Functional Organization of Single and Paired V(D)J Cleavage Complexes

Abstract

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.