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Molecular and Cellular Biology, April 1999, p. 3010-3017, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Roles of the "Dispensable" Portions of RAG-1 and RAG-2 in V(D)J Recombination

S. B. Steen,1,dagger J.-O. Han,2 C. Mundy,3,4 M. A. Oettinger,3,4 and D. B. Roth1,2,5,*

Program in Cell and Molecular Biology,1 Department of Microbiology and Immunology,2 and Howard Hughes Medical Institute,5 Baylor College of Medicine, Houston, Texas 77030; Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 021143; and Department of Genetics, Harvard Medical School, Boston, Massachusetts 021154

Received 14 August 1998/Returned for modification 29 September 1998/Accepted 22 January 1999

V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the "dispensable" regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination.

* Corresponding author. Mailing address: HHMI/Baylor College of Medicine, Immunology - M929/DeBakey Bldg., One Baylor Plaza, Houston, TX 77030-3498. Phone: (713) 798-8145. Fax: (713) 798-3700. E-mail: davidbr{at}bcm.tmc.edu.

dagger Present address: Department of Molecular Biology, Princeton University, Princeton, NJ 08544.

Molecular and Cellular Biology, April 1999, p. 3010-3017, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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