53BP1 orchestrates sequence feature of RAG targets to balance DNA repair outcomes during V(D)J recombination [ChIP-seq]

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. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.