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Biophys J, May 2000, p. 2614-2627, Vol. 78, No. 5

Restrained Torsional Dynamics of Nuclear DNA in Living Proliferative Mammalian Cells

Marc Tramier,* Klaus Kemnitz,dagger Christiane Durieux,* Jacques Coppey,* Patrick Denjean,Dagger Robert B. Pansu,Dagger and Maïté Coppey-Moisan*

 *Institut Jacques Monod, UMR 7592, CNRS, Universités P 6/P 7, 75251 Paris cedex 05, France,  dagger EuroPhoton GmbH, D-12247 Berlin, Germany, and  Dagger Laboratoire de Photophysique et Photochimie Macromoléculaire et Supramoléculaire, Département de Chimie/UMR 85 31, ENS de Cachan/CNRS, 94235 Cachan Cedex, France

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.

Biophys J, May 2000, p. 2614-2627, Vol. 78, No. 5
© 2000 by the Biophysical Society   0006-3495/00/05/2614/14  $2.00


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