Evidence for a triplex DNA conformation at the bcl-2 major breakpoint region of the t(14;18) translocation.
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.
Raghavan
Sathees C
SC
Norris Comprehensive Cancer Center, Zilka Neurogenetics Institute, University of Southern California Keck School of Medicine, Los Angeles, 90033, USA.
Chastain
Paul
P
Lee
Jeremy S
JS
Hegde
Balachandra G
BG
Houston
Sabrina
S
Langen
Ralf
R
Hsieh
Chih-Lin
CL
Haworth
Ian S
IS
Lieber
Michael R
MR
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001483:Base Sequence; D045744:Cell Line, Tumor; D002883:Chromosomes, Human, Pair 14; D002887:Chromosomes, Human, Pair 18; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D006801:Humans; D006860:Hydrogen Bonding; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D021381:Protein Transport; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D012984:Software; D013447:Sulfites / Q000494:pharmacology; D014178:Translocation, Genetic
D000911:Antibodies, Monoclonal; D019253:Proto-Oncogene Proteins c-bcl-2; D013447:Sulfites; C051802:triplex DNA; D004247:DNA