Transient electric dichroism studies of nucleosomal particles

Crothers, Donald M.; Dattagupta, Nanibhushan; Hogan, Michael E.; Klevan, Leonard; Lee, K. S.

doi:10.1021/bi00614a026

Cited by 75 publications

(80 citation statements)

References 19 publications

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“…However, at the same time, a change appears in the birefringence sign, indicating a modification of the orientation axis [6]. Such a reversal of sign has been very recently observed by Crothers and his group: they found positive electric dichroism for compact chromatin fibers containing at least 45 nucleosomes [50] in contrast to the negative dichroism of mononucleosomes and dinucleosomes [51].…”

Section: Conformation Qf Oligomers In Solutionmentioning

confidence: 79%

Conformation of Chromatin Oligomers

Marion¹,

Bezot²,

Hesse-Bezot³

et al. 1981

European Journal of Biochemistry

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Quasielastic laser light scattering measurements have been made on chromatin oligomers to obtain information on the transition in their electrooptical properties, previously observed for the hexameric structures [Marion, C. and Roux, B. (1978) Nucleic Acids Res. 5, 4431 -44491. Translational diffusion coefficients were determined for mononucleosomes to octanucleosomes containing histone HI over a range of ionic strength. At high ionic strength, oligomers show a linear dependence of the logarithm of diffusion coefficient upon the logarithm of number of nucleosomes. At low ionic strength a change occurs between hexamer and heptamer. Our results agree well with the recent sedimentation data of Osipova et al. [Eur. J . Biochem. (1980) 113, 183 -1881 and of Butler and Thomas [J. Mol. Biol. (1980) 140, 505-5291 showing a change in stability with hexamer. Various models for the arrangements of nucleosomes in the superstructure of chromatin are discussed. All calculations clearly indicate a conformational change with the hexanucleosome and the results suggest that, at low ionic strength, the chromatin adopts a loosely helical structure of 28-nm diameter and 22-nm pitch. These results are also consistent with a discontinuity every sixth nucleosome, corresponding to a turn of the helix. This discontinuity may explain the recent electric dichroism data of Lee et al. [Biochemistry (1981) 20, 1438 -14451. The hexanucleosome structure which we have previously suggested, with the faces of nucleosomes arranged radially to the helical axis has been recently confirmed by Mc Ghee et al. [Cell (1980) 22, 87-96]. With an increase of ionic strength, the helix becomes more regular and compact with a slightly reduced outer diameter and a decreased pitch, the dimensions resembling those proposed for solenoid models.A detailed knowledge of the structural organization of chromatin is required to understand the mechanisms involved in gene expression. In order to account for the highly condensed state of chromosomes, the chromatin is organized into higher-order structures whose diameter (20 -30 nm) is about twice that of nucleosomes, the basic chromatin units (for reviews see [I]). It is not clear how nucleosome chains are folded or coiled in the chromosome fiber and this packing of nucleosomes has been discussed in terms of either solenoidal structure or helical arrays having about 6 -10 nucleosomes per turn, or of 'superbeads' or 'clustered arrays' containing several nucleosomes. The evidence in favour of superbeads has. however, been questioned recently [ 2 ] . Much of the data supporting these models comes from electron microscopy and it appeared recently that this technique may be subject to possible artefacts, especially at low ionic strengths [3 -51. Evidence from other methods is thus needed. In previous papers [6,7], we reported transient electric birefringence studies of rat liver chromatin oligomers. They showed a change in conformation with hexamer. This result is confirmed by recent sedimentation data [5] and birefringence ...

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Section: Conformation Qf Oligomers In Solutionmentioning

confidence: 79%

Conformation of Chromatin Oligomers

Marion¹,

Bezot²,

Hesse-Bezot³

et al. 1981

European Journal of Biochemistry

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“…As seen in Table 1 'Orientation of the dye absorption dipole relative to the helix axis at the dye binding site. a was calculated from data with nucleosomes by using methods described elsewhere (19 ends of the helix in the nucleosome because such specific binding would lead to averaged dichroism for MB, which would be much larger than the (solenoidally averaged) value measured for the base planes in the nucleosome (19).…”

Section: Resultsmentioning

confidence: 99%

DNA motions in the nucleosome core particle.

Wang¹,

Hogan²,

Austin³

1982

Proc. Natl. Acad. Sci. U.S.A.

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We have used time-resolved triplet state anisotropy decay techniques to measure the conformational flexibility of DNA in the nucleosome. From these measurements we conclude that, in a nucleosome, the DNA helix experiences substantial internal flexibility, which occurs with a time constant near 30 nsec. We find that our data can be fit well by a modified version of the Barkley6-Zimm model for DNA motion, allowing only DNA twisting motions and the overall tumbling of the nucleosome. That fit yields a calculated torsional rigidity equal to 1.8 x 109. erg-cm, a value equal to that measured for uncomplexed DNA. We conclude from such similarity that large, fast twisting motions of the DNA helix persist, nearly unaltered, when DNA is wrapped to form a nucleosome.The nucleosome, the basic structural unit of eukaryotic chromatin,. consists of 146 base pairs (bp) of DNA helix complexed with an octamer of histones (H3, .H4, H2A, and H2B). The structure of the nucleosome has been studied extensively. As revealed by low-resolution x-ray diffraction (1) and low~angle neutron scattering (2), the nucleosome can be described as a stubby cylinder with radius of 55 A and length of 60 A. The DNA helix wraps around the octamer core to form approximately 13/4 turns of left-handed superhelix.The thermal stability and conformation of the nucleosome are known to be affected by solvent conditions and temperature (3); however, very little is known about the dynamic properties of the nucleosome.Earlier, we showed that triplet state anisotropy decay can be used to monitor nanosecond time scale twisting motions that occur in the DNA helix (4). DNA motions ofthis kind have been detected by using NMR (5, 6), fluorescence anisotropy decay (7-9), and ESR techniques (10) and are of considerable interest because such motions are a direct measure of the elasticity of DNA structure and because large-amplitude conformational flexibility is sure to affect the specific binding of proteins.Because in eukaryotes most DNA exists as a nucleosome complex in chromatin, it is of importance to understand the internal flexibility ofnucleosomal DNA. On the basis of 31P NMR measurements, Klevan et al. (11) suggested that a large motion with a time.constant near 1 nsec.occurs in the phosphate backbone ofDNA in the nucleosome. On the basis ofmore extensive 31P relaxation measurements Shindo and McGhee (12) came to similar conclusions concerning DNA motion; their best fit to the data suggested that the phosphate. backbone expresses motions in the nucleosome with time constants nearer to 30 nsec.However,. on the basis of 'H NMR linewidth measurements, Feigon and Kearns (13) concluded that DNA is in fact held rigidly in the nucleosome. Because of such controversy in the interpretation ofNMR data, we have chosen to study DNA conformational flexibility by using triplet anisotropy decay, a technique that can measure motion over a very wide, time range. *Here, we describe time-dependent triplet state anisotropy measurements ofthe nucleosome core particle, tagged ...

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“…5). Electric dichroism studies of isolated nucleosomal particles also indicate that the DNA straightens as it exits the nucleosome (26).…”

Section: Plot D)mentioning

confidence: 99%

Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin.

Pehrson

1989

Proc. Natl. Acad. Sci. U.S.A.

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Photo-induced thymine dimer formation was used to probe nucleosome structure in nuclei. The distribution of thymine dimers in the nucleosome and recent studies of the structure of thymine dimer-containing DNA suggest that the rate of thymine dimer formation is affected by the direction and degree of DNA bending. This premise was used to construct a model of the path of DNA in the nucleosome, which has the following features. (i) There are four regions of sharp bending, two which have been seen previously by x-ray crystallography of the core particle. (i) The DNA in Hi-containing nucleosomes deviates from its superhelical path near the midpoint; this is not seen with Hl-stripped chromatin. (ii) The internucleosomal (linker) DNA appears to be relatively straight.Much of the knowledge about nucleosome structure comes from studies of isolated nucleosome core particles. Such studies provide only limited information about native chromatin structure since these particles lack histone H1, the internucleosomal (linker) DNA, and any constraints imposed by higher order chromatin structure. Therefore, it is valuable to develop methods to study nucleosome structure in situ. One such method is photodimerization of pyrimidines. Recently, the distribution of pyrimidine dimers (PDs) in core particles isolated from irradiated chromatin was described in careful detail (1, 2). Peaks of PD formation were found to occur with an average periodicity of 10.3 bases and were interpreted in terms of a footprint of histone-DNA interactions. Recent reports indicate that DNA in the immediate vicinity of a thymine dimer (TD) is bent toward the major groove (3, 4). Therefore, TDs should form preferentially wherever the DNA is already bent toward the major groove, and their distribution might reveal a great deal about the path of DNA in the nucleosome. Very little is known about the effect of H1 on the path of DNA in the nucleosome or about the path of DNA in the internucleosomal (linker) region. As this information is important for understanding higher order chromatin structure and the role of H1 in nucleosome structure, I have explored the possibility of using TD formation as an approach to gain such information. (5) except for the peak at 4.726; instead they reported a peak at 2.246. Also, the melting point differs from the one they reported, 283-284TC. However, the NMR spectrum and elemental analysis given here are consistent with the structure of AcOD, and the compound was found to be effective in greatly stimulating the production of TDs in DNA irradiated with light >310 nm. MATERIALS AND METHODSIsolation of Nuclei. All steps were carried out at 0-40C unless otherwise stated. Frozen rat livers (Pel-Freez Biologicals) were homogenized with a Waring blender in buffer A [0.34 M mannitol/60 mM KCl/15 mM NaCl/0.15 mM spermine/0.5 mM spermidine/2 mM EDTA/0.5 mM EGTA/15 mM triethanolamine/1% thiodiglycol/0.25 mM phenylmethanesulfonyl fluoride, pH 7.3] and filtered through cheese cloth, and nuclei were isolated essentially as described (8...

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Transient electric dichroism studies of nucleosomal particles

Cited by 75 publications

References 19 publications

Conformation of Chromatin Oligomers

Conformation of Chromatin Oligomers

DNA motions in the nucleosome core particle.

Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin.

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