The DDE motif in RAG-1 is contributed in trans to a single active site that catalyzes the nicking and transesterification steps of V(D)J recombination

The process of assembling immunoglobulin and T-cell receptor genes from var iable (V), diversity (D), and joining (J) gene segments, called V(D)J recom bination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first- strand nicking, followed by hairpin formation via direct transesterificatio n. In vitro, these reactions minimally proceed in discrete protein-DNA comp lexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a sin gle recombination signal sequence. Recently, a DDE triad of carboxylate res idues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral i ntegrase active sites. To investigate which RAG-1 subunit contributes the r esidues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing R AG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA bin ding. The results show that the DDE triad is contributed to a single recomb inase active site, which catalyzes the nicking and transesterification step s of V(D)J recombination by a single RAG-1 subunit opposite the one bound t o the nonamer of the recombination signal undergoing cleavage (cleavage in trans). The implications of a trans cleavage mode observed in these complex es on the organization of the V(D)J synaptic complex are discussed.