Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells.
The rate of covalent photobinding of trimethylpsoralen to DNA is greater when the DNA is wound with negative superhelical tension than when it is relaxed. In vitro the rate of photobinding is directly proportional to the negative superhelical density of the DNA. Thus measurement of the rate of photobinding provides an assay for probing in vivo unrestrained tension in the winding of the DNA double helix. This approach has been applied to measure torsional tension in DNA as it is packaged in living E. coli. Drosophila and HeLa cells. A method is described for measuring the rate of photobinding to intracellular DNA and rRNA, and for using the latter measurement as an internal control of the rate of me3-psoralen photobinding in vivo. This permits more accurate and reproducible measurement of changes in the DNA-psoralen photobinding reaction. The me3-psoralen probe interacts with intracellular bacterial DNA as expected for a purified DNA duplex wound with superhelical density sigma = -0.05 +/- 0.01. This superhelical tension is relaxed in cells when multiple single-strand breaks are introduced into the chromosomal DNA by gamma-irradiation. Similar relaxation occurs when cells are treated with the DNA gyrase inhibitor coumermycin. The results suggest that the DNA double helix is wound with torsional tension in vivo and that DNA supercoils which are equilibrated with this tension are not completely restrained in nucleosome-like structures. Torsional tension in the DNA of eucaryotic cells is not detectable in analogous measurements of the packaged DNA of HeLa and Drosophila cells. The simplest interpretation of this finding is that, within the limits of detection, all superhelical turns in the DNA are restrained in nucleosomes or nucleosome-like structures in these eucaryotic cells.
D000818:Animals; D004269:DNA, Bacterial / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004331:Drosophila melanogaster; D004926:Escherichia coli; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009994:Osmolar Concentration; D059005:Topoisomerase II Inhibitors; D014307:Trioxsalen / Q000378:metabolism
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D059005:Topoisomerase II Inhibitors; D014307:Trioxsalen