NHEJ-deficient DT40 cells have increased levels of immunoglobulin gene conversion: evidence for a double strand break intermediate.
Activation-induced cytidine deaminase (AID) likely initiates immunoglobulin gene-conversion (GC) by deaminating cytidines within the V-region of chicken B-cells. However, the intervening DNA lesion required to initiate GC remains elusive. GC could be initiated by a single strand break or a double strand break (DSB). To distinguish between these possibilities, we examined GC in the chicken DT40 B cell line deficient in non-homologous end joining (NHEJ). It is known that the NHEJ and homologous recombination DNA repair pathways compete for DSBs. In light of this, if a DSB is the major intermediate, deficiency in NHEJ should result in increased levels of GC. Here we show that DNA-PKcs(-/-/-) and Ku70(-/-) DT40 cells had 5- to 10-fold higher levels of GC relative to wildtype DT40 as measured by surface IgM reversion and sequencing of the V-region. These data suggest that a DSB is the major DNA lesion that initiates GC.
Tang
Ephraim S
ES
Department of Immunology, 5265 Medical Sciences Building, University of Toronto, Ontario, Toronto, Canada M5S 1A8.
Martin
Alberto
A
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics; D002460:Cell Line; D002645:Chickens / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005785:Gene Conversion; D017353:Gene Deletion; D005803:Genes, Immunoglobulin; D007075:Immunoglobulin M / Q000378:metabolism; D000072200:Ku Autoantigen
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D007075:Immunoglobulin M; D051747:DNA-Activated Protein Kinase; D000072200:Ku Autoantigen