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Stability and Strand Asymmetry in the Non-B DNA Structure at the bcl-2 Major Breakpoint Region
The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakag...
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Published in: | The Journal of biological chemistry 2004-10, Vol.279 (44), p.46213-46225 |
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Main Authors: | , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions
(hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we
could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing.
Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2
Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing
the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure
observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer
probing, a bisulfite modification assay, or a P1 nuclease assay. We provide evidence that two different non-B conformations
exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation
to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location
and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M406280200 |