53BP1 orchestrates sequence feature of RAG targets to balance DNA repair outcomes during V(D)J recombination.

RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Loss of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. This phenotype is partially recapitulated by RIF1 or Shieldin depletion, implicating the 53BP1-mediated end-joining axis. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, fully reverts 53BP1 deficiency-mediated increase of junctional MH during cRSS recombination. RAG mutant and RSS score analyses indicate that the sequence strength of RAG targets dictates the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair at RAG-cleaved on- and off-targets during V(D)J recombination.