| Antigen recognition by the adaptive immune system is mediated by an extensive repertoire of immunoglobulin and T cell receptors that are generated through the rearrangement of a limited number of V (variable), D (diversity) and J (joining) gene segments. The broad array of antigen binding pockets is determined by the combinatorial diversity produced through the recombination of these gene segments as well as by the introduction of junctional diversity at the sites where these genetic elements are joined. This recombination process is directed by the lymphoid-specific proteins RAG1 and RAG2 that form the core components of the V(D)J recombinase. In this work, the nuclease properties of RAG1/RAG2 are explored. It is demonstrated that the RAG recombinase functions as both a sequence-specific DNA nuclease capable of introducing the double-strand breaks that initiate the recombination process as well as a sequence-independent, structure specific nuclease that processes many of the 3' flap/branched intermediates that are likely central to the generation of junctional diversity during the late stages of the rearrangement reaction. A mechanistic link is proposed between these seemingly disparate nuclease activities. The bacterial transposase Tn 10 can also nick 3' overhangs hence highlighting mechanistic conservation between the active sites of the V(D)J recombinase and of other transposases.;The indispensable role of RAG1/RAG2 in the recombination process has been previously revealed by targeted deletion of the genes in mice. Accordingly, in humans, null mutations in RAG1/RAG2 abolish initiation of recombination and lead to severe combined immunodeficiency (SCID) with a total absence of mature lymphocytes. We demonstrate here that Omenn syndrome, a variant of SCID characterized by oligoclonal T-lymphocytes and an absence of detectable circulating B-lymphocytes, results from multiple genetic lesions that render RAG1/RAG2 only partially active. These pleiotropic lesions include mutations within the RAG1 nonamer-binding domain, mutations within the kelch-repeats of RAG2 that likely form the core of a beta-propeller fold and frameshift mutations in the 5' end of RAG1 that are compensated by internal methionine usage leading to the generation of truncated/partially active proteins. In all, the data offer insights into the biology of RAG1/RAG2 in both V(D)J recombination and immunodeficiency. |
