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.

ACKNOWLEDGMENTS

Monica Calicchio and Wei-han Kan provided technical assistance, and we thank Suzanne Robertson and Denise Guzman for secretarial support. We are grateful to Vicky Brandt for editorial help and to Tania Baker and Ilana Goldhaber-Gordon for helpful discussions. Mary Purugganan, Heather Schultz, Leslie Huye, Sundeep Shah, and Matt Neiditch provided critical suggestions on the manuscript.

This work was supported by a grant from the National Institutes of Health (AI-36420). M.A.L. was supported by a National Institutes of Health Predoctoral Fellowship (T32-AI07495). D.B.R. is an Assistant Investigator of the Howard Hughes Medical Institute.