Effect of Z-DNA on nucleosome placement

Histone octamers were reconstituted on plasmids carrying the alternating nucleotide sequence (G-C)15. The plasmids, radioactively labeled at one of two neighboring sites near the (G-C) insert, were digested with micrococcal nuclease. Nucleosome core particles were isolated and the monomer DNA subjected to restriction analysis. Quite different results are obtained if the reconstitution is carried out with relaxed plasmids, in which the (G-C) insert is in the B form, or with supercoiled plasmids, where it is in the Z form. With supercoiled plasmids, there is a marked reduction (compared with relaxed plasmids) in the abundance of labeled monomers, the result of a large decrease in core particles carrying any (G-C) sequence. Some core particles formed on supercoiled (Z) plasmids are positioned either just outside the (G-C) sequence, or with the sequence occupying the terminal position within the core particle. In contrast, monomers obtained from relaxed plasmids incorporate the (G-C) sequence in the B form more or less randomly in the interior of the core particle; species showing discrete positioning make only a minor contribution. We conclude that DNA in the Z form cannot be incorporated within core particles, except at their termini, and that a transition from the B to the Z form in vivo might result in a significantly altered local placement of nucleosomes.     

Reconstitution of mononucleosomes: characterization of distinct particles that differ in the position of the histone core

Reconstitution of mononucleosomes from DNA and core histones was carried out to study the positioning of histone octamers on the DNA. Using random DNA molecules in the 200 to 250 bp size range we found that the reconstitution products consisted of a mixture of three different types of particles that could be separated by low ionic strength gel electrophoresis. In one particle, DNA was complexed with histones along its entire length indicating the binding of more than one histone octamer. The second particle contained only one histone core that was always associated, however, with the terminal 145 bp of the DNA regardless of its sequence which can be ascribed to a DNA end effect. Only the third particle consisted of histone octamers bound at internal positions of the DNA and is therefore the only particle suitable for investigating the influence of the DNA sequence on the positioning of the histone cores. A defined 154 bp pBR 322 restriction fragment that contains three BspRI restriction sites was also reconstituted with core histones. The accessibility of these sites to BspRI was measured in order to delineate the utility of restriction nucleases as probes for the structure of chromatin. Two sites located close to the center of the DNA were less susceptible by at least a factor of 1000 as compared to free DNA while the susceptibility of the third site in the terminal section of the DNA decreased about 50 fold after reconstitution.     

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.     

H2A and H2B tails are essential to properly reconstitute nucleosome core particles

The conformation of recombinant Nucleosome Core Particles (NCPs) lacking H2A and H2B histone tails (gH2AgH2B) are studied. The migration of these particles in acrylamide native gels is slowed down compared to intact reconstituted NCPs. gH2AgH2B NCPs are also much more sensitive to nuclease digestion than intact NCPs. Small angle X-ray scattering (SAXS) experiments point out that the absence of H2A and H2B tails produces small but significant conformational changes of the octamers conformation (without wrapped DNA), whereas gH2AgH2B NCP conformations are significantly altered. A separation of about 25–30 bp from the core could account for the experimental curves, but other types of DNA superhelix deformation cannot be excluded. The distorted gH2AgH2B octamer may not allow the correct winding of DNA around the core. The absence of the H2A and H2B tails would further prevent the secondary sliding of the DNA around the core and therefore impedes the stabilisation of the particle. Cryo-electron microscopy on the same particles also shows a detachment of DNA portions from the particle core. The effect is even stronger because the vitrification of the samples worsens the instability of gH2AgH2B NCPs.     

X-ray scattering study of the shape of the DNA region in nucleosome core particle with synchrotron radiation.

The structure of the DNA region in rat thymus nucleosome core particle has been studied by synchrotron X-ray scattering analysis and the contrast-variation technique has been applied to determine the contribution of the DNA to the total scatterings. Small-angle contrast-matching measurements show that the entire core particle and isolated histone octamers are contrast-matched by solvents containing 64 and 54% (w/w) sucrose, respectively. At a contrast of 54% sucrose, where the scattering of the DNA dominates, the scattering data extending to higher angle of about 0.05 A-1 have been collected from relatively concentrated solutions (10 mg/ml) of core particles and interpreted on the basis of the regular helical model for the DNA region. The model calculations show that the shape of the DNA around the histone core is approximately by 1.8 turns of regular helix of 42 A radius and 28 A pitch. These values for helical parameters of our model are in good agreement with those of the structure of DNA in crystallized nucleosome cores shown by earlier diffraction studies.     

X-ray diffraction study of a new crystal form of the nucleosome core showing higher resolution

Crystals have been grown of intact (unproteolysed) nucleosome cores from a variety of sources. The unit cells are all very similar, with one core particle per asymmetric unit. The X-ray diffraction patterns extend to about 5 Å in the direction perpendicular to the plane of the flat particle, and to somewhat less than this in other directions. The arrangement of particles in the unit cell has been deduced from Patterson projection maps, which also indicate the presence of a particle dyad. The data are consistent with the earlier proposed model for the core particle in which the 146 base-pairs of DNA are wound in about $\text{1}\text{3}\text{4}$ turns of superhelix about a histone octamer core.High angle diffuse X-ray scattering from the crystals shows that the DNA of the core particle is in the B form. The anisotropy of the diffuse scattering shows that the DNA is not firmly fixed to the histone core all along the superhelix path, but only over limited regions whose location correlates well with those in which the DNA is differentially protected against nuclease digestion.     

Effect of glycerol on the separation of nucleosomes and bent DNA in low ionic strength polyacrylamide gel electrophoresis.

We report that glycerol changes the separation characteristics of polyacrylamide nucleoprotein gels in which it is included as a stabilizing agent. Polyacrylamide gel electrophoresis fractionates DNA and nucleosomes according to net negative charge, mass and conformation. With glycerol included, fractionation seems to be largely based on particle mass and charge. The conformation factor in separation is progressively lost with increasing glycerol concentrations. Nucleosome positions on the same DNA fragment are no longer resolved, while the difference in electrophoretic mobility between core particles and nucleosomes carrying longer DNA becomes smaller and is eventually lost. The retardation of bent DNA is also much reduced. Using the differences in separation characteristics between glycerol-containing and regular nucleoprotein gels could be a new means to obtain information on macromolecules in solution.     

Isolation and characterization of acetylated histones H3 and H4 and their assembly into nucleosomes

Nucleosome and chromatin structure/function relationships of histone acetylations are not understood. To address these questions we have developed chromatographic procedures that separate subtypes of H3 and the acetylated states of histone H3 and H4 in exceptionally pure forms. The sites of acetylation of the intermediately acetylated states of H3 have been determined and show a specific pattern of acetylation. An unexpected finding was the identification of a fifth site of acetylation in H3 at lysine 27. Nucleosome particles with fully acetylated H3 and H4 have been assembled on the Lytechinus variegatus 5 S rRNA DNA phasing sequence and characterized. These defined acetylated H3 and H4 particles migrate more slowly in polyacrylamide nucleoprotein particle gels than the control particles indicating a subtle effect of acetylation in nucleosome structure. However, DNA footprinting of these particles using DNase I show only small changes when compared to control particles over the core particle DNA length. It is shown further that H3 cysteines in the particle containing fully acetylated H3 and H4 were not accessible to iodoacetamide indicating that protein factors additional to H3 and H4 acetylation are required to make H3 cysteines accessible to the label. These findings are consistent with the proposal that histones H3, H4 acetylations exert their major effects outside of the core particle 146-base pair DNA, either on the DNA segment entering and leaving the nucleosome or possibly on the internucleosome interactions that involve the amino-terminal domains of the core histones in organization and stability of higher order chromatin structures.     

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.     

lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

Crystallization of the DpnM methylase from the DpnII restriction system of Streptococcus pneumoniae

Three proteins, two DNA methylases and an endonuclease, from the DpnII restriction system of Streptococcus pneumoniae recognize the DNA sequence 5' GATC 3' but have very different amino acid sequences, which make them interesting subjects for structural determination. A purification procedure was developed that conveniently yields milligram amounts of the DpnM methylase. The DpnM protein tends to precipitate at reduced ionic strength, and this property was exploited to yield well-formed bipyramidal crystals. By X-ray diffraction, the crystals of DpnM were found to be orthorhombic, with cell dimensions a = 56.9 A, b = 68.2 A, c = 84.5 A; systematic absences identify the space group as P2(1)2(1)2(1). Diffraction extends beyond 3 A, so the crystals may allow structural determination at atomic resolution.     

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.     

Supercoiled DNA is interwound in liquid crystalline solutions.

Two structures have been proposed for supercoiled DNA: it is idealized either as a toroidal ring or as a rod of two interwound duplex chains. The latter model is the most widely depicted but the evidence remains controversial. We have worked with monomers and dimers of two plasmids, pUC8 and pKS414, of similar size and natural superhelical density. pKS414 contains a bend promoting sequence whereas pUC8 does not. In concentrated solutions these plasmids form a partially ordered liquid crystalline phase which is found, using neutron diffraction, to consist of a hexagonally packed assembly of parallel rod-like particles. This shape strongly suggests an interwound conformation for which some structural parameters are deduced. The mass/unit length obtained by combining the area of the hexagonal lattice and the concentration is approximately 3.6 times that of linear DNA. This implies a shallow superhelical pitch angle approximately 36 degrees which, when combined with the known number of supercoil turns, yields the pitch approximately 360 A and radius approximately 80 A for the supercoil. Oriented X-ray fibre diffraction patterns at 92% relative humidity indicate a B type duplex structure. Nicked circular plasmids also form liquid crystals but their behaviour, as a function of concentration, differs from that of the superhelical plasmids.     

Crystal Structures of Nucleosome Core Particles Containing the ‘601’ Strong Positioning Sequence

Nucleosome positioning plays a key role in genomic regulation by defining histone-DNA context and by modulating access to specific sites. Moreover, the histone-DNA register influences the double-helix structure, which in turn can affect the association of small molecules and protein factors. Analysis of genomic and synthetic DNA has revealed sequence motifs that direct nucleosome positioning in vitro; thus, establishing the basis for the DNA sequence dependence of positioning would shed light on the mechanics of the double helix and its contribution to chromatin structure in vivo. However, acquisition of well-diffracting nucleosome core particle (NCP) crystals is extremely dependent on the DNA fragment used for assembly, and all previous NCP crystal structures have been based on human α-satellite sequences. Here, we describe the crystal structures of Xenopus NCPs containing one of the strongest known histone octamer binding and positioning sequences, the so-called ‘601’ DNA.Two distinct 145-bp 601 crystal forms display the same histone-DNA register, which coincides with the occurrence of DNA stretching-overtwisting in both halves of the particle around five double-helical turns from the nucleosome center, giving the DNA an ‘effective length’ of 147 bp. As we have found previously with stretching around two turns from the nucleosome center for a centromere-based sequence, the terminal stretching observed in the 601 constructs is associated with extreme kinking into the minor groove at purine-purine (pyrimidine-pyrimidine) dinucleotide steps. In other contexts, these step types display an overall nonflexible behavior, which raises the possibility that DNA stretching in the nucleosome or extreme distortions in general have unique sequence dependency characteristics. Our findings indicate that DNA stretching is an intrinsically predisposed site-specific property of the nucleosome and suggest how NCP crystal structures with diverse DNA sequences can be obtained.     

Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism

A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library. A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library. Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone. Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6. Individual-specific complex pattern of DNA bands was produced. TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism.     

Nucleosomes are positioned on mouse satellite DNA in multiple highly specific frames that are correlated with a diverged subrepeat of nine base-pairs

Nucleosome phasing on highly repetitive DNA was investigated using a novel strategy. Nucleosome cores were prepared from mouse liver nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population that contains satellite sequences was then purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling, the termini of the satellite core DNA fragments were mapped with an accuracy of +/- 1 base-pair relative to known restriction sites on the satellite DNA. Sixteen dominant nucleosome positions were detected. There is a striking correlation between these nucleosome frames and an internal highly diverged 9 base-pair subrepeat of the satellite DNA. The results are consistent with a sequence-dependent association of histone octamers with the satellite DNA. Our finding that histone octamers can interact with a given DNA in a number of different defined frames has important implications for the possible biological significance of nucleosome phasing.     

Site-specific aflatoxin B1 adduction of sequence-positioned nucleosome core particles

The question of how the presence of nucleosomal packing of DNA modifies carcinogen interaction at specific sites cannot be answered by studies on whole chromatin or bulk nucleosomes because of the heterogeneity of DNA sequences in the particles. We have circumvented this problem by using nucleosomes that are homogenous in DNA sequence and hence in DNA-histone contact points. A cloned DNA fragment containing a sea urchin 5 S gene which precisely positions a histone octamer was employed. By using 32P end-labeled DNA and genotoxins that allow cleavage at sites of attack, the frequency of adduction at every susceptible nucleotide can be determined on sequencing gels. The small methylating agent dimethyl sulfate and the bulky alkylating agent aflatoxin B1-dichloride (AFB1-Cl2) were used to probe the influence of DNA-histone interactions on DNA alkylation patterns in the sequence-positioned core particle. We find dimethyl sulfate to bind with equal preference to naked or nucleosomal DNA. In contrast, AFB1-Cl2 binding is suppressed an average of 2.4-fold at guanyl sites within nucleosomes compared with AFB1-Cl2 affinity at the corresponding site in naked DNA. The DNA is more accessible in regions near the particle boundary. We observe no other histone-imposed localized changes in AFB1-Cl2 sequence specificity. Further, sites of DNase I cleavage or proposed DNA bending show neither enhanced nor reduced AFB1-Cl2 adduction to N7-guanine. Since AFB1-Cl2 binding sites lie in the major groove, nucleosomal DNA appears accessible to AFB1-Cl2 at all points of analysis but with an access which is uniformly restricted in the central 100 nucleotides of the core particle. The data available do not indicate further localized or site-specific perturbations in DNA interactions with the two carcinogens studied.     

Transcriptional and translational mapping and nucleotide sequence analysis of a vaccinia virus gene encoding the precursor of the major core polypeptide 4b

We prepared antiserum that reacted with a major core polypeptide of approximately 62,000 daltons (62K polypeptide), designated 4b, and its 74K precursor, designated P4b. A cell-free translation product of vaccinia virus late mRNA that comigrated with P4b was specifically immunoprecipitated. The late mRNA encoding P4b hybridized to restriction fragments derived from the left end of the HindIII A fragment and to a lesser extent from the right side of the HindIII D fragment. A polypeptide that comigrated with P4a, the precursor of another major core polypeptide, was synthesized by mRNA that hybridized to DNA segments upstream of the P4b gene. Complete nucleotide sequence analysis of the P4b gene revealed an open reading frame, entirely within the HindIII A fragment, that was sufficient to encode a 644-amino-acid polypeptide of 73K. The 5' end of the P4b mRNA was located at or just above the translational initiation site.