Magnetic tweezers are used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin 3-D architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, which are determined by the crossing status of the entry/exit DNAs (positive, null or negative). Torsional strain, in displacing that equilibrium, extensively reorganizes the fiber architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin, and may provide the ground to better understand the dynamic binding of most chromatin-associated proteins.

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Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the [H3-H4](2) tetramer bound with approximately 50 bp of DNA) obtained by depletion of H2A-H2B dimers suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented right-handed tetrasome. In view of its low energy, <8 kT, we propose that this transition is physiologically relevant and serves to break the docking of the dimers on the tetramer that in the absence of other factors exerts a strong block against elongation of transcription by the main RNA polymerase.

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We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two 113s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a leftto a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame Nucleosome dynamics is a necessary requirement of DNA function in chromatin, including transcription, replication, or repair. It has long been thought to be mediated by the tripartite organization of the histone octamer, made of an (H3-H4)2 tetramer bound with two H2A-H2B dimers (1). Thermodynamic studies of octamer assembly and disassembly showed that the forces holding the tetramer and dimers together are of a different nature and much stronger than the forces binding the dimers to the tetramer (1, 2), inspite of an extensive dimer-tetramer interface. This interface is disrupted in the first step of octamer disassembly.The crystal structure of the nucleosome core particle (3), and even more so, of the histone octamer (4), subsequently confirmed this tripartite organization. Moreover, one of the H2A-H2B dimers in the crystal structure of the core particle appears significantly displaced from its original position (3), which probably reflects the effects of crystal-packing forces and the relative ease with which the dimers can move relative to the tetramer. Such dimer lability may also be important in vivo, as is emphasized by the observations of an H2A-H2B deficit in nucleosome cores originating from transcriptionally active chromatin (5), and of H2A-H2B exchange with the endogenous histone pool upon chromatin transcription (6-8).Here we provide evidence for a potential conformational flexibility of the (H3-H4)2 tetramer, a tribute to the role of this tripartite organization of the histone octamer in nucleosome dynamics. This conformational flexibility is suggested by the observation of a similar affinity of the tetramer for negatively and positively supercoiled DNA minicircles, which apparently arises from the ability of the wrapped DNA to switch from a left-to a right-handed superhelix. This transition is found to require only modest positive torsional stress in the DNA, and can be triggered by DN...

Two methods have been used to measure the single-strand lengths of the DNA fragments produced by deoxyribonuclease I digestion of chromatin. The average lengths obtained are muliples of about 10.4 bases, significantly different from the value of 10 previously reported. This periodicity in fragment lengths is closely related to the periodicity of the DNA double helix in chromatin, but the two values need not be exactly the same.

The linking number paradox of DNA in chromatin (two negative crossings around the octamer, associated with a unit linking number reduction), which is 21 years old this year, has come of age. After stirring much debate in the past, the initially hypothetical explanation of the paradox by DNA overtwisting on the nucleosome surface is now presented as a hard fact in recent textbooks. The first part of this article presents a historical perspective of the problem and details the numerous attempts to measure DNA local periodicity, which in one remarkable example sowed the seeds for the discovery of DNA bending. The second part is devoted to the DNA minicircle system, which has been developed in the author's laboratory as an alternative to the local-periodicity-measurement approach. It offers a simple proposal: a unit linking number reduction associated with a single crossing. This conclusion is contrasted with the latest high-resolution crystallographic data of the nucleosome in the third part of the article, and the fourth part examines the available evidence supporting an extension of these results to nucleosomes in chromatin. The last part addresses another basic question pertaining to nucleosome dynamics, the conformational flexibility of the histone tetramer.

Chromatin was reconstituted from core histones and recombinant plasmid DNAs carrying poly(dA) . poly(dT) inserts of various lengths. A 97‐bp insert was found to occupy discrete and regularly‐spaced positions on the edges of the nucleosome. This insert cannot, however, be entirely included due to a block in the center of the particle. In contrast, nucleosomes reconstitute on a shorter 20‐bp insert. In this case, the insert shows a marked preference for the edges of the particle. Possible structural and physiological implications of these observations are discussed.