Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase.
The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG proteins requires a divalent metal ion but does not involve a covalent protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two chemical steps of V(D)J cleavage (nicking and hairpin formation) is severely impaired. Because both chemical steps are affected by the same mutations, a single active site appears responsible for both reactions. Two independent lines of evidence demonstrate that at least two of these acidic residues are directly involved in coordinating a divalent metal ion: The substitution of Cys for Asp allows rescue of some catalytic function, whereas an alanine substitution is no longer subject to iron-induced hydroxyl radical cleavage. Our results support a model in which the RAG1 protein contains the active site of the V(D)J recombinase and are interpreted in light of predictions about the structure of RAG1.
Kim
D R
DR
Molecular Biology Department, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Dai
Y
Y
Mundy
C L
CL
Yang
W
W
Oettinger
M A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins; D015321:Gene Rearrangement / Q000502:physiology*; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017665:Hydroxyl Radical; D007501:Iron / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010641:Phenotype; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013329:Structure-Activity Relationship; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D001224:Aspartic Acid; D017665:Hydroxyl Radical; D008345:Manganese; D007501:Iron; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases