ATM and 53BP1 regulate alternative end joining-mediated V(D)J recombination

The critical role of the alternative end joining (AEJ) repair mechanism in V(D)J recombination for B and T lymphocyte development, particularly in the absence of the key non-homologous end joining (NHEJ) component Ku70, is not well understood. AEJ involves functionally redundant factors such as Parp1 vs Pol? and Lig3 vs Lig1, making it unclear which factors are responsible for repairing V(D)J recombination. Additionally, the regulatory mechanism of AEJ remains enigmatic. In this study, we utilized the murine Ig? antigen locus as a model and employed gene editing and HTGTS-Seq pipelines to systematically evaluate potential factors involved in V(D)J recombination. We assessed various aspects of recombination, including efficiency, V gene usage, end resection, joining fidelity, repair patterns, and inter-chromosome translocation. Our findings highlight the Parp1-XRCC1-Lig3 axis as a key mediator of V(D)J recombination, regulated by 53BP1 and ATM. This study advances our understanding of AEJ in DNA damage repair and contributes to our knowledge of lymphocyte development in immunology. Overall design: Various murine proB cells including WT, Ku70-/-, Ku70-/- 53BP1-/-, Ku70-/- XRCC1-/-, Ku70-/- Parp1-/-, Ku70-/- ATM-/-, Ku70-/- Exo1-/-, etc, with/without indicated inhibitors were arrested by STI-571 to induce V(D)J recombination, the V-J recombination in IgK locus were systematically captured using JK1CE and JK1SE by HTGTS.