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 DSB-induced 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.

Physiological Roles of DNA Double-Strand Breaks

“…AID-mediated deamination on the opposite strand introduces a second nick in close proximity, thereby generating a staggered DSB [ 89 , 96 ]. Processing of these overhangs forms blunt free DNA ends which are permissive for shuffling [ 87 , 97 ].…”

Diversification of AID/APOBEC-like deaminases in metazoa: multiplicity of clades and widespread roles in immunity

“…This process is central to class-switch recombination ( 24 ). Additionally, at least in some jawed vertebrates, AID-catalyzed deamination causes somatic hypermutation and/or gene conversion, which drives antibody diversification, for instance as part of affinity maturation during an immune response ( 28 , 29 ). The AADs expressed in agnathan (jawless vertebrate) lymphocytes (PmCDA1 and PmCDA2) are implicated in gene conversion-based diversification of their antigen receptors ( 20 ), the variable lymphocyte receptors (VLRs), which are structurally unrelated to antibodies (review ref.…”

Integrity of immunoglobulin variable regions is supported by GANP during AID-induced somatic hypermutation in germinal center B cells

“…In contrast, NHEJ roughly reconnects broken ends, which probably induces frameshifts and jeopardizes the whole structure of the IgV region. This could explain why the IgV region must be protected from NHEJ ( 50 ). The role of GANP in protecting the IgV genomic structure may be part of a more global role in protecting DNA from damage and is probably associated with transcription-coupled DNA repair.…”