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Rad52 competes with Ku70/Ku86 for binding to switch region DSB ends to modulate immunoglobulin class switch DNA recombination

  • Author(s): Tat, Connie
  • Advisor(s): Casali, Paolo
  • Hertel, Klemens
  • et al.
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Public License
Abstract

Class switch DNA recombination (CSR) diversifies the biological effector functions of antibodies and is critical for the maturation of the antibody response. This process is initiated by AID-mediated introduction of double-strand DNA breaks (DSBs) in the upstream and downstream switch (S) regions that will be recombined as effected by intervention of Ku70/Ku86 and the non-homologous end joining (NHEJ) DNA repair pathway. However, Ku-deficient B cells undergo CSR, albeit at a reduced rate, suggesting the involvement of other DNA repair mechanisms. We have demonstrated that B cell deficiency of Rad52, a critical component of the homologous recombination (HR) DNA repair pathway, resulted in increased CSR. Conversely, enforced Rad52 expression impaired CSR. This elevation of CSR in Rad52−/− B cells was associated with increased recruitment of Ku70/Ku86 to S region DSB ends. Indeed, Rad52 competed with Ku70/Ku86 for binding to blunt and staggered DSB ends, as shown by in vitro competition using purified Rad52 and Ku70/Ku86 molecules, and chromatin immunoprecipitation (ChIP) assays involving anti-Rad52 antibody (Ab) and anti-Ku70/Ku86 monoclonal antibody (mAb). In Rad52−/− B cells, the increased role of Ku70/Ku86 in S region recombination was emphasized by decreased occurrence of microhomologies in Sμ-Sα and Sμ-Sγ1 junctions, decreased inter-chromosomal c-Myc/IgH translocations and reduced intra-Sμ recombination. Our findings demonstrate that Rad52 binds to S region DSB ends to facilitate a Ku-independent repair pathway, which favors intra-switch (S) region re-joining and can also resolve inter-switch (S) region DSBs, possibly as part of an alternative NHEJ pathway.

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