DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination.
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining C(H) exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs-deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs(-/-) B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.
Franco
Sonia
S
Howard Hughes Medical Institute, the Children's Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Murphy
Michael M
MM
Li
Gang
G
Borjeson
Tiffany
T
Boboila
Cristian
C
Alt
Frederick W
FW
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D015342:DNA Probes / Q000235:genetics; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases; D042822:Genomic Instability; D017578:Immunoglobulin Class Switching; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014178:Translocation, Genetic
D015342:DNA Probes; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497264:Prkdc protein, mouse; D004720:Endonucleases; C473337:Dclre1c protein, mouse