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

G 0 -G 1 phase alternative end joining (A-EJ) is a recently defined mutagenic pathway characterized by resected deletion and translocation joints that are predominantly direct and are distinguished from A-EJ in cycling cells that rely much more on microhomology-mediated end joining (MMEJ). Using chemical and genetic approaches, we systematically evaluate potential A-EJ factors and DNA damage response (DDR) genes to support this mechanism by mapping the repair fates of RAG1/2-initiated double-strand breaks in the context of Igκ locus V-J recombination and chromosome translocation. Our findings highlight a polymerase theta-independent Parp1-XRCC1/LigIII axis as central A-EJ components, supported by 53BP1 in the context of an Ataxia-telangiectasia mutated (ATM)-activated DDR. Mechanistically, we demonstrate varied changes in short-range resection, MMEJ, and translocation, imposed by compromising specific DDR activities, which include polymerase alpha, Ataxia-telangiectasia and Rad3-related (ATR), DNA2, and Mre11. This study advances our understanding of DNA damage repair within the 53BP1 regulatory domain and the RAG1/2 postcleavage complex.