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© The Rockefeller University Press, 0022-1007/1997/6/2025/ $5.00
The Journal of Experimental Medicine, Volume 185, Number 11, June 2, 1997 2025-2032

Article

V(D)J Recombination: Modulation of RAG1 and RAG2 Cleavage Activity on 12/23 Substrates by Whole Cell Extract and DNA-bending Proteins

Dennis J. Sawchuk*, Frances Weis-Garcia*, Sohail Malik{ddagger}, Eva Besmer*,§, Michael Bustin||, Michel C. Nussenzweig*,§, and Patricia Cortes*

From the * Laboratory of Molecular Immunology, and {ddagger} Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York 10021; § Howard Hughes Medical Institute Research Laboratory, The Rockefeller University, New York 10021; and the || Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.

Address correspondence to Patricia Cortes, Laboratory of Molecular Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021.

D.J. Sawchuk is supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) PGS-A fellowship. M.C. Nussenzweig is an associate investigator of the Howard Hughes Medical Institute. This work was supported in part by grants from the National Institutes of Health to M.C. Nussenzweig.

1Abbreviations used in this paper: HMG, high mobility group protein; IHF, integration host factor; RSS, recombination signal sequences; WCE, whole cell extract.


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