General and Molecular Genetics

Protein and DNA contributions in the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4) 2 tetramer) to a right-handed conformation have been investigated in a recent article from this laboratory. As the evidence for a protein contribution, a sterical hindrance introduced at the H3/H3 interface of the two constituent H3-H4 dimers by oxidation of H3 cysteine 110 blocked the tetramer in a half-left-handed or semi-right-handed conformation, depending on the SH-reagent used. The DNA contributed at the level of the dyad region, which appeared to act through its sequencedependent deformability in modulating both the loop threshold positive constraint required to trigger the transition, and the tetrasome lateral opening. This opening, which electron microscopic visualizations directly showed to be associated with the transition, is expected to help remove the clash between the entering and exiting DNAs. In this work, the transition mechanism was further investigated by applying a positive constraint in the loop through ethidium bromide (EtBr) intercalation. This technique, including the determination of binding isotherms, has ®rst been used with mononucleosomes on DNA minicircles, and has revealed that these particles could tolerate large positive supercoilings without disruption, owing to the loop ability to cross positively in a histone taildependent manner. The transition of 359 bp tetrasomes was found to go to completion in lower salt (10 mM), but not in higher salt (100 mM), whereas the transition of 256 bp tetrasomes was already hindered in lower salt. Histone acetylation relieved that lower salt hindrance but enhanced the higher salt hindrances. These data again pointed to the DNA in the dyad region as a regulator of the transition. The block was indeed expected to originate from a local EtBr intercalation in that DNA, which opposed its overtwisting during the transition. The occurrence of the block, or its relief, then depended on the outcome of the competition between the tails and EtBr for binding to that region, that is, on whether the tails could prevent EtBr intercalation before the ongoing transition hampered both bindings. Destabilization of the tails in the course of the transition is documented in an accompanying article through a relaxation study of a 351-366 bp tetrasome series.

We have recently described the relaxation of mononucleosomes on an homologous series of 351-366 bp DNA minicircles, as a tool to study nucleosome structure and dynamics in vitro. Nucleosomes were found to have a tail-regulated access to three distinct DNA conformations, depending on the crossing between the entering and exiting DNAs, and its polarity. This approach was now used to explore tetrasome chiral transition, and the in¯uence of the histone tails. The data con®rmed the existence of two states, with linking number differences ÁLk t À 0.74(AE0.01) and 0.51(AE0.06). As expected, the particle free energy is higher in the right-handed state (ÁG t 1.9(AE0.I) kT), but it decreased (to 1.3(AE0.1) kT) upon histone acetylation and the addition of phosphate, a potent tail destabilizer. Removal of the tails with trypsin further decreased ÁG t (to 0.6 kT), and also induced a loss of supercoiling in both states, to ÁLk t À 0.64(AE0.03) and 0.35(AE0.05). The loop endconditions, and hence the parameters of the DNA superhelix, were then calculated for both states using the explicit solutions to the equations of the mechanical equilibrium in the theory of elastic rod model for DNA. Whereas the pitch of the DNA superhelix may be approximately equal and opposite in the two conformations, its radius (r) was 20 % larger in the right-handed conformation, con®rming previous observations by electron microscopy of a tetrasome lateral opening in that conformation. The above supercoiling losses were found to re¯ect a further 3 % increase in r (to 23 %) upon removal of the tails in the right-handed conformation, and a 14 % increase in the left-handed conformation. The use of composite tetramers with one histone tail intact and the other removed showed these effects to be essentially due to the H3 tails. Altogether, these results show that the H3 tails oppose the tetrasome opening which is expected to be required to relieve the clash between the entering and exiting DNAs in the course of the transition, but which also appears to be intrinsic to the protein reorientation mechanism. We propose that the block against opening results from the H3 tails intercalating into the small groove of the double helix at AE10 bp from the dyad, and acting as wedges against local DNA straightening. The tails (especially H3) may therefore regulate tetrasome chiral transition in vivo.

The mean linking number (hLki) of the topoisomer equilibrium distribution obtained upon relaxation of DNA minicircles with topoisomerase I did not increase linearly, but rather in a step wise fashion, with DNA size between 351 and 366 bp. As a consequence, the corresponding linking number difference (hÁLki) did not remain equal to 0, but rather oscillated between AE0.3 with the periodicity of the double helix. This oscillation, not observed with plasmid-size DNA, is an expected consequence of the stiffness of short DNA. When minicircles were reconstituted with a nucleosome, the associated hÁLk n i oscillated between $ À 1.4 AE0.2. This oscillation appears to result from the combined effects of DNA stiffness, and nucleosome ability to thermally¯uctuate between three distinct DNA conformational states. Two of these states, a closed $1.75-turn DNA conformation with negatively crossed entering and exiting DNAs, and an open $1.4-turn conformation with uncrossed DNAs, are well known, whereas the third state, with a closed DNA conformation and DNAs tending to cross positively rather than negatively, is less familiar. Access to both closed``negative'' and``positive'' states appears to be mediated by histone N-terminal tails, as shown by speci®c alterations to the hÁLk n i oscillation caused by histone acetylation and phosphate ions, a potent tail destabilizator. These results extend previous observations of ethidium bromide¯uorescence titration in the accompanying article, which have pointed to an histone tail-dependent¯exibility of entering and exiting DNAs to positive crossing. They also show that DNA wrapping around the histones occurred without twist alteration compared to the DNA free in solution, and reveal an intriguing new facet of the``linking-number-paradox'' problem: the possibility for linkers in chromatin to adopt different crossing status within an overall dynamic equilibrium which may be regulated by histone acetylation.

Relaxation of nucleosomes on an homologous series (pBR) of ca 350 -370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also "closed", but with these DNAs positively crossed; and the third was "open", with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by , 0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a , 1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance (, 20 bp) on H3-tail interactions with the small groove at the nucleosome entry -exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them.

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 mononucleosomes reconstituted on DNA minicircles. The results of gel electrophoresis,¯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 octamer. 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.

A DNA sequence-dependent nucleosome structural and dynamic polymorphism was recently uncovered through topoisomerase I relaxation of mononucleosomes on two homologous approximately 350-370 bp DNA minicircle series, one originating from pBR322, the other from the 5S nucleosome positioning sequence. Whereas both pBR and 5S nucleosomes had access to the closed, negatively crossed conformation, only the pBR nucleosome had access to the positively crossed conformation. Simulation suggested this discrepancy was the result of a reorientation of entry/exit DNAs, itself proposed to be the consequence of specific DNA untwistings occurring in pBR nucleosome where H2B N-terminal tails pass between the two gyres. The present work investigates the behavior of the same two nucleosomes after binding of linker histone H5, its globular domain, GH5, and engineered H5 C-tail deletion mutants. Nucleosome access to the open uncrossed conformation was suppressed and, more surprisingly, the ability of 5S nucleosome to positively cross was largely restored. This, together with the paradoxical observation of a less extensive crossing in the negative conformation with GH5 than without, favored an asymmetrical location of the globular domain in interaction with the central gyre and only entry (or exit) DNA, and raised the possibility of the domain physical rotation as a mechanism assisting nucleosome fluctuation from one conformation to the other. Moreover, both negative and positive conformations showed a high degree of loop conformational flexibility in the presence of the full-length H5 C-tail, which the simulation suggested to reflect the unique feature of the resulting stem to bring entry/exit DNAs in contact and parallel. The results point to the stem being a fundamental structural motif directing chromatin higher order folding, as well as a major player in its dynamics.

CENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-Aand H3-containing nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry-exit relative to canonical nucleosomes on both 5 S DNA and the α-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(±2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4) 2 tetramer was also more readily destabilized with heparin than the (H3-H4) 2 tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, especially as is seen following CENP-A transient over-expression.

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Heparin, a strong acidic polyelectrolyte, releases H2A-H2B dimers sequentially from mononucleosomes reconstituted on DNA minicircles, giving rise to hexasomes and tetrasomes (Figure S1A and legend). Heparin concentrations from 0.01 to 100 µg/mL were initially tested in the single-fiber experiment. The fiber length was not much affected up to 2 µg/mL, but it increased above 5 µg/mL (not shown), and nucleosomes were destroyed at 100 µg/mL (Bancaud et al., 2006). We tentatively chose a mild treatment at 1 µg/mL and obtained the red curve in Figure S1B, panel 2, not much different from the initial forward curve (blue). Following the excursion at large positive torsion, a reproducible curve quite similar to the initial green curve was generated, which could be run back and forth several times without hysteresis (purple in panel 3). The merging of purple and blue curves at negative torsions, like the merging of green and blue curves, indicates that canonical nucleosomes reform, and thus that dimers are still present. These nucleosomes, however, switch to the altered form as soon as the constraint becomes positive, presumably as a result of an heparin-facilitated transition. Nucleosome core particles were subsequently added

Heparin, a strong acidic polyelectrolyte, releases H2A-H2B dimers sequentially from mononucleosomes reconstituted on DNA minicircles, giving rise to hexasomes and tetrasomes (Figure S1A and legend). Heparin concentrations from 0.01 to 100 µg/mL were initially tested in the single-fiber experiment. The fiber length was not much affected up to 2 µg/mL, but it increased above 5 µg/mL (not shown), and nucleosomes were destroyed at 100 µg/mL (Bancaud et al., 2006). We tentatively chose a mild treatment at 1 µg/mL and obtained the red curve in Figure S1B, panel 2, not much different from the initial forward curve (blue). Following the excursion at large positive torsion, a reproducible curve quite similar to the initial green curve was generated, which could be run back and forth several times without hysteresis (purple in panel 3). The merging of purple and blue curves at negative torsions, like the merging of green and blue curves, indicates that canonical nucleosomes reform, and thus that dimers are still present. These nucleosomes, however, switch to the altered form as soon as the constraint becomes positive, presumably as a result of an heparin-facilitated transition. Nucleosome core particles were subsequently added

The influence of ionic strength on DNA-histone and histone-histone interactions in reconstituted nucleosomes was studied by measuring the parameters of histone tyrosine fluorescence: fluorescence intensity and l position. The first max parameter is sensitive to histone-DNA interactions. The changes of the second one accrue due to hydrogen bond Ž. formationrdisruption between tyrosines in the histone H2A-H2B dimer and the H3-H4 tetramer. The simultaneous 2 measurement of these parameters permits the recording of both the dissociation of histone complexes from DNA, as well as changes in histone-histone interactions. As ionic strength is increased, the H2A-H2B histone dimer dissociated first, Ž. w Ž. followed by dissociation of the H3-H4 tetramer Yager, T.G., McMurray, C.T. and Van Holde, K.E. 1989 Biochemistry 2 x 28, 2271-2276. The H2A-H2B dimer is dissociated in two stages: first, the ionic bonds with DNA were disrupted, followed by the dissociation of the histone dimer from the tetramer. And secondly, the disruption of dimer-tetramer specific H-bonds. It was established that the energy of electrostatic interactions of the histone dimer with DNA within the nucleosome is much less than the energy of interaction of the histone dimer with the tetramer.

Studies on the chromatin nucleosome organization play an ever increasing role in our comprehension of mechanisms of the gene activity regulation. This minireview describes the results on the nucleosome conformational flexibility, which were obtained using magnetic tweezers to apply torsion to oligonucleosome fibers reconstituted on single DNA molecules. Such an approach revealed a new structural form of the nucleosome, the reversome, in which DNA is wrapped in a right-handed superhelix around a distorted histone octamer. Molecular mechanisms of the nucleosome structural flexibility and its biological relevance are discussed.

We investigated the mechanisms of DNA exit during single-cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single-strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.