B cell Rad52 deficiency enhances immunoglobulin class switching through increased recruitment of Ku70/Ku86 to S region double-strand DNA breaks (IRM10P.618)

Abstract

Class switch DNA recombination (CSR) diversifies the biological effector functions of antibodies and is critical for the maturation of the immune response. It is initiated by AID-mediated generation of DSBs in the switch (S) regions that will undergo recombination, mainly through Ku70/Ku86-mediated nonhomologous end-joining. It has been shown, however, that Ku-deficient B cells can undergo CSR, suggesting that other DNA repair mechanisms are at work. Herein, we show that deficiency of Rad52, an important component of the homologous recombination DNA repair pathway, resulted in significantly increased CSR and is associated with increased recruitment of Ku70/Ku86 to the S region DSB ends. In Rad52-/- B cells, the increased role of Ku70/Ku86 in S region DNA recombination was further emphasized by decreased occurrence of microhomologies in S-S junctions and inter-chromosomal c-Myc/IgH translocations. Conversely, enforced expression of Rad52 in B cells impaired CSR and decreased Ku70/Ku86 recruitment to S region DNA, as revealed by ChIP assays. Accordingly, EMSAs using Sμ probes with blunt-ends or short overhangs, revealed that in CSR, Ku70/Ku86 and Rad52 bind to different types of DSB ends. Our findings show that Rad52 modulates CSR by binding to S region DSB ends. This may facilitate a Ku-independent repair pathway, which would resolve AID-mediated S region DSBs, thereby explaining the residual CSR that has been shown to occur in a conditional Ku70/Ku86 B cell knockout.