Nucleosome Dynamics. II{. High Flexibility of Nucleosome Entering and Exiting DNAs to Positive Crossing. An Ethidium Bromide Fluorescence Study of Mononucleosomes on DNA Minicircles Andrei Sivolob 1,3 , Filomena De Lucia 1,4 , Bernard Re  vet 2 and Ariel Prunell 1 * 1 Institut Jacques Monod Centre National de la Recherche Scienti®que et Universite  Denis Diderot Paris 7, 2 place Jussieu 75251 Paris Ce Âdex 05, France 2 Laboratoire de Microscopie Cellulaire et Mole Âculaire Institut Gustave Roussy rue Camille Desmoulins 94805 Villejuif Ce Âdex, France 3 Department of General and Molecular Genetics, National Shevchenko University 252601 Kiev, Ukraine 4 Dipartimento di Chimica Organica e Biologica University of Naples 16 Via Mezzocannone, Naples 80134, Italy H2A-H2B exchange with the intranuclear histone pool upon chromatin transcription in vivo is generally viewed as being triggered by the DNA positive supercoiling wave pushed by the elongating polymerase. This notion was tested here by investigating a potential release of H2A-H2B by ethidium bromide-induced positive supercoiling in the loop of mono- nucleosomes reconstituted on DNA minicircles. The results of gel electro- phoresis, ¯uorescence titration and electron microscopy showed that such a positive supercoiling was not able to release H2A-H2B, nor to unfold the nucleosome to any detectable extent. The reason appeared to be the ease with which the loop could undergo a positive crossing, a surprising observation in view of the DNA left-handed wrapping around the octa- mer. Moreover, the in¯uence of histone acetylation suggested that such loop ¯exibility to positive crossing is mediated by histone N-terminal tails which, by interacting with entering and exiting DNAs, reduce their electrostatic repulsion. These conclusions are con®rmed and extended in the accompanying article through relaxation with topoisomerase I. # 1999 Academic Press Keywords: ethidium bromide binding isotherms; DNA minicircles; DNA ¯exibility; histone tails; electron microscopy of nucleosomes *Corresponding author Introduction DNA transactions in vivo require not only chro- matin, but also nucleosomes, to be dynamic struc- tures. Nucleosome dynamics have long been thought to be mediated by the tripartite organiz- ation of the histone octamer made of a (H3-H4) 2 tetramer ¯anked by two H2A-H2B dimers (Eickbush & Moudrianakis, 1978). Physico-chemi- cal assays showed that the cohesion forces holding the dimers and the tetramer together are of a different nature and much stronger than the forces linking the dimers to the tetramer, in spite of an extensive dimer-tetramer interface (Eickbush & Moudrianakis, 1978; Baxevanis et al., 1991). Two main observations suggested that this lability of H2A-H2B dimers played a physiological role. The ®rst one is the de®cit in H2A-H2B observed in core particles originating from transcriptionally active chromatin (Baer & Rhodes, 1983) and the second, H2A-H2B exchange with the intranuclear histone pool observed upon transcription in vivo (Louters & Chalkley, 1985; Schwager et al., 1985; Jackson, 1990). The ®rst step in this process, H2A-H2B destabilization and release, has been thought to be triggered by the positive supercoiling wave pushed in front of the polymerase, whereas nucleosome regeneration by recapture of H2A-H2B was simi- larly supposed to be the consequence of the nega- tive supercoiling wave in the wake of the polymerase (Liu & Wang, 1987; Jackson, 1990, 1995). {Article I in this series is Hamiche et al. (1996b). Abbreviations used: EtBr, ethidium bromide; BSA, bovine serum albumin. E-mail address of the corresponding author: prunell@ccr.jussieu.fr Article No. jmbi.1998.2380 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 285, 1081±1099 0022-2836/99/031081±19 $30.00/0 # 1999 Academic Press