Molecular mechanisms of the RAG recombinase in V(D)J recombination and disease

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ABSTRACT A hallmark of vertebrate immunity is the diverse repertoire of antigen-receptor genes that results from combinatorial splicing of gene coding segments by V(D)J recombination, which cleaves and splices variable (V), diversity (D) and joining (J) non-contiguous immunoglobulin (Ig) segments in the genome. The critical cleavage step in V(D)J recombination is executed by the lymphocyte specific enzyme containing the multi-domain proteins recombination-activating gene 1 and 2 (RAG1-RAG2). The RAG recombinase recognizes specific recombination signal sequences (RSSs) flanking the 3' end of the V, D, and J segments, which are composed of a conserved heptamer, a spacer of either 12 or 23 base pairs, and a conserved nonamer. These RSSs are designated as 12-RSS or 23-RSS after the length of the spacer. Splicing can only occur between one gene coding segment flanked by a 12-RSS and another segment flanked by a 23-RSS, establishing the 12/23 rule. Because V, D and J segments are flanked by different RSSs such as in the IgH locus, the 12/23 rule helps to ensure recombination between V, D and J, but not within homotypic gene segments. The RAG complex catalyzes two consecutive reactions, nicking (strand cleavage) and hairpin formation (strand transfer), without dissociation, generating cleaved RSSs and coding end hairpins. Subsequently, proteins in the classical nonhomologous end joining (NHEJ) DNA repair pathway are recruited to the RAG complex to process and join the coding segments. Human RAG mutations are associated with a spectrum of genetic disorders ranging from severe combined immunodeficiency (SCID) to milder variants, such as Omenn syndrome and RAG deficiency with γδ T cell expansion, granuloma formation, or maternofetal engraftment. Aberrant V(D)J recombination is an important mechanism responsible for chromosomal translocations in lymphoid malignancies. RAG genes are supposed to be active only during development. RAG1 and RAG2 re-expression is often linked to autoimmune states and cancers, such as in systemic lupus erythematosus, colorectal cancer and colon cancer. Here we propose a series of structural and functional studies on the RAG recombinase using cryo-electron microscopy and crystallography. A molecular understanding on the functions and regulatory mechanisms of the RAG complex will contribute to the understanding and the potential therapeutic strategies for these human diseases.