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Molecular and Cellular Biology, July 2005, p. 5904-5919, Vol. 25, No. 14
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.14.5904-5919.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Double-Strand Break Formation by the RAG Complex at the Bcl-2 Major Breakpoint Region and at Other Non-B DNA Structures In Vitro{dagger}

Sathees C. Raghavan,1,2,3,4,5 Patrick C. Swanson,6 Yunmei Ma,1,2,3,4,5 and Michael R. Lieber1,2,3,4,5*

Norris Comprehensive Cancer Center and Departments of,1 Pathology,2 Biochemistry & Molecular Biology,3 Biological Sciences,4 Molecular Microbiology & Immunology, University of Southern California Keck School of Medicine, 1441 Eastlake Ave., Los Angeles, California 90033,5 Department of Medical Microbiology and Immunology, Creighton University Medical Center, 2500 California Plaza, Omaha, Nebraska 681786

Received 22 November 2004/ Returned for modification 30 December 2004/ Accepted 10 April 2005

The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.

* Corresponding author. Mailing address: Norris Comprehensive Cancer Center, Rm. 5428, University of Southern California, 1441 Eastlake Ave., MC9176, Los Angeles, CA 90033. Phone: (323) 865-0568. Fax: (323) 865-3019. E-mail: lieber{at}usc.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, July 2005, p. 5904-5919, Vol. 25, No. 14
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.14.5904-5919.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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