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.     

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.     

Effects of sequence alterations in a DNA segment containing the 5 S RNA gene from Lytechinus variegatus on positioning of a nucleosome core particle in vitro

Reassociation of a 260-base pair cloned fragment of Lytechinus variegatus DNA with core histones has been shown to give rise to a uniquely positioned nucleosome (Simpson, R. T., and Stafford, D. W. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 51-55). In an attempt to define the features that dictate the unique positioning of the nucleosome, we have constructed a number of mutants of this DNA sequence. The ability of these mutants to form positioned nucleosomes was analyzed by DNase I digestion of the DNA after reassociation with chicken erythrocyte core histones. While all the mutants were efficiently incorporated into core particles, not all of these modified sequences were capable of forming a positioned nucleosome. Of the 13 mutants examined, 7 fell into a class that gave rise to nucleosomes in which no unique positioning could be demonstrated. While no specific feature of the DNA sequences has been identified as the critical factor in allowing, or dictating, the formation of positioned nucleosomes, our results do indicate that the region 20-30 bases either side of the center of the core particle appears to contain the major elements necessary for positioning. Additionally, these studies clearly show that differences in the digestion of naked and core particle DNA are related to specific interactions of the DNA and histones rather than to an altered specificity of the enzyme induced by the presence of the proteins.     

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.     

cis- and trans-diamminedichloroplatinum(II) interstrand cross-linking of a defined sequence nucleosomal core particle

Interstrand cross-linking studies with the antitumor drug cis-diamminedichloroplatinum(II) and its clinically inactive isomer, trans-diamminedichloroplatinum(II), were performed on a fragment of the 5S rRNA gene of Xenopus borealis in the free and nucleosomal state. 5S nucleosomes were formed via histone octamer exchange from chicken erythrocyte core particles. Native polyacrylamide gel electrophoresis was used to probe the ability of platinated DNA to reconstitute into core particles. Both isomers negatively impacted reconstitution when histones were present during incubation with the drug. When histones were not present during the drug treatment, platinated DNA was successfully reconstituted into core particles. These results suggest that platination of histones impedes reconstitution of free DNA. However, already-formed core particles were not disrupted upon platination. Sites of interstrand cross-linking were probed through denaturing polyacrylamide gel electrophoresis and quantitative phosphorimagery. We found both site-specific enhancement and depression of cis-diamminedichloroplatinum(II) cross-linking in the nucleosomal samples relative to free DNA at both drug concentrations that were tested (0.01 and 0.0025 mM). trans-Diamminedichloroplatinum(II) exhibited no detectable differences in the interstrand cross-linking of free and nucleosomal samples.     

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn²⁺ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.     

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.     

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.     

A bulky DNA lesion derived from a highly potent polycyclic aromatic tumorigen stabilizes nucleosome core particle structure

The impact of a bulky DNA lesion on the structure and dynamics of a nucleosome core particle (NCP) containing a lesion derived from the unusually potent tumorigen dibenzo[a,l]pyrene that resists nucleotide excision repair (NER) in free DNA was investigated using 65 ns molecular dynamics simulations. Our results reveal that, relative to unmodified NCP, the lesion stabilizes the nucleosome via stacking interactions, improved Watson-Crick base pairing, hydrogen bonding between DNA and histones, and damped dynamics. These findings suggest that such lesions should be as resistant to NER in the nucleosome environment as they are in free DNA.     

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.     

Maintenance DNA methylation of nucleosome core particles

The enzyme responsible for maintenance methylation of CpG dinucleotides in vertebrates is DNMT1. The presence of DNMT1 in DNA replication foci raises the issue of whether this enzyme needs to gain access to nascent DNA before its packaging into nucleosomes, which occurs very rapidly behind the replication fork. Using nucleosomes positioned along the 5 S rRNA gene, we find that DNMT1 is able to methylate a number of CpG sites even when the DNA major groove is oriented toward the histone surface. However, we also find that the ability of DNMT1 to methylate nucleosomal sites is highly dependent on the nature of the DNA substrate. Although nucleosomes containing the Air promoter are refractory to methylation irrespective of target cytosine location, nucleosomes reconstituted onto the H19 imprinting control region are more accessible. These results argue that although DNMT1 is intrinsically capable of methylating some DNA sequences even after their packaging into nucleosomes, this is not the case for at least a fraction of DNA sequences whose function is regulated by DNA methylation.     

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.     

Sequence periodicities in chicken nucleosome core DNA

The rotational positioning of DNA about the histone octamer appears to be determined by certain sequence-dependent modulations of DNA structure. To establish the detailed nature of these interactions, we have analysed the sequences of 177 different DNA molecules from chicken erythrocyte core particles. All variations in the sequence content of these molecules, which may be attributed to sequence-dependent preferences for DNA bending, correlate well with the detailed path of the DNA as it wraps around the histone octamer in the crystal structure of the nucleosome core. The sequence-dependent preferences that correlate most closely with the rotational orientation of the DNA, relative to the surface of the protein, are of two kinds: ApApA/TpTpT and ApApT/ApTpT, the minor grooves of which face predominantly in towards the protein; and also GpGpC/GpCpC and ApGpC/GpCpT, whose minor grooves face outward. Fourier analysis has been used to obtain fractional variations in occurrence for all ten dinucleotide and all 32 trinucleotide arrangements. These sequence preferences should apply generally to many other cases of protein-DNA recognition, where the DNA wraps around a protein. In addition, it is observed that long runs of homopolymer (dA) X (dT) prefer to occupy the ends of core DNA, five to six turns away from the dyad. These same sequences are apparently excluded from the near-centre of core DNA, two to three turns from the dyad. Hence, the translational positioning of any single histone octamer along a DNA molecule of defined sequence may be strongly influenced by the placement of (dA) X (dT) sequences. It may also be influenced by any aversion of the protein for sequences in the "linker" region, the sequence content of which remains to be determined.     

Characteristics of nucleosome core DNA and their applications in predicting nucleosome positions

By analyzing dinucleotide position-frequency data of yeast nucleosome-bound DNA sequences, dinucleotide periodicities of core DNA sequences were investigated. Within frequency domains, weakly bound dinucleotides (AA, AT, and the combinations AA-TT-TA and AA-TT-TA-AT) present doublet peaks in a periodicity range of 10-11 bp, and strongly bound dinucleotides present a single peak. A time-frequency analysis, based on wavelet transformation, indicated that weakly bound dinucleotides of core DNA sequences were spaced smaller (∼10.3 bp) at the two ends, with larger (∼11.1 bp) spacing in the middle section. The finding was supported by DNA curvature and was prevalent in all core DNA sequences. Therefore, three approaches were developed to predict nucleosome positions. After analyzing a 2200-bp DNA sequence, results indicated that the predictions were feasible; areas near protein-DNA binding sites resulted in periodicity profiles with irregular signals. The effects of five dinucleotide patterns were evaluated, indicating that the AA-TT pattern exhibited better performance. A chromosome-scale prediction demonstrated that periodicity profiles perform better than previously described, with up to 59% accuracy. Based on predictions, nucleosome distributions near the beginning and end of open reading frames were analyzed. Results indicated that the majority of open reading frames’ start and end sites were occupied by nucleosomes.