Nucleosome mononucleosome

In the absence of conclusive data on the role of a positive supercoiling wave, static positive supercoiling elicited by nucleosome reconstitution on relaxed or slightly positively-supercoiled plasmids [51] or by ethidium bromide intercalation in the loop of mononucleosomes on DNA minicircles [52] did not succeed either in releasing dimers. Moreover, circular dichroism, histone chemical modi-flcation and H3-thiol accessibility failed to detect an even slight alteration in the structure of such torsionally-stressed nucleosomes [51]. The reason was later found to lie in the ability of nucleosome entry/exit DNAs to form a positive crossing [52]. 

Fig. 4. Nucleosome relaxation, and influence of histone N-terminal tails. Example of nucleosomes on 356 bp ALk= —2.9 topoisomer from the pBR DNA minicircle series [28]. (a) Mononucleosomes (Mo) were reconstituted with control (Control) or acetylated (Acetyl) histones, incubated at 37 °C in Tris buffer [T 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 50 mM KCl, 5 mM MgC, and 0.5 mM dithiothreitol] or phosphate buffer [P same as Tris buffer with 50 mM potassium phosphate (pH 7.5) instead of 50 mM Tris-HCl] in the absence (Topo I —) or presence (Topo I +) of topoisomerase I, and electrophoresed in a native polyacrylamide gel at room temperature. Note the splitting of nucleosome relaxation products in two bands. TE starting chromatin in TE buffer, (b) Gel slices (brackets) were cut out, and eluted DNAs were electrophoresed in a chloroquine-containing native polyacrylamide gel, together with control naked topoisomers (C1-C4). Lanes were numbered as in the (a) gel. Autoradiograms are shown, (c) Radioactivity profiles of lanes 2 and 5 in the (b) gel. Topoisomers are indicated by their ALk values. (Adapted from Fig. 2 in Ref. [28].)...

De Lucia, F., Alilat, M., Sivolob, A., and Prunell, A. (1999) Nucleosome dynamics. III. Histone-tail dependent fluctuation of nucleosomes between open and closed DNA conformations implications for chromatin dynamics and the linking number paradox. A relaxation study of mononucleosomes on DNA minicircles. J. Mol. Biol. 285, 1101-1119. 

The interaction of the abundant HMGB proteins with both nucleosomal particles lacking linker histones and with linker histones themselves in vitro is well documented. A soluble chromatin fraction released from mouse myeloma nuclei contained mononucleosomes associated with almost stoichiometric amounts of HMGB1,2 but lacking histone HI [81]. Nhp6ap binds directly to nucleosomal particles reconstituted with chicken histones (i.e., a single histone octamer... 

HeLa mononucleosomes Nucleosome arrays reconstituted from modified 208-12 and core histones The arrays remodeled with hSWI/ SNF... 

The interaction between nucleosomes plays an important role for the stability of the 30 nm fiber recent experiments on liquid crystals of mononucleosomes [44-47] and also less concentrated mononucleosome solutions [48,49] show an attractive interaction that can be parameterized by an anisotropic Leonard-Jones type potential [50]. Also, an electrostatic interaction potential has been computed using the crystallographic structure of the nucleosome [51]. The influence of these potentials on the structure of the fiber is discussed below together with the corresponding models. 

Nucleosome-nucleosome interaction potentials can be calibrated by comparison with the characteristics of liquid crystals of mononucleosomes at high concentrations. Under suitable conditions, nucleosome core particles form a hexagonal-columnar phase with a distance of 11.55 1 nm between the columns and a mean distance of 7.16 0.65 nm between the particles in one column [44,46]. These distances may be assumed to correspond to the positions of the minima of an attractive internucleosomal potential. The depth of the interaction potential (i.e., the binding energy per nucleosome) was estimated in the stretching experiments of Cui and Bustamante [66] to 2.6-3.4 kT. A slightly lower potential minimum of 1.25 kT is obtained by a comparison of the stability of the nucleosome liquid crystal phase with simulations [50]. 

An important addition compared to previous models was the parameterization of the internucleosomal interaction potential in the form of an anisotropic attractive potential of the Lennard-Jones form, the so-called Gay-Berne potential [90]. Here, the depth and location of the potential minimum can be set independently for radial and axial interactions, effectively allowing the use of an ellipsoid as a good first-order approximation of the shape of the nucleosome. The potential had to be calibrated from independent experimental data, which exists, e.g., from the studies of mononucleosome liquid crystals by the Livolant group [44,46] (see above). The position of the potential minima in axial and radial direction were obtained from the periodicity of the liquid crystal in these directions, and the depth of the potential minimum was estimated from a simulation of liquid crystals using the same potential. 

In the experiment of Fig. 6 we examined whether poly(ADP-ribose) would induce complete dissociation of DNA from isolated nucleosome particles. Fig. 6 suggests that, after addition of poly(ADP-ribose), part of the core DNA molecules were released as they moved in front of the mononucleosome zone. We are, however, aware that more detailed analyses are required in order to determine the type and degree of dissociation. 

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