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Molecular and Cellular Biology, July 2001, p. 4256-4264, Vol. 21, No. 13
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.13.4256-4264.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Mark A. Landree,1 Sam B. Kale,2 and David B. Roth1,2,3,*

Interdepartmental Program in Cell and Molecular Biology,1 Department of Immunology,2 and Howard Hughes Medical Institute,3 Baylor College of Medicine, Houston, Texas 77030

Received 17 January 2001/Returned for modification 22 February 2001/Accepted 4 April 2001

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.

* Corresponding author. Mailing address: Department of Immunology, Immunology M929, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. Phone: (713) 798-8145. Fax: (512) 857-0178. E-mail: davidbr{at}bcm.tmc.edu.

Molecular and Cellular Biology, July 2001, p. 4256-4264, Vol. 21, No. 13
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.13.4256-4264.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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