title: Definition of minimal domains of interaction within the recombination-activating genes 1 and 2 recombinase complex.
abstract: During V(D)J recombination, recognition and cleavage of the recombination signal sequences (RSSs) requires the coordinated action of the recombination-activating genes 1 and 2 (RAG1/RAG2) recombinase complex. In this report, we use deletion mapping and site-directed mutagenesis to determine the minimal domains critical for interaction between RAG1 and RAG2. We define the active core of RAG2 required for RSS cleavage as aa 1-371 and demonstrate that the C-terminal 57 aa of this core provide a dominant surface for RAG1 interaction. This region corresponds to the last of six predicted kelch repeat motifs that have been proposed by sequence analysis to fold RAG2 into a six-bladed beta-propeller structure. Residue W317 within this sixth repeat is shown to be critical for mediating contact with RAG1 and concurrently for stabilizing binding and directing cleavage of the RSS. We also show that zinc finger B (aa 727-750) of RAG1 provides a dominant interaction domain for recruiting RAG2. In all, the data support a model of RAG2 as a multimodular protein that utilizes one of its six faces for establishing productive contacts with RAG1.
authors.affiliation: Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: V
authors.initials: V
authors.lastname: Aidinis
authors.forename: D C
authors.initials: DC
authors.lastname: Dias
authors.forename: C A
authors.initials: CA
authors.lastname: Gomez
authors.forename: D
authors.initials: D
authors.lastname: Bhattacharyya
authors.forename: E
authors.initials: E
authors.lastname: Spanopoulou
authors.forename: S
authors.initials: S
authors.lastname: Santagata
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D011232:Chemical Precipitation; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D016654:Genes, RAG-1 / Q000276:immunology*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D045567:VDJ Recombinases; D016335:Zinc Fingers / Q000276:immunology
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; C499311:RAG2 protein, human; C064655:V(D)J recombination activating protein 2; C082035:Zfa protein, mouse; C068058:Zfb protein, chicken; C064658:RAG-1 protein; D014364:Tryptophan; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases