Polymerase theta-mediated end-joining repairs persistent G1-induced DNA breaks in S/G2.

In homologous recombination deficient cells, polymerase theta (Pol ?)-mediated end-joining (TMEJ) repairs replicative DNA breaks during mitosis through interactions with RHINO and TOPBP1. V(D)J recombination and class switch recombination (CSR) rely on DNA breakage in G1-phase B lymphocytes and repair by non-homologous end-joining (NHEJ). In this study, we assayed CSR in primary B cells deficient in XRCC4, SHLD1, and Pol ?. We found that B cells deficient in both XRCC4 and Pol ?, or SHLD1 and Pol ?, exhibit an increased rate of IgH breaks, while retaining CSR levels similar to those of B cells deficient in XRCC4 or SHLD1. We show that in XRCC4- or SHLD1-deficient B cells, Pol ? promotes the formation of aberrant recombination products characterized by exacerbated double-strand break (DSB) end resection, inversion and microhomology, and that these end-joining events occur independently of RHINO. Furthermore, we find that TMEJ of RAG-generated DSBs is also independent of RHINO and concomitant to S/G2 entry. Based on these results, we propose that in the absence of NHEJ, TMEJ repairs persistent G1-phase DSBs in S/G2, rather than in mitosis. Overall design: CD19-enriched murine splenic B cells proficient or deficient in Xrcc4 (with CD21-Cre), Polq, Shld1, or in combination were stimulated for IgH class switch reocmbination and subjected to LAM-HTGTS JoinT-seq using Smu bait priming as described in PMIDs: 26308889, 27031497, 34006647.