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J. Biol. Chem., Vol. 280, Issue 24, 22749-22760, June 17, 2005

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Evidence for a Triplex DNA Conformation at the bcl-2 Major Breakpoint Region of the t(14;18) Translocation^*

From the ^aNorris Comprehensive Cancer Center, Departments of ^bPathology, ^cBiochemistry and Molecular Biology, ^dBiological Sciences, ^eMolecular Microbiology and Immunology, and ⁱUrology, ^hZilka Neurogenetics Institute, and ^jDepartment of Pharmaceutical Sciences, University of Southern California Keck School of Medicine, Los Angeles, California 90033, the ^fDepartments of Biochemistry and of Pathology, University of North Carolina, Chapel Hill, North Carolina 27599, and the ^gDepartment of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N5E5, Canada

The most common chromosomal translocation in cancer, t(14;18), occurs at the bcl-2 major breakpoint region (Mbr) in follicular lymphomas. The 150-bp bcl-2 Mbr, which contains three breakage hotspots (peaks), has a single-stranded character and, hence, a non-B DNA conformation both in vivo and in vitro. Here, we use gel assays and electron microscopy to show that a triplex-specific antibody binds to the bcl-2 Mbr in vitro. Bisulfite reactivity shows that the non-B DNA structure is favored by, but not dependent upon, supercoiling and suggests a possible triplex conformation at one portion of the Mbr (peak I). We have used circular dichroism to test whether the predicted third strand of that suggested structure can indeed form a triplex with the duplex at peak I, and it does so with 1:1 stoichiometry. Using an intracellular minichromosomal assay, we show that the non-B DNA structure formation is critical for the breakage at the bcl-2 Mbr, because a 3-bp mutation that disrupts the putative peak I triplex also markedly reduces the recombination of the Mbr. A three-dimensional model of such a triplex is consistent with bond length, bond angle, and energetic restrictions (stacking and hydrogen bonding). We infer that an imperfect purine/purine/pyrimidine (R.R.Y) triplex likely forms at the bcl-2 Mbr in vitro, and in vivo recombination data favor this as the major DNA conformation in vivo as well.

Received for publication, March 17, 2005 , and in revised form, April 14, 2005.

^* These studies were supported by grants from the National Institutes of Health (to M. R. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material, including an additional six figures and one table.

^k To whom correspondence should be addressed: Norris Cancer Center, Rm. 5428, 1441 Eastlake Ave., MC9176, Los Angeles, CA 90033. Tel.: 323-865-0568; Fax: 323-865-3019; E-mail: lieber{at}usc.edu.

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