lymphocytes generates DNA double-stranded breaks intermediates that are repaired by non-homologous end joining (NHEJ). This repair pathway requires the sequential recruitment and activation onto coding and signal DNA ends of several proteins, including the DNA-dependent protein kinase and the nuclease Artemis. Artemis activity, triggered by the DNA-dependent protein kinase, is necessary to process the genes hairpin-sealed coding ends but appears dispensable for the ligation of the reciprocal phosphorylated, blunt-ended signal ends into a signal joint. The DNA-dependent protein kinase is however present on signal ends and could potentially recruit and activate Artemis during signal joint formation. To determine whether Artemis plays a role during the resolution of signal ends during V(D)J recombination, we analyzed the structure of signal joints generated in developing thymocytes during the rearrangement of T cell receptor genes in wild type mice and mice mutated for NHEJ factors. These joints exhibit junctional diversity resulting from N nucleotide polymerization by the terminal nucleotidyl transferase and nucleotide loss from one or both of the signal ends before they are ligated. Our results show that Artemis participates in the repair of signal ends in vivo. Furthermore, our results also show that while the DNA-dependent protein kinase complex protects signal ends from processing, including deletions, Artemis seems on the opposite to promote their accessibility to modifying enzymes. In addition, these data suggest that Artemis might be the nuclease responsible for nucleotide loss from signal ends during the repair process.
Touvrey
Cédric
C
CEA, DSV, DRDC, Laboratoire d'Immunochimie, INSERM U548, Université Joseph Fourier, Grenoble F-38054, France.
Couedel
Chrystelle
C
Soulas
Pauline
P
Couderc
Rachel
R
Jasin
Maria
M
de Villartay
Jean-Pierre
JP
Marche
Patrice N
PN
Jouvin-Marche
Evelyne
E
Candéias
Serge M
SM
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000378:metabolism; D004720:Endonucleases; D004789:Enzyme Activation; D000072200:Ku Autoantigen; D008214:Lymphocytes / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000172:deficiency / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; D004720:Endonucleases; C473337:Dclre1c protein, mouse; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen