Functional organization of single and paired V(D)J cleavage complexes

RAG-1 and RAG-2 initiate V(D)J recombination binding to specific recognitio n 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 on e or two monomers of RAG-2 bind to a single RSS, but the enzymatic contribu tions of the RAG molecules within this nucleoprotein complex and its functi onal organization have not been elucidated. Using heterodimeric protein pre parations containing both wild-type and catalytically deficient RAG-1 molec ules, we found that one active monomer is sufficient for both nicking and h airpin formation at a single RSS, demonstrating that a single active site c an carry out both cleavage steps. Furthermore, the mutant heterodimers effi ciently cleaved both RSS in a synaptic complex. These results strongly sugg est that two RAG-I dimers are responsible for RSS cleavage in a synaptic co mplex, with one monomer of each dimer catalyzing both nicking and hairpin f ormation at each RSS.