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1.
Tóth K  Brun N  Langowski J 《Biochemistry》2006,45(6):1591-1598
Using a previously described FRET technique, we measured the distance between the ends of DNA fragments on which nucleosomes were reconstituted from recombinant and native histones. This distance was analyzed in its dependence on the DNA fragment length, concentration of mono- and divalent counterions, presence of linker histone H1, and histone modifications. We found that the linker DNA arms do not cross under all conditions studied but diverge slightly as they leave the histone core surface. Histone H1 leads to a global approach of the linker DNA arms, confirming the notion of a "stem structure". Increasing salt concentration also leads to an approach of the linker DNAs. To study the effect of acetylation, we compared chemically acetylated recombinant histones with histones prepared from HeLa cells, characterizing the sites of acetylation by mass spectroscopy. Nucleosomes from chemically acetylated histones have few modifications in the core domain and form nucleosomes normally. Acetylating all histones or selectively only H3 causes an opening of the nucleosome structure, indicated by the larger distances between the linker DNA ends. Selective acetylation of H4 distances the linker ends for short fragments but causes them to approach each other for fragments longer than 180 bp.  相似文献   

2.
We previously documented condensation of the H1 CTD consistent with adoption of a defined structure upon nucleosome binding using a bulk FRET assay, supporting proposals that the CTD behaves as an intrinsically disordered domain. In the present study, by determining the distances between two different pairs of sites in the C-terminal domain of full length H1 by FRET, we confirm that nucleosome binding directs folding of the disordered H1 C-terminal domain and provide additional distance constraints for the condensed state. In contrast to nucleosomes, FRET observed upon H1 binding to naked DNA fragments includes both intra- and inter-molecular resonance energy transfer. By eliminating inter-molecular transfer, we find that CTD condensation induced upon H1-binding naked DNA is distinct from that induced by nucleosomes. Moreover, analysis of fluorescence quenching indicates that H1 residues at either end of the CTD experience distinct environments when bound to nucleosomes, and suggest that the penultimate residue in the CTD (K195) is juxtaposed between the two linker DNA helices, proposed to form a stem structure in the H1-bound nucleosome.  相似文献   

3.
Bussiek M  Tóth K  Schwarz N  Langowski J 《Biochemistry》2006,45(36):10838-10846
The effect of the salt concentration, linker histone H1, and histone acetylation on the structure of trinucleosomes reconstituted on a 608 bp DNA containing one centered nucleosome positioning signal was studied. Fluorescence resonance energy transfer (FRET) in solution and scanning force microscopy (SFM) measurements in liquid were done on the same samples. The distance between the DNA ends decreases under the effect of an increasing salt concentration and also by the incorporation of the H1 linker histone. A decrease of internucleosomal center-to-center (cc) distances by H1 was observed that was limited to a minimal value of about 20 nm. The distribution of the angle formed between consecutive nucleosomes was narrowed by H1. The effect of acetylation of all histones leads to decompaction, measured as an increased distance between the DNA ends, and also increased the internucleosomal distances. Selective acetylation of histone H4, however, compacts the structure as measured by FRET.  相似文献   

4.
Linker histones play essential roles in the chromatin structure of higher eukaryotes. While binding to the surface of nucleosomes is directed by an ~ 80-amino-acid-residue globular domain, the structure and interactions of the lysine-rich ~ 100-residue C-terminal domain (CTD), primarily responsible for the chromatin-condensing functions of linker histones, are poorly understood. By quantitatively analyzing binding of a set of H1 CTD deletion mutants to nucleosomes containing various lengths of linker DNA, we have identified interactions between distinct regions of the CTD and nucleosome linker DNA at least 21 bp from the edge of the nucleosome core. Importantly, partial CTD truncations caused increases in H1 binding affinity, suggesting that significant entropic costs are incurred upon binding due to CTD folding. van't Hoff entropy/enthalpy analysis and intramolecular fluorescent resonance energy transfer (FRET) studies indicate that the CTD undergoes substantial nucleosome-directed folding, in a manner that is distinct from that which occurs upon H1 binding to naked DNA. In addition to defining critical interactions between the H1 CTD and linker DNA, our data indicate that the H1 CTD is an intrinsically disordered domain and provide important insights into the biological function of this protein.  相似文献   

5.
Physiologically spaced nucleosome formation in HeLa cell extracts is ATP dependent. ATP hydrolysis is required for chromatin assembly on both linear and covalently closed circular DNA. The link between the phosphorylation state of histones and nucleosome formation has been examined and we demonstrate that in the absence of histone phosphorylation no stable and regularly spaced nucleosomes are formed. Phosphorylated H3 stabilizes the nucleosome core; while phosphorylation of histone H2a is necessary to increase the linker length between nucleosomes from 0 to approximately 45 bp. Histone H1 alone, whether phosphorylated or unphosphorylated, does not increase the nucleosome repeat length in the absence of core histone phosphorylation. Phosphorylations of H1 and H3 correlate with condensation of chromatin. Maximum ATP hydrolysis which is necessary to increase the periodicity of nucleosomes from approximately 150 to approximately 185 bp, not only inhibits H1 and H3 phosphorylation but facilitates their dephosphorylation.  相似文献   

6.
Organization of 5S genes in chromatin of Xenopus laevis.   总被引:5,自引:2,他引:3       下载免费PDF全文
The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.  相似文献   

7.
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.  相似文献   

8.
9.
Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.  相似文献   

10.
A minimal amount of extranucleosomal DNA was required for nucleosome mobilization by ISW2 as shown by using a photochemical histone mapping approach to analyze nucleosome movement on a set of nucleosomes with varied lengths of extranucleosomal DNA. ISW2 was ineffective in repositioning or mobilizing nucleosomes with 相似文献   

11.
The mode of interaction of histone H1 with the nucleosome is governed by the relative distribution of the linker with respect to the core DNA. Preliminary experiments (Simpson, R.T. 1978, Biochemistry, 17, 5524-5531) and tentative models (Thoma, F. et al. (1979), J. Cell. Biol., 83, 403-427) suggest that part of the linker complete two full turns of DNA around the histone core, probably by adding 10 base pairs at each end of the core DNA. In the present study Exonuclease III has been utilized to digest the 3' ends of H1 depleted nucleosomes. (i.e. the 195 base pair particle). The analysis of the resulting DNA fragments under denaturing conditions shows that the whole linker is distributed symmetrically with respect to the core DNA.  相似文献   

12.
Using a combination of small-angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET) measurements we have determined the role of the H3 and H4 histone tails, independently, in stabilizing the nucleosome DNA terminal ends from unwrapping from the nucleosome core. We have performed solution scattering experiments on recombinant wild-type, H3 and H4 tail-removed mutants and fit all scattering data with predictions from PDB models and compared these experiments to complementary DNA-end FRET experiments. Based on these combined SAXS and FRET studies, we find that while all nucleosomes exhibited DNA unwrapping, the extent of this unwrapping is increased for nucleosomes with the H3 tails removed but, surprisingly, decreased in nucleosomes with the H4 tails removed. Studies of salt concentration effects show a minimum amount of DNA unwrapping for all complexes around 50-100mM of monovalent ions. These data exhibit opposite roles for the positively-charged nucleosome tails, with the ability to decrease access (in the case of the H3 histone) or increase access (in the case of the H4 histone) to the DNA surrounding the nucleosome. In the range of salt concentrations studied (0-200mM KCl), the data point to the H4 tail-removed mutant at physiological (50-100mM) monovalent salt concentration as the mononucleosome with the least amount of DNA unwrapping.  相似文献   

13.
B Wittig  S Wittig 《Cell》1979,18(4):1173-1183
DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.  相似文献   

14.
15.
DNase I was used to footprint the 147 bp DNA fragment of the nucleosome in whole chicken erythrocyte nuclei. It was found that the higher-order structure imposes an additional protection on nucleosomes at sites close to the entry and exit points of the linker DNA, around the dyad axis (site S 0). The observed protection is extended up to 20 bp on either side of S 0. It is partial (∼50%) and most probably reflects a full protection of different regions in alternatively oriented nucleosomes. These are the same regions which interact with linker histones. The results strongly support the findings by simulation of DNase I digests of unlabelled oligonucleosome fragments in the 30 nm fibre that in all nucleosomes sites S −5 to S −3 and S +3 to S +5 ara on the outside of the fibre exposed to DNase I.  相似文献   

16.
CENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-A- and H3-containing nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry-exit relative to canonical nucleosomes on both 5 S DNA and the alpha-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(+/-2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4)2 tetramer was also more readily destabilized with heparin than the (H3-H4)2 tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, especially as is seen following CENP-A transient over-expression.  相似文献   

17.
The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.  相似文献   

18.
Nucleosome dimers containing, on average, a single molecule of histone H5 have been isolated from chicken erythrocyte nuclei and the associated DNA fragments cloned and sequenced. The average sequence organization of at least one of the two nucleosomes in the dimers is highly asymmetric and suggests that the torsional, as well as the axial, flexibility of DNA is a determinant of nucleosome positioning. On average the nucleosome dimer is a polar structure containing linker DNA of variable lengths. The sequences associated with H5 containing nucleosomes and core particles are sufficiently different to indicate that removal of histone H5 (or H1) from chromatin may result in the migration of the histone octamer and a consequent exposure of sites for regulatory proteins.  相似文献   

19.
Histone acetylation reduces nucleosome core particle linking number change   总被引:28,自引:0,他引:28  
V G Norton  B S Imai  P Yau  E M Bradbury 《Cell》1989,57(3):449-457
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.  相似文献   

20.
X Y Zhang  F Fittler    W Hrz 《Nucleic acids research》1983,11(13):4287-4306
The question of nucleosome phasing on African Green Monkey (AGM) alpha-satellite DNA has been addressed by employing a new approach. Nucleosome cores were prepared from AGM nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population derived from alpha-satellite DNA containing chromatin was 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 alpha-satellite containing core DNA fragments were mapped by high resolution gel electrophoresis relative to known restriction sites along the 172 bp repeat unit of the satellite DNA. The results show that nucleosomes occupy eight strictly defined positions on the alpha-satellite DNA which could be determined with an accuracy of +/- 1 base pair. Approximately 35% of all nucleosomes are organized in one of these frames while the other seven registers contribute about 10% each.  相似文献   

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