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Molecular and Cellular Biology, November 2002, p. 7790-7801, Vol. 22, No. 22
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.22.7790-7801.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

Patrick C. Swanson*

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

Received 18 July 2002/ Returned for modification 13 August 2002/ Accepted 21 August 2002

Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.

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

Molecular and Cellular Biology, November 2002, p. 7790-7801, Vol. 22, No. 22
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.22.7790-7801.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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