Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4.
The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; "MH-mediated" joins) or no homologies ("direct" joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via "alternative" end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.
Boboila
Cristian
C
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
Yan
Catherine
C
Wesemann
Duane R
DR
Jankovic
Mila
M
Wang
Jing H
JH
Manis
John
J
Nussenzweig
Andre
A
Nussenzweig
Michel
M
Alt
Frederick W
FW
D000818:Animals; D034961:Antigens, Nuclear / Q000276:immunology / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000276:immunology / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D017578:Immunoglobulin Class Switching; D007136:Immunoglobulins / Q000235:genetics* / Q000378:metabolism; D000072200:Ku Autoantigen; D051379:Mice
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D007136:Immunoglobulins; C000607893:LIG4 protein, human; C101467:XRCC4 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP