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Molecular and Cellular Biology, July 2001, p. 4256-4264, Vol. 21, No. 13
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.
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
*
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.
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