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Molecular and Cellular Biology, January 2001, p. 449-458, Vol. 21, No. 2
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.2.449-458.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

The DDE Motif in RAG-1 Is Contributed in trans to a Single Active Site That Catalyzes the Nicking and Transesterification Steps of V(D)J Recombination

Patrick C. Swanson*

Department of Medical Microbiology and Immunology, Creighton University, School of Medicine, Omaha, Nebraska 68178

Received 30 June 2000/Returned for modification 18 August 2000/Accepted 23 October 2000

The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first-strand nicking, followed by hairpin formation via direct transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage in trans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.

* Mailing address: Department of Medical Microbiology and Immunology, Creighton University, School of Medicine, 2500 California Plaza, Omaha, NE 68178. Phone: (402) 280-2716. Fax: (402) 280-1875. E-mail: pswanson{at}creighton.edu.

Molecular and Cellular Biology, January 2001, p. 449-458, Vol. 21, No. 2
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.2.449-458.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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