RAG Proteins Shepherd Double-Strand Breaks to a Specific Pathway, Suppressing Error-Prone Repair, but RAG Nicking Initiates Homologous Recombination

The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex, making ends available to alternative DNA repair pathways

V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC’s ability to restrict repair pathway choice