A double-strand break can trigger immunoglobulin gene conversion

Abstract

All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSBinduced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries.