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Molecular and Cellular Biology, November 1998, p. 6408-6415, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step

Robert B. West and Michael R. Lieber*

Norris Comprehensive Cancer Center, Departments of Pathology, Biochemistry and Molecular Biology, and Molecular Microbiology and Immunology, University of Southern California School of Medicine, Los Angeles, California 90033

Received 19 May 1998/Returned for modification 27 July 1998/Accepted 19 August 1998

A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals only on 12×23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.

* Corresponding author. Mailing address: Norris Comprehensive Cancer Center, Room 5428, Departments of Pathology and Biochemistry, University of Southern California School of Medicine, 1441 Eastlake Ave., Mail Stop 73, Los Angeles, CA 90033. Phone: (323) 865-0568. Fax: (323) 865-3019. E-mail: lieber_m{at}froggy.hsc.usc.edu.

Molecular and Cellular Biology, November 1998, p. 6408-6415, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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