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J. Biol. Chem., Vol. 276, Issue 40, 37093-37101, October 5, 2001
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From the V(D)J recombination is instigated by the
recombination-activating proteins RAG1 and RAG2, which catalyze
site-specific DNA cleavage at the border of the recombination signal
sequence (RSS). Although both proteins are required for
activity, core RAG1 (the catalytically active region containing
residues 384-1008 of 1040) alone displays binding specificity for the
conserved heptamer and nonamer sequences of the RSS. The
nonamer-binding region lies near the N terminus of core RAG1, whereas
the heptamer-binding region has not been identified. Here, potential
domains within core RAG1 were identified using limited proteolysis
studies. An iterative procedure of DNA cloning, protein expression, and
characterization revealed the presence of two topologically independent
domains within core RAG1, referred to as the central domain (residues 528-760) and the C-terminal domain (residues 761-980). The domains do
not include the nonamer-binding region but rather largely span the
remaining relatively uncharacterized region of core RAG1. Characterization of macromolecular interactions revealed that the
central domain bound to the RSS with specificity for the heptamer and
contained the predominant binding site for RAG2. The C-terminal domain bound DNA cooperatively but did not show specificity for either
conserved RSS element. This domain was also found to self-associate, implicating it as a dimerization domain within RAG1.
Identification of Two Topologically Independent Domains in RAG1
and Their Role in Macromolecular Interactions Relevant to V(D)J
Recombination*
,,§,
, and**
Department of Biochemistry and Molecular
Biology, The University of Oklahoma Health Sciences Center, Oklahoma
City, Oklahoma 73190 and the ¶ Ruttenberg Cancer Center, Mount
Sinai School of Medicine of New York University, New York, New York
10029
*
This work was supported by Research Project Grant
RPG-00-032-01-CIM from the American Cancer Society, an Oklahoma Center
for Advancement in Science and Technology award for project
number HR99-040 and funds from the Presbyterian Health Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Section of Immunobiology, Yale University
School of Medicine, New Haven, CT 06520.
**
To whom correspondence should be addressed. Tel.: 405-271-2227, Ext. 1248; Fax: 405-271-3139; E-mail: Karla-Rodgers@ouhsc.edu.
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