The impact of DNA-PKcs in V(D)J recombination of mouse IgK antigen locus
DNA-PKcs is a critical component of the non-homologous end joining (NHEJ) pathway, playing a role in the re-ligation of DNA double-strand breaks (DSBs) generated by RAG1/2 during V(D)J recombination in antigen loci. When NHEJ is deficient, DSBs can be repaired through alternative end joining (A-EJ). In this study, we investigated the consequences of DNA-PKcs deletion on V-J recombination at the IgK antigen locus. Our results reveal that DNA-PKcs deletion leads to inefficient V-J recombination, exhibiting phenotypes such as reduced efficiency, increased end resection, and decreased micro-insertions in the repair pattern. These findings are consistent with previously reported functions of Ku70, another key player in NHEJ. However, unlike Ku70, additional deletion of DNA-PKcs does not restore the efficiency of repair in the absence of Lig4. Instead, it reduces both end resection and microhomology-mediated repair. These observations highlight the context-dependent role of DNA-PKcs in end processing. The presence or absence of Lig4 appears to influence the function of DNA-PKcs, further emphasizing the intricate interplay between repair factors in the DNA damage response. Overall, our findings provide insights into the distinct functions of DNA-PKcs and Ku70 in V(D)J recombination and underscore the complex regulatory mechanisms underlying repair efficiency in different genetic backgrounds. Overall design: Various murine proB cells including WT, Lig4-/-, PKcs-/-, Ku70-/-, Lig4-/- PKcs-/- and Lig4-/- Ku70 (with/without DNA-PKcs inhibitors Nu7441 and Nu7026)  were arrested by STI-571 to induce V(D)J recombination, the V-J recombination in IgK locus were systematically captured using all four JK baits (JK1, JK2, JK4 and JK5) by HTGTS.