Leukemias and lymphomas are common cancers that affect human health and longevity. There is evidence that aberrant V(D)J recombination, the process of antigen receptor gene rearrangement in lymphocytes, plays a role in the incidence of chromosome translocation events that lead to these cancers. The V(D)J recombinase, consisting of the RAG-1 and RAG-2 proteins, normally introduces double-strand DNA breaks once the complex recognizes and binds to a pair of recognition signal sequences (RSSs) in the antigen receptor loci. The nicked DNA intermediates generated by RAGs during cleavage have not been thought to be particularly dangerous even though unpaired RSSs as well as RSS-like sequences can be nicked in vitro and are thought to lack the protection of the post-cleavage complex, in which the RAGs remain bound to the DNA ends until joining factors complete the reaction. If the RAGs can generate single-strand nicks at RSS-like sequences in vivo, these lesions may be able to participate in aberrant translocation events. RSS-like sequences have been found near translocation breakpoints in lymphoid tumors and recent work from the Roth lab has shown that RAG-generated nicks are quite reactive in a homologous recombination assay. Thus, RAG-generated nicks at RSS-like sequences could be a major contributor to aberrant rearrangements in lymphoid cells. This hypothesis will be tested by determining 1) the minimal RSS features that support RAG-mediated nicking in vivo; 2) whether nicks are converted to double-strand breaks in vivo; and 3) the effects of a nick-only RAG mutation on immune system development and lymphomagenesis in a knock-in mouse model. ? ?
