Secondary Structure of Histones and DNA in Chromatin

A relatively stable specific complex of the chromatin core histones H2A, H2B, H3, and H4 has been obtained in 2 M -NaCl/25 mM sodium phosphate buffer, pH 7.0. The histone core complex has an apparent specific volume of 0.73 ml/g. Its sedimentation coefficient was dependent on rotor speed (angular velocity, w) and attained different stable values at low and high rotor speeds. The drop in sedimentation coefficient occurred sharply between w2 values of about 9 X 106 and 1

mentioning

confidence: 47%

Nucleosome core histone complex isolated gently and rapidly in 2 M NaCl is octameric.

Philip¹,

Jamaluddin²,

Sastry³

et al. 1979

“…Moreover, high salt concentrations may provide screening for the highly charged histones similar to that provided by DNA in chromatin. There is experimental evidence that the conformations of core histones in 2 M NaCl are similar to those that exist in chromatin (20). Also, at this salt concentration histones are completely dissociated from DNA (21).…”

Section: Methodsmentioning

confidence: 76%

Stability of DNA in nucleosomes.

Bina¹,

Sturtevant²,

Stein³

1980

Heats of thermal denaturation of chromatin core particles and core particles with covalently crosslinked histones were measured by differential scanning calorimetry. The additional stabilization of the nucleoprotein complex by crosslinking is not reflected in the transition enthalpy. The contribution of protein denaturation to the total heat was estimated by comparison of core particles with core particle DNA in a hifh-alt solution. By taking into account the temperature dependence of the transition entialpy of DNA, we conclude that the enthalpy change for denaturation of DNA in core particles is nearly the same as that for naked DNA in solution. The nature of the forces responsible for maintaining DNA in a condensed state in the cell nucleus is not yet understood. It is established that the first order of DNA compaction is achieved by the wrapping of from 145 to about 200 base pair length of DNA around histone cores containing two molecules each of the four smaller histones (1-3). These structures, called nucleosomes, are the fundamental repeating units of chromatin (1-3). As a first step toward understanding the DNA compaction process, it is of interest to know the stability of nucleosomal DNA relative to that of naked DNA, as well as the enthalpic and entropic contributions to the free energy of stabilization. In this paper we report the results of a thermodynamic study of the stability of nucleosome core particles, employing differential scanning calorimetry. MATERIALS AND METHODSPreparation of Core Particles. Chicken erythrocyte nuclei were prepared as described by Hymer and Kuff (4). The nuclei were swollen in 0.25 mM EDTA and the very lysine rich histones (Hi, H5) were removed by repeated suspension of the chromatin in 0.7 M NaCl/0.01 M Tris-HCI, pH 8.0/1 mM EDTA (5). The chromatin was subsequently digested with micrococcal nuclease (Worthington) and the digest was fractionated by sucrose gradient centrifugation as described (5). Core particles thus purified contain the four smaller histones in equal amounts, approximately 145 base pairs of DNA, and no HI or H5 histones.

“…There is some evidence suggesting that under such conditions the histones exist as heterotypic tetramers, containing one molecule of each of the four core histones (19). Because these tetramers retain much of the histone secondary structure (19,45), Olins et al (44) have suggested that the cysteine residues are buried between the contact surfaces of two heterotypic tetramers in the intact nucleosome and that dissociation into separate tetramers exposes the SH groups.…”

Section: Resultsmentioning

confidence: 99%

Histone H3 disulfide dimers and nucleosome structure.

Camerini-Otero¹,

Felsenfeld²

1977

The arginine-rich histone, H3, isolated from avian erythrocytes, can dimerize by forming a disulfide linkage between the single cysteine sulfhydryl residues at position 110 of the H3 polypeptide chain. The H3 dimer can be substituted for undimerized H3 in experiments in which the nucleosome is reconstituted from DNA and mixtures of the four "core" histones, H2A, H2B, H3, and 114. We report here that reconstituted nucleosomes containing H3 dimer are indistinguishable, by a number of criteria, either from native nucleosomes or from reconstitutes containing H3 monomer. The criteria include the pattern of susceptibility of the complex to nucleases, the amount of DNA supercoiling induced by histone binding, and the hydrodynamic properties of reconstituted nucleosome "core" preparations.The results suggest that the residues in the neighborhood of position 110 on each H3 molecule are in close contact in the nucleosome. If, as has been proposed, the nucleosome has a dyad axis, then the disulfide bridge between H3 molecules must lie on this axis.An understanding of the biological activity of chromatin will require detailed knowledge of the internal architecture of its fundamental repeating subunit, the nucleosome.Considerable evidence has accumulated that the DNA in the 140-base-pair nucleosome "core" is wrapped around (1, 2) a histone octamer consisting of two of each of the core histones H2A, H2B, H3, and H4 (3). Studies of histone-DNA interactions have shown that the arginine-rich histones, H3 and H4, play a central role in nucleosome organization (4-10). We are therefore interested in obtaining further information about the way in which these histones interact with each other within the nucleosome. An obvious approach is to use chemical crosslinking reagents to estimate the proximity of functional groups in the proteins; there have been many such studies of histone interactions in chromatin (3). However, most crosslinking reagents can react at more than one site, and so far it has not been possible to identify the specific amino acid residues within the histones that are involved in the crosslinking.There is one crosslinking reaction that may avoid some of these problems: the formation of intermolecular disulfide bonds between cysteine residues. This is a particularly useful reaction for the study of histones because H3 is the only histone that has cysteine and in organisms other than higher mammals there is only one such residue per molecule (11)(12)(13)(14). The cysteine at this one position, residue 110 of H3, has been preserved, with the single exception of yeast H3 (15), throughout evolution.It is well known (11,12,16) that H3 molecules can dimerize in vitro through a disulfide bond between the cysteine residues at position 110. We report here that such a dimer is capable of entering into the formation of nucleosomes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §...