Biomedical subjects

A Sivolob

Publications and source records attributed to A Sivolob.

6 recordsLinked to original sources

PubMed2000

Nucleosome dynamics V. Ethidium bromide versus histone tails in modulating ethidium bromide-driven tetrasome chiral transition. A fluorescence study of tetrasomes on DNA minicircles.

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 sequence-dependent 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 first 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 tail-dependent 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.

Acetylation↗

PubMed2000

Nucleosome dynamics. VI. Histone tail regulation of tetrasome chiral transition. A relaxation study of tetrasomes on DNA minicircles.

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 influence of the histone tails. The data confirmed the existence of two states, with linking number differences DeltaLk(t)=-0.74(+/-0.01) and +0.51(+/-0.06). As expected, the particle free energy is higher in the right-handed state (DeltaG(t)=1.9(+/-0.I) kT), but it decreased (to 1.3(+/-0.1) kT) upon histone acetylation and the addition of phosphate, a potent tail destabilizer. Removal of the tails with trypsin further decreased DeltaG(t) (to 0.6 kT), and also induced a loss of supercoiling in both states, to DeltaLk(t)=-0.64(+/-0.03) and +0. 35(+/-0.05). The loop end-conditions, 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, confirming previous observations by electron microscopy of a tetrasome lateral opening in that conformation. The above supercoiling losses were found to reflect 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 +/-10 bp from the dyad, and acting as wedges against local DNA straightening. The tails (especially H3) may therefore regulate tetrasome chiral transition in vivo.

Acetylation↗

PubMed1999

Nucleosome dynamics. Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)2 tetramer-DNA particle.

Our laboratory has previously reported the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)2tetramer). This transition was induced by DNA positive torsional constraint, generated either by initial supercoiling of the loop or by its thermal fluctuations during topoisomerase relaxation. Taking into account the wrapping of the DNA around the histones into less than a turn, and its negative crossing at the entry-exit, the transition was proposed to involve a 360 degrees rotation of the loop around the particle dyad axis, and the formation of a positive crossing. The tetramer horseshoe-shaped conformation within the octamer further suggested that this process could be mediated by a reorientation of the two sector-like H3-H4 dimers about their H3/H3 interface, which would switch the overall handedness of the proteinaceous superhelix from left to right-handed. We now provide additional evidence for such a contribution of the protein by showing, through gel electrophoresis, topoisomerase relaxation and electron microscopy, that a sterical hindrance at the H3/H3 interface, introduced by covalent linking of bulky adducts through thiol oxidation of H3 cysteine 110, interferes with the transition. Such interference varies, depending on the particular SH-reagent used; but the most remarkable effect was obtained with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB), which displaces the preferred conformation of the tetrasomes from left-handed to semi-right-handed, and at the same time preserves a significant degree of chiral flexibility. DNA contribution was evidenced by a specific fractionation of circular tetrasomes in gel electrophoresis which, together with a different positioning of control and DTNB tetrasomes on linear DNA, pointed to an interdependence between tetrasome conformation and positions. Moreover, linear tetrasomes fluctuate between crossed and uncrossed conformations in a salt-dependent equilibrium which appears to vary with their positions on the DNA. These data suggest a modulatable role of the DNA around the dyad in the transition, depending primarily on its sequence-dependent deformability. This role is played at both levels of H3-H4 dimer reorientation and lateral opening, a mechanism by which the particle may relieve the clash between its entering and exiting DNAs. These properties make the tetrasome an attractive potential intermediate in nucleosome dynamics in vivo, in particular duringX transcriptional activation and elongation.

Animals↗

PubMed1999

Nucleosome dynamics. II. High flexibility of nucleosome entering and exiting DNAs to positive crossing. An ethidium bromide fluorescence study of mononucleosomes on DNA minicircles.

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, fluorescence 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 influence of histone acetylation suggested that such loop flexibility 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 confirmed and extended in the accompanying article through relaxation with topoisomerase I.

Animals↗

PubMed1999

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.

The mean linking number ( ) 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 ( ) did not remain equal to 0, but rather oscillated between +/-0.3 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 oscillated between approximately -1.4 +/-0. 2. This oscillation appears to result from the combined effects of DNA stiffness, and nucleosome ability to thermally fluctuate between three distinct DNA conformational states. Two of these states, a closed approximately 1.75-turn DNA conformation with negatively crossed entering and exiting DNAs, and an open approximately 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 specific alterations to the oscillation caused by histone acetylation and phosphate ions, a potent tail destabilizator. These results extend previous observations of ethidium bromide fluorescence titration in the accompanying article, which have pointed to an histone tail-dependent flexibility 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.

Acetylation↗

PubMed1997

Electrostatic contribution to the bending of DNA.

A model is derived that accounts for the short-range electrostatic contribution to the bending of DNA molecule in solution and in complexes with proteins in terms of the non-linear Poisson-Boltzmann equation. We defined that the short-range electrostatic interactions depend on the changes of the polyion surface charge density under deformation, while the long-range interactions depend on the bending-induced changes in distances between each two points along the polyion axis. After an appropriate simplification of the Poisson-Boltzmann equation, the short-range term is calculated separately giving the lower limit for the electrostatic contribution to the DNA persistence length. The result is compared with the theoretical approaches developed earlier [M. Fixman, J. Chem. Phys. 76 (1982) 6346; M. Le Bret, J. Chem. Phys. 76 (1982) 6243] and with the experimental data. The conclusion is made that the results of Fixman-Le Bret, which took into account both types of the electrostatic interactions for a uniformly bent polyion, give the upper limit for the electrostatic persistence length at low ionic strength, and the actual behavior of the DNA persistence length lies between two theoretical limits. Only the short-range term is significant at moderate-to-high ionic strength where our results coincide with the predictions of Fixman-Le Bret. The bending of DNA on the protein surface that is accompanied by an asymmetric neutralization of the DNA charge is also analyzed. In this case, the electrostatic bending energy gives a significant favorite contribution to the total bending energy of DNA. Important implications to the mechanisms of DNA-protein interactions, particularly in the nucleosome particle, are discussed.

Journal Article↗