Structure of the nucleosome core particle at 8 A resolution

The x-ray crystallographic structure of the nucleosome core particle has been determined using 8 A resolution diffraction data. The particle has a mean diameter of 106 A and a maximum thickness of 65 A in the superhelical axis direction. The longest chord through the histone core measures 85 A and is in a non-axial direction. The 1.87 turn superhelix consists of B-DNA with about 78 base pairs or 7.6 helical repeats per superhelical turn. The mean DNA helical repeat contains 10.2 +/- 0.05 base pairs and spans 35 A, slightly more than standard B-DNA. The superhelix varies several Angstroms in radius and pitch, and has three distinct domains of curvature (with radii of curvature of 60, 45 and 51 A). These regions are separated by localized sharper bends +/- 10 and +/- 40 base pairs from the center of the particle, resulting in an overall radius of curvature about 43 A. Compression of superhelical DNA grooves on the inner surface and expansion on the outer surface can be seen throughout the DNA electron density. This density has been fit with a double helical ribbon model providing groove width estimates of 12 +/- 1 A inside vs. 19 +/- 1 A outside for the major groove, and 8 +/- 1 A inside vs. 13 +/- 1 A outside for the minor groove. The histone core is primarily contained within the bounds defined by the superhelical DNA, contacting the DNA where the phosphate backbone faces in toward the core. Possible extensions of density between the gyres have been located, but these are below the significance level of the electron density map. In cross-section, a tripartite organization of the histone octamer is apparent, with the tetramer occupying the central region and the dimers at the extremes. Several extensions of histone density are present which form contacts between nucleosomes in the crystal, perhaps representing flexible or "tail" histone regions. The radius of gyration of the histone portion of the electron density is calculated to be 30.4 A (in reasonable agreement with solution scattering values), and the histone core volume in the map is 93% of its theoretical volume.     

X-ray structure of the nucleosome core particle

Two monoclinic crystal forms (P2(1),C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2(1) crystal form has been solved to 15 A resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2(1) asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions

Chromatin is composed of nucleosomes, the universally repeating protein-DNA complex in eukaryotic cells. The crystal structure of the nucleosome core particle from Saccharomyces cerevisiae reveals that the structure and function of this fundamental complex is conserved between single-cell organisms and metazoans. Our results show that yeast nucleosomes are likely to be subtly destabilized as compared with nucleosomes from higher eukaryotes, consistent with the idea that much of the yeast genome remains constitutively open during much of its life cycle. Importantly, minor sequence variations lead to dramatic changes in the way in which nucleosomes pack against each other within the crystal lattice. This has important implications for our understanding of the formation of higher order chromatin structure and its modulation by post-translational modifications. Finally, the yeast nucleosome core particle provides a structural context by which to interpret genetic data obtained from yeast. Coordinates have been deposited with the Protein Data Bank under accession number 1ID3.     

ESR spin label studies of the nucleosome core particle and histone core

An imidazole spin label has been used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosyl residues in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tryosyls in the nucleosome core particle can be labeled even at 200- to 300-fold M excess of label. The effect of urea on the conformational state of the spin-labeled tyrosyls in both the nuclesome core particles and the histone core has been studied. Ionic effects on the spin-labeled nucleosome core have been investigated. Several conformational transitions are observed in the range of 1 mM NaCl to 2.5 M NaCl. Three major transitions are found at 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl, respectively. The observed changes can be interpreted as swelling and conformational change of the inner histone core, gradual separation of DNA from the histone core, and tightening of the histone core.     

Helical repeat of DNA in the nucleosome core particle

Although the crystal structure of nucleosome core particle is essentially symmetrical in the vicinity of the dyad, the linker histone binds asymmetrically in this region to select a single high-affinity site from potentially two equivalent sites. To try to resolve this apparent paradox we mapped to base-pair resolution the dyads and rotational settings of nucleosome core particles reassembled on synthetic tandemly repeating 20 bp DNA sequences. In agreement with previous observations, we observed (1) that the helical repeat on each side of the dyad cluster is 10 bp maintaining register with the sequence repeat and (2) that this register changes by 2 bp in the vicinity of the dyad. The additional 2 bp required to effect the change in the rotational settings is accommodated by an adjustment immediately adjacent to the dyad. At the dyad the hydroxyl radical cleavage is asymmetric and we suggest that the inferred structural asymmetry could direct the binding of the linker histone to a single preferred site.     

Histone acetylation reduces nucleosome core particle linking number change

Nucleosome core particles differing in their levels of histone acetylation have been formed on a closed circular DNA that contains a tandemly repeated 207 bp nucleosome positioning sequence. The effect of acetylation on the linking number per nucleosome particle has been determined. With increasing levels of acetylation, the negative linking number change per nucleosome decreases from -1.04 +/- 0.08 for control to -0.82 +/- 0.05 for highly acetylated nucleosomes. These results indicate that histone acetylation has the ability to release negative supercoils previously constrained by nucleosomes into a closed chromatin loop and in effect function as a eukaryotic gyrase.     

Crystals of a nucleosome core particle containing defined sequence DNA

Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystallized, and the crystals examined by X-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a symmetrical arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange high-pressure liquid chromatography. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base-pairs from the molecular dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a approximately equal to 4 A, b approximately equal to 5 A and c approximately equal to 3 A, giving about a twofold increase in the number of measurable X-ray reflections over previous crystals containing mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.     

Redefinition of the cleavage sites of DNase I on the nucleosome core particle

DNase I has been widely used for the footprinting of DNA-protein interactions including analyses of nucleosome core particle (NCP) structure. Our understanding of the relationship between the footprint and the structure of the nucleosome complex comes mainly from digestion studies of NCPs, since they have a well-defined quasi-symmetrical structure and have been widely investigated. However, several recent results suggest that the established consensus of opinion regarding the mode of digestion of NCPs by DNase I may be based on erroneous interpretation of results concerning the relationship between the NCP ends and the dyad axis. Here, we have used reconstituted NCPs with defined ends, bulk NCPs prepared with micrococcal nuclease and molecular modelling to reassess the mode of DNase I digestion. Our results indicate that DNase I cuts the two strands of the nucleosomal DNA independently with an average stagger of 4 nt with the 3'-ends protruding. The previously accepted value of 2 nt stagger is explained by the finding that micrococcal nuclease produces NCPs not with flush ends, but with approximately 1 nt 5'-recessed ends. Furthermore we explain why the DNA stagger is an even and not an odd number of nucleotides. These results are important for studies using DNase I to probe nucleosome structure in complex with other proteins or any DNA-protein complex containing B-form DNA. We also determine the origin of the 10n +/- 5 nt periodicity found in the internucleosomal ladder of DNase I digests of chromatin from various species. The explanation of the 10n +/- 5 nt ladder may have implications for the structure of the 30 nm fibre.     

Spatial organization of histones and DNA in the nucleosome core particle: A model

We present here an attempt to build up a space-filling model of the nucleosome core particle based on the chemical crosslinking data of Mirzabekov and coworkers (23). It is shown that the models proposed earlier are inconsistent with the results of these authors. The main characteristics of our model are as follows: a) the DNA superhelix contains at least 90 base pairs (bp) per turn; b) the particle has a dyad axis of symmetry; c) the histone octamer may be regarded as consisting of two heterotypic tetramers. The possible shape and function of core histones are discussed in the light of the model.     

Neutron-scattering studies of accurately reconstituted nucleosome core particles and the effect of ionic strength on core particle structure

Chicken erythrocyte nucleosome core particles can be dissociated quantitatively into histones (H3, H4)2 bound to 146 base pairs of DNA, and 2(H2A, H2B). Reconstitution of core particles from the two components produces an 85% yield of particles which neutron scattering studies show to be accurate stoichiometrically and indistinguishable from native core particles: the radii of gyration of the shape, the protein components and the DNA components of the particles are 4.02 nm, 3.3 nm and 4.95 nm respectively. The largest distance and most probable distance which can be drawn in the particles are 11.5 nm and 4.3 nm respectively. The molecular weight of the particles is identical to that of control 'native' core particles. All of these values, within limits of error, are the same as known values for 'native' core particles. These experiments confirm the essential role of histones H3 and H4 in the initial organisation of core-particle structure, make possible the manufacture of perfectly pure and homogeneous core-particle preparations and allow the 100% incorporation of labelled or modified histones. Neutron scattering studies of core particles at high contrast (in D2O and H2O) have been carried out over a range of ionic strengths and pH. No change in structure is detected down to pH 5.5 in 20 mM NaCl or down to ionic strength 2.0 mM at pH 7.     

Histone hyperacetylation. Its effects on nucleosome core particle transitions.

Effects of histone hyperacetylation on transitions of HeLa cell nucleosome core particles were studied. The transitions examined were induced by low salt concentrations, pH, temperature, and nondissociating high salt. Effects of salt dissociation were also examined. The low-salt transition was found to shift to higher ionic strength by approximately three fold for hyperacetylated particles, a change which may be due simply to the increased overall negative charge on the particles caused by acetylation of lysine residues. Some differences were also seen in the way in which core particles refold after exposure to very low salt (which induces a nonreversible change in the particles). Otherwise no significant effects of hyperacetylation were observed.     

Interaction of rat testis protein, TP, with nucleosome core particle

Circular dichroism studies have revealed that addition of testis specific protein, TP in vitro, to rat testes nucleosome core particle resulted in a decrease in the compaction of the core particle DNA. This was also corroborated by thermal denaturation analysis. Addition of TP to nucleosome core particle resulted in the conversion of a biphasic transition towards a single phase. However, at the same time there was a 20% reduction in the overall hyperchromicity of core particle DNA at core particle to TP molar ratios of 1:2 and 1:3. These observations along with our earlier report, showing the DNA melting properties of TP, suggest that TP may play an important role in the disassembly process of nucleosome core particle during spermiogenesis.     

Attenuation of DNA charge transport by compaction into a nucleosome core particle

The nucleosome core particle (NCP) is the fundamental building block of chromatin which compacts ~146 bp of DNA around a core histone protein octamer. The effects of NCP packaging on long-range DNA charge transport reactions have not been adequately assessed to date. Here we study DNA hole transport reactions in a 157 bp DNA duplex (AQ-157TG) incorporating multiple repeats of the DNA TG-motif, a strong NCP positioning sequence and a covalently attached Anthraquinone photooxidant. Following a thorough biophysical characterization of the structure of AQ-157TG NCPs by Exonuclease III and hydroxyl radical footprinting, we compared the dynamics of DNA charge transport in ultraviolet-irradiated free and NCP-incorporated AQ-157TG. Compaction into a NCP changes the charge transport dynamics in AQ-157TG drastically. Not only is the overall yield of oxidative lesions decreased in the NCPs, but the preferred sites of oxidative damage change as well. This NCP-dependent attenuation of DNA charge transport is attributed to DNA–protein interactions involving the folded histone core since removal of the histone tails did not perturb the charge transport dynamics in AQ-157TG NCPs.     

Strategies for crystallizing a chromatin protein in complex with the nucleosome core particle

The molecular details of how chromatin factors and enzymes interact with the nucleosome are critical to understanding fundamental genetic processes including cell division and gene regulation. A structural understanding of such processes has been hindered by the difficulty in producing diffraction-quality crystals of chromatin proteins in complex with the nucleosome. We describe here the steps used to grow crystals of the 300-kDa RCC1 chromatin factor/nucleosome core particle complex that diffract to 2.9-Å resolution. These steps include both pre- and postcrystallization strategies potentially useful to other complexes. We screened multiple variant RCC1/nucleosome core particle complexes assembled using different RCC1 homologs and deletion variants, and nucleosomes containing nucleosomal DNA with different sequences and lengths, as well as histone deletion variants. We found that using RCC1 from different species produced different crystal forms of the RCC1/nucleosome complex consistent with key crystal packing interactions mediated by RCC1. Optimization of postcrystallization soaks to dehydrate the crystals dramatically improved the diffraction quality of the RCC1/nucleosome crystal from 5.0- to 2.9-Å resolution.     

The structure of duplex DNA in the nucleosome core particle: An alternative model

Many studies indirectly indicate that the conformation ofin vivo duplex DNA is the double helix. The most direct view, from the X-ray analysis of the nucleosome core particle, has also been interpreted in terms of the double helix structure. However, an alternative possibility exists; that the duplex adopts a metastable side-by-side conformation which readily converts to the double helix on removal of protein. Evidence for the existence of this conformation has been obtained from a reanalysis of the electron density map for the nucleosome particle.     

Binding of ethidium to the nucleosome core particle. 1. Binding and dissociation reactions

We have examined binding properties of and dissociation induced by the intercalating dye ethidium bromide when it interacts with the nucleosome core particle under low ionic strength conditions. Ethidium binding to the core particle results in a reversible dissociation which requires the critical binding of 14 ethidium molecules. Under low ionic strength conditions, dissociation is about 90% completed in 5 h. The observed ethidium binding isotherm was corrected for the presence of free DNA due to particle dissociation. The corrected curve reveals that the binding of ethidium to the core particle itself is a highly cooperative process characterized by a low intrinsic binding constant of KA = 2.4 X 10(4) M-1 and a cooperativity parameter of omega = approximately 140. The number of base pairs excluded to another dye molecule by each bound dye molecule (n) is 4.5. Through the use of a chemical probe, methidiumpropyl-EDTA (MPE), we have localized the initial binding sites of ethidium in the core particle to consist of an average of 27 +/- 4 bp of DNA that are distributed near both ends of the DNA termini. MPE footprint analysis has also revealed that, prior to dissociation, the fractional population of core particles which bind the dye (f) may be as low as 50%. Comparison of the binding and dissociation data showed that the cooperative maximum of the binding curve occurred at or near the critical value, i.e., at the point where dissociation began. The data were used to generate a detailed model for the association of ethidium with chromatin at the level of the nucleosome.