title: 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.
abstract: The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, 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 transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-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 binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage in trans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.
authors.affiliation: Department of Medical Microbiology and Immunology, Creighton University, School of Medicine, Omaha, Nebraska 68178, USA. pswanson@creighton.edu
authors.forename: P C
authors.initials: PC
authors.lastname: Swanson
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D015321:Gene Rearrangement / Q000235:genetics*; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D009154:Mutation; D009687:Nuclear Proteins; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D011995:Recombination, Genetic / Q000235:genetics; D045567:VDJ Recombinases
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007136:Immunoglobulins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D021122:Protein Subunits; C499311:RAG2 protein, human; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases