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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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