Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres.

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.     

Subunit structure of simian-virus-40 minichromosome.

Electron microscopic evidence indicates that Simian virus 40 (SV40) minichromosomes extracted from infected cells consist of 20 +/- 2 nucleosomes, each containing 190 -- 200 base pairs of DNA. About 50% of the nucleosomes are not close together, but connected by segments of DNA of irregular lengths which correspond to about 15% of the viral genome, irrespective of the ionic strength. Micrococcal nuclease digestion studies show that there is about 200 base pairs of DNA in the biochemical unit of SV40 chromatin. Therefore, the visible internucleosomal DNA of the SV40 minichromosome does not arise from an unfolding of a fraction of the 190 - 200 base pairs of DNA initially wound in the nucleosome. These results support the chromatin model which proposes that the same DNA length is contained in the nucleosome and the biochemical unit. Results from extensive micrococcal nuclease digestion suggest that an SV40 nucleosome consists of a 'core' containing a DNA segment of about 135 base pairs associated to a DNA fragment more susceptible to nuclease attack. The addition of histone H1 results in a striking condensation of the SV40 minichromosome, which supports the assumption that histone H1 is involved in the folding of chromatin fibers.     

The helical periodicity of DNA on the nucleosome

The precise number of base pairs per turn of the DNA double helix in the nucleosome core particle has been the subject of controversy. In this paper the positions of nuclease cutting sites are analysed in three dimensions. Using this midpoint of the DNA on the nucleosome dyad as origin, the cutting site locations measured along a strand of DNA are mapped onto models of the nucleosome core containing DNA of different helical periodicities. It is found that a helical periodicity of 10.5 base pairs per turn leads to cutting site positions which are sterically inaccessible. In contrast, a periodicity of 10.0 base pairs per turn leads to cutting site positions which are not only sterically sound, but which fall into a pattern such as would be expected when the access of the nuclease to the DNA is restricted by the presence of the histone core on one side and of the adjacent superhelical turn of DNA on the other. As proposed earlier by us (1), a value for the helical periodicity close to 10 base pairs per turn on the nucleosome, taken together with a periodicity close to 10.5 for DNA in solution - a value now established - resolves the so-called linkage number paradox.     

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.     

DNA sequence directs placement of histone cores on restriction fragments during nucleosome formation.

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored.     

Heterogeneity of chromatin subunits in vitro and location of histone H1.

Chromatin subunits ("nucleosomes") which were purified by sucrose gradient centrifugation of a staphylococcal nuclease digest of chromatin have been studied. We found that such a preparation contains nucleosomes of two discrete types which can be separated from each other by polyacrylamide gel electrophoresis. Nucleosome of the first type contains all five histones and a DNA segment of approximately 200 base pairs long, whereas nucleosome of the second type lacks histone H1 and its DNA segment is approximately 170 base pairs long, i.e., about 30 base pairs shorter than the DNA segment of the nucleosome of the first type. Purified dimer of the nucleosome also can be fractionated by gel electrophoresis into three discrete bands which correspond to dinucleosomes containing two molecules of histone H1, one and no H1. These and related findings strongly suggest that the H1 molecule is bound to a short (approximately 30 base pairs) terminal stretch of the nucleosomal DNA segment which can be removed by nuclease (possibly in the form of H1-DNA complex) without any significant disturbance of main structural features of the nucleosome.     

Isolation and characterization of a spacerless dinucleosome from H1-deleted chromatin.

Calf thymus chromatin, depleted in histone H1, was digested with micrococcal nuclease and fractionated by column chromatography. 140 base pair nucleosome core particles were isolated along with an unusual particle containing 2 histone octamers and 240 base pairs of DNA. Evidence is presented that the spacer DNA region is absent from these modified dinucleosomes, which appear as stable products of the digestion process. The physical properties of both particles are presented along with brief speculation on their possible origin and function.     

Self-assembly of single and closely spaced nucleosome core particles.

Self-assembly of DNA with the four core histones but in the absence of H1 generates nucleosome core particles which are spaced randomly over large distances. Closely spaced core particles, however, exhibit a preferred short linkage which is not a multiple of 10 base pairs. They bind about 140 base pairs whereas apparently shorter DNA lengths per nucleosome observed after digestion with micrococcal nuclease are the result of degradation from the ends. The DNA length of one superhelical turn in the core particle is 83 +/- 4 base pairs. Single core particles may bind more DNA than closely spaced core particles but probably less than two full turns of 168 base pairs. The internal structures of single and of native core particles are very similar as judged by their amount of DNA, sedimentation coefficient, appearance in the electron microscope, and digestion with DNase I. In addition to core particles, a particle is described which sediments at 9 S and consists of 108 base pairs of DNA bound to the histone octamer. It appears to be the smallest stable "core particle" but it is not a degradation product of the 146-base-pair core particle. Digestion of end-labeled 9 S and nucleosome core particles with DNase I shows distinct differences.     

Control of RNA synthesis by chromatin proteins.

The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA.     

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.     

Unpaired bases in phage DNA after gamma-irradiation in-situ and in-vitro

Summary Phage Lambda DNA, gamma-irradiated in-situ and in-vitro, has been analyzed for unpaired bases by melting, reannealing, and cleavage with Sl nuclease which is specific for single-stranded DNA. DNA, irradiated in-situ, i.e., in the phage particle, contained sites being sensitive to Sl nuclease. These single-stranded lesions were passed over and conserved during reannealing, whereas adjacent DNA regions reannealed specifically. Complementary base-pairing was restored after Sl nuclease treatment. Comparison of the T_m,-data before and after Sl nuclease treatment indicated that the single-stranded regions were removed by the enzyme. In contrast, DNA irradiated in-vitro, i.e., gamma-irradiated in aqueous solution, failed to match complementarily and was not sensitive to Sl nuclease. Thus it appears that lesions leading to unpaired bases were randomly distributed in DNA irradiated in-vitro, but occurred in clusters after irradiation in-situ. Most probably these clusters contain damaged bases which in turn caused localized disruption of the hydrogen bonds between complementary base pairs.     

Unique positioning of reconstituted nucleosomes occurs in one region of simian virus 40 DNA

A direct end label method was used to study the positioning of nucleosome arrays on several long (greater than 2200 base pairs) SV40 DNA fragments reconstituted in vitro with core histones. Comparison of micrococcal nuclease cutting sites in reconstituted and naked DNA fragments revealed substantial differences in one DNA region. When sufficient core histones were annealed with the DNA to form closely spaced nucleosomes over most of the molecule, a uniquely positioned array of four nucleosomes could be assigned, by strict criteria, to a 610-base pair portion of the SV40 "late region," with a precision of about +/- 20 base pairs. In some other DNA regions, a number of alternative nucleosome positions were indicated. The uniquely positioned four-nucleosome array spanned the same 610 nucleotides on two different DNA fragments that possessed different ends. Removal of a DNA region that had contained a terminal nucleosome of the array, by truncation of the fragment before reconstitution, did not affect the positioning of the other three nucleosomes. As the core histone to DNA ratio was lowered, evidence for specific positioning of nucleosomes diminished, except within the region where the four uniquely positioned nucleosomes formed. This region, however, does not appear to have a higher affinity for core histones than other regions of the DNA.     

The use of micrococcal nuclease as a probe for drug-binding sites on DNA

The cutting pattern produced by micrococcal nuclease on three DNA fragments has been determined in the absence and presence of various DNA-binding drugs. The enzyme itself cuts almost exclusively at pA and pT bonds, showing a greater activity at (A-T)n than in homopolymeric runs of A and T. Each drug produces distinct changes in the cleavage pattern. The protected regions can not be pinpointed with sufficient precision to assess the exact drug-binding sites on account of the sequence selectivity of the enzyme, although where a direct comparison is possible these include most of those seen as DNAase I footprints. The enzyme is most useful for assessing the selectivity of drugs which bind to AT-rich regions. Several drugs protect the DNA from micrococcal nuclease attack in regions which do not contain their acknowledged best binding sites. It appears that micrococcal nuclease is sensitive to the existence of secondary drug-binding sites which are not evident with other footprinting techniques.     

Extended C-terminal tail of wheat histone H2A interacts with DNA of the "linker" region

The preparation of hybrid histone octamers with wheat histone H2A variants replacing chicken H2A in the chicken octamer is described. The fidelity of the reconstituted hybrid octamers was confirmed by dimethyl suberimidate cross-linking. Polyglutamic-acid-mediated assembly of these octamers on long DNA and subsequent micrococcal nuclease (MNase) digestion demonstrated that, whereas chicken octamers protected 167 base-pairs (representing 2 full turns of DNA), hybrid histone octamers containing wheat histone H2A(1) with its 19 amino acid residue C-terminal extension protected an additional 16 base pairs of DNA against nuclease digestion. The protection observed by hybrid histone octamers containing wheat histone H2A(3) with both a 15 residue N-terminal and a 19 residue C-terminal extension was identical with that observed with H2A(1)-containing hybrid histone octamers with only the 19 residue C-terminal extension. These results suggest that the role of the C-terminal extension is to bind to DNA of the "linker" region. The thermal denaturation of chicken and hybrid core particles was identical in 10 mM-Tris.HCl.20 mM-NaCl, 0.1 mM-EDTA, confirming that there was no interaction between the basic C-terminal extension and DNA of the core particle. Denaturation in EDTA, however, showed that hybrid core particles had enhanced stability, suggesting that the known conformational change of core particles at very low ionic strength allows the C-terminal extension to bind to core particle DNA under these conditions. A model accounting for the observed MNase protection is presented.     

Structure of subnucleosomal particles. Tetrameric (H3/H4)2 146 base pair DNA and hexameric (H3/H4)2(H2A/H2B)1 146 base pair DNA complexes

The tetrameric (H3/H4)2 146 base pair (bp) DNA and hexameric (H3/H4)2(H2A/H2B)1 146 bp DNA subnucleosomal particles have been prepared by depletion of chicken erythrocyte core particles using 3 or 4 M urea, 250 mM sodium chloride, and a cation-exchange resin. The particles have been characterized by cross-linking and sedimentation equilibrium. The structures of the particles, particularly the tetrameric, have been studied by sedimentation velocity, low-angle neutron scattering, circular dichroism, optical melting, and nuclease digestion with DNase I, micrococcal nuclease, and exonuclease III. It is concluded that since the radius of gyration of the DNA in the tetramer particle and its maximum dimension are very close to those of the core particle, no expansion occurs on removal of all the H2A and H2B. Nuclease digestion results indicate that histones H3/H4 in the tetramer particle protect a total of 70 bp of DNA that are centrally located within the 146 bp. Within the 70 bp DNA length, the two terminal regions of 10 bp are, however, not strongly protected from digestion. The optical melting profile of both particles can be resolved into three components and is consistent with the model of histone protection of DNA proposed from nuclease digestion. The structure proposed for the tetrameric histone complex bound to DNA is that of a compact particle containing 1.75 superhelical turns of DNA, in which the H3 and H4 histone location is the same as found for the core particle in chromatin by histone/DNA cross-linking [Shick, V. V., Belyavsky, A. V., Bavykin, S. G., & Mirzabekov, A. D. (1980) J. Mol. Biol. 139, 491-517]. Optical melting of the hexamer particle shows that each (H2A/H2B)1 dimer of the core particle protects about 22 base pairs of DNA.     

Photochemical cross-linking of histones to DNA nucleosomes.

Ultraviolet (UV)-induced cross-linking was utilized in order to identify histone-DNA interacting regions in the chromatin repeating unit. Fractionated mononucleosomes which contained 185 base pairs of DNA and a full complement of the histones, including histone H1, were irradiated with light of lambda greater than 290nm in the presence of a photosensitizer. Equimolar amounts of histones H2A and H2B were found, by two independent labeling experiments, to be cross-linked to the DNA. Based on previous finding that the UV irradiation specifically cross-links residues which are in close proximity, irrespective of the nature of the amino acid side chain or the nucleotide involved, our results indicate that the four core histones are not positioned equivalently with respect to the DNA. This arrangement allows histones H2A and H2B to preferentially cross-link to the DNA. A water soluble covalent complex of DNA and histones was isolated. This complex was partially resistant to mild nuclease digestion, it exhibited a CD spectrum similar to that of chromatin, and was found to contain histone H1. These results are compatible with a model which suggests that histone H1, though anchored to the linker, is bound to the DNA at additional sites. By doing so it spans the whole length of the nucleosome and clamps together the DNA fold around the histone core.     

DNA repeat lengths of erythrocyte chromatins differing in content of histones H1 and H5

Among the erythrocytes of chicken, trout, carp, and sucker, the relative proportion of the lysine-rich histone H5 varied from 20 to 0% of the total histones. Following digestion of nuclear chromatin with micrococcal nuclease, each of them displayed a longer DNA repeat length and greater repeat length heterogeneity than found in liver chromatin. Fish erythrocytes possessed similar repeat lengths of 207-209 base pairs which was 10-12 base pairs shorter than in chicken erythrocyte chromatin and approximately 10 base pairs longer than in liver chromatin. No correlation existed between the DNA repeat length or repeat length heterogeneity and the relative proportion of H5.     

Relaxation of chromatin structure induced by ethidium binding. Involvement of the intercalation process

In this paper we study the effects of the binding of ethidium on the structure of chromatin, using micrococcal nuclease as a structural probe. This binding induces two structural changes of chromatin either isolated or in the nuclei. (a) An unfolding of the overall structure which results in an activation of the rate of degradation by the nuclease. (b) A disorganisation of the core particle structure which has the effect of unwrapping the DNA from the histone core, this disruption can go on so far as to leave only 90 base pairs. By comparing the bindings of ethidium and tetramethylethidium, we conclude that the first type of structural change is due to an electrostatic effect and does not depend upon intercalation. On the other hand, the second one is due to the intercalation process and to the change of topological constraints on the DNA that such a process involves.