Elucidating Pathways of Classical and Alternative Pathways of Non-Homologous End-Joining in B-Lymphocytes

Abstract

DNA double-stranded breaks (DSBs), if not properly repaired, drive genomic instability. Accordingly, mammalian cells employ classical non-homologous end-joining (C-NHEJ) to repair DSBs generated throughout interphase, including those generated during V(D)J recombination in G1-phase progenitor lymphocytes and immunoglobublin heavy-chain Class Switch Recombination (CSR) in activated mature B-lymphocytes. The Ku70/Ku80 complex, which senses DSBs, and the XRCC4/Ligase4 complex, which joins them, are “core” C-NHEJ factors required for joining of most known DSB ends by this pathway. In cells deficient for core C-NHEJ, alternative end-joining (A-EJ) pathway(s) join DSBs in cycling cells, including those generated during CSR. Potential contributions of candidate factors to C-NHEJ and A-EJ pathways, as well as the relative contributions of C-NHEJ and A-EJ during the G1 cell-cycle phase, were unknown. We first investigated the contribution of the PAXX repair factor to C-NHEJ. We found that PAXX is dispensable for V(D)J recombination and for normal ionizing radiation resistance. However, we further found that combined deficiency for PAXX and XLF abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure in G1-arrested pro-B lines. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro–B-cell lines. We further investigated A-EJ activity in G1-arrested pro-B cell lines. In this context, we found that Ku excludes A-EJ of both RAG-generated and general DSBs. Thus, Cas9 DSBs can robustly re-ligate as well as translocate to RAG-initiated DSBs in WT and Ku70-deficient, but not Lig4-deficient, lines. Strikingly, deletion of Ku70 in Ligase4-deficient cells restores A-EJ to Ku70-deficient levels, indicating that Ku70 blocks A-EJ in this context. Thus, our findings indicate that the G1-phase substantially contributes to restricting V(D)J joining to C-NHEJ. Finally, we tested whether the XRCC1 single strand break repair factor had A-EJ functions in the absence of Ku70 during CSR. While loss of XRCC1 in the absence of Ku70 has no impact on end-joining efficiency during CSR, XRCC1 has a modest role in promoting direct join usage for both switch-switch junctions as well as junctions between general DSBs. Thus, we define an impact of XRCC1 on A-EJ during CSR.