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Molecular and Cellular Biology, May 1999, p. 3674-3683, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

RAG-2 Promotes Heptamer Occupancy by RAG-1 in the Assembly of a V(D)J Initiation Complexdagger

Patrick C. Swanson and Stephen Desiderio*

Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 28 December 1998/Returned for modification 3 February 1999/Accepted 12 February 1999

V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. Phone: (410) 955-4735. Fax: (410) 955-9124. E-mail: sdesider{at}jhmi.edu.

dagger We dedicate this paper to the memory of our friend and colleague Eugenia Spanopoulou.

Molecular and Cellular Biology, May 1999, p. 3674-3683, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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