Polymerase theta repairs persistent G1-induced DNA breaks in S-phase during class switch recombination

Non-homologous end joining (NHEJ) is the primary pathway for repairing G1 phase-induced DNA double-strand breaks (DSBs) during immunoglobulin heavy chain (Igh) class switch recombination (CSR) in B lymphocytes. In B cells lacking NHEJ (XRCC4) or DSB end protection (SHLD1), end joining during CSR proceeds through an alternative end-joining pathway. Polymerase theta (Pol θ) is widely regarded as a mediator of this pathway, essential for repairing replication-associated DSBs during mitosis when homologous recombination is unavailable. In this study, we examined CSR in primary B cells lacking XRCC4, SHLD1, and/or Pol θ, revealing two repair pathways: Pol θ-independent productive switching and Pol θ-dependent unproductive switching characterized by end resection, inversion and microhomology. Furthermore, we show that Pol θ-mediated repair under NHEJ-deficiency coincides with G1-to-S phase transition and occurs independently of RHINO and PLK1. Thus, in the absence of NHEJ, Pol θ repairs persistent G1-phase DSBs during S-phase rather than mitosis.