Functional organization of single and paired V(D)J cleavage complexes.
RAG-1 and RAG-2 initiate V(D)J recombination by binding to specific recognition sequences (RSS) and then cleave the DNA in two steps: nicking and hairpin formation. Recent work has established that a dimer of RAG-1 and either one or two monomers of RAG-2 bind to a single RSS, but the enzymatic contributions of the RAG molecules within this nucleoprotein complex and its functional organization have not been elucidated. Using heterodimeric protein preparations containing both wild-type and catalytically deficient RAG-1 molecules, we found that one active monomer is sufficient for both nicking and hairpin formation at a single RSS, demonstrating that a single active site can carry out both cleavage steps. Furthermore, the mutant heterodimers efficiently cleaved both RSS in a synaptic complex. These results strongly suggest that two RAG-1 dimers are responsible for RSS cleavage in a synaptic complex, with one monomer of each dimer catalyzing both nicking and hairpin formation at each RSS.
Landree
M A
MA
Interdepartmental Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Kale
S B
SB
Roth
D B
DB
D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D008345:Manganese; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D008274:Magnesium