| 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 (RSS) adjoining V, D, and J gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends in a post-cleavage complex (PCC), which is critical for their proper joining by "classical" nonhomologous end-joining (NHEJ). Certain mutations in RAG1/2 destabilize the post-cleavage complex, allowing DNA ends to access inappropriate repair pathways such as "alternative" NHEJ, an error-prone pathway implicated in chromosomal translocations. The post-cleavage complex 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 RAGsignal end complex, I hypothesized that nonconsensus heptamer sequences might affect post-cleavage complex stability.;I find that certain nonconsensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the post-cleavage complex, allowing coding and signal ends to be repaired by nonstandard pathways, including "alternative" nonhomologous end-joining. These data suggest that some nonconsensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the post-cleavage complex's ability to restrict repair pathway choice. I also found a novel regulatory function for the C-terminus of RAG2. Full-length RAG2 suppresses uncoupled cleavage at a single RSS by the V(D)J recombinase and stabilizes the RAG post-cleavage complex in vitro. |
