The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
Karolin Luger - One of the best experts on this subject based on the ideXlab platform.
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A New Fluorescence Resonance Energy Transfer Approach Demonstrates That the Histone Variant H2AZ Stabilizes the Histone Octamer within the Nucleosome
The Journal of biological chemistry, 2004Co-Authors: Young-jun Park, Pamela N. Dyer, David J. Tremethick, Karolin LugerAbstract:Abstract Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major Histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the Histone Octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)2 tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential Histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z.
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Blocking transcription through a nucleosome with synthetic DNA ligands
Journal of molecular biology, 2002Co-Authors: Joel M. Gottesfeld, Christian Melander, Peter B. Dervan, Jason M. Belitsky, Karolin LugerAbstract:Pyrrole-imidazole (Py-Im) polyamides are synthetic ligands that bind in the minor groove of DNA. Previous studies have established that sites on nucleosomal DNA facing away from the Histone Octamer, or even partially facing the Histone Octamer, are fully accessible for molecular recognition by Py-Im polyamides, and that nucleosomes remain fully folded upon ligand binding. Two polyamides that bind within the sea urchin 5S gene nucleosome positioning sequence inhibit both heat-induced nucleosome sliding and transcription by bacteriophage T7 RNA polymerase from the nucleosomal template, but not from Histone-free DNA. These polyamides prevent repositioning of the Histone Octamer by RNA polymerase, and thereby inhibit passage of the elongating polymerase through nucleosomal DNA. These results establish unambiguously the requirement for Octamer mobility for transcription of nucleosomal templates by T7 RNA polymerase.
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Energetics and affinity of the Histone Octamer for defined DNA sequences.
Biochemistry, 2001Co-Authors: Joel M. Gottesfeld, Karolin LugerAbstract:Previous studies have compared the relative free energies for Histone Octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the Octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and Histone Octamers (or free Histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (
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energetics and affinity of the Histone Octamer for defined dna sequences
Biochemistry, 2001Co-Authors: Joel M. Gottesfeld, Karolin LugerAbstract:Previous studies have compared the relative free energies for Histone Octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the Octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and Histone Octamers (or free Histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (<400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome reconstitution studies, human alpha-satellite DNA and Lytechinus variegatus 5S DNA, and find that the Octamer exhibits K(d)s of 0.03 and 0.06 nM, respectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are approximately 2 kcal/mol more stable than total NCPs isolated from cellular chromatin. As for mixed-sequence NCPs, increasing ionic strength or temperature promotes dissociation. van't Hoff plots of K(a)s versus temperature reveal that the difference in binding free energy for alpha-satellite and 5S NCPs compared to bulk NCPs is due almost entirely to a more favorable entropic component for NCPs formed on the unique sequences compared to mixed-sequence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of Histones H3 and H4 to Octamer affinity through the use of recombinant tailless Histones.
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Sequence-specific recognition of DNA in the nucleosome by pyrrole-imidazole polyamides.
Journal of molecular biology, 2001Co-Authors: Joel M. Gottesfeld, Karolin Luger, Christian Melander, Robert K. Suto, Holger Raviol, Peter B. DervanAbstract:The ability of DNA-binding proteins to recognize their cognate sites in chromatin is restricted by the structure and dynamics of nucleosomal DNA, and by the translational and rotational positioning of the Histone Octamer. Here, we use six different pyrrole-imidazole polyamides as sequence-specific molecular probes for DNA accessibility in nucleosomes. We show that sites on nucleosomal DNA facing away from the Histone Octamer, or even partially facing the Histone Octamer, are fully accessible and that nucleosomes remain fully folded upon ligand binding. Polyamides only failed to bind where sites are completely blocked by interactions with the Histone Octamer. Removal of the amino-terminal tails of either Histone H3 or Histone H4 allowed these polyamides to bind. These results demonstrate that much of the DNA in the nucleosome is freely accessible for molecular recognition in the minor groove, and also support a role for the amino-terminal tails of H3 and H4 in modulating accessibility of nucleosomal DNA.
Mario Halic - One of the best experts on this subject based on the ideXlab platform.
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Structural rearrangements of the Histone Octamer translocate DNA.
Nature communications, 2018Co-Authors: Silvija Bilokapic, Mike Strauss, Mario HalicAbstract:Nucleosomes, the basic unit of chromatin, package and regulate expression of eukaryotic genomes. Nucleosomes are highly dynamic and are remodeled with the help of ATP-dependent remodeling factors. Yet, the mechanism of DNA translocation around the Histone Octamer is poorly understood. In this study, we present several nucleosome structures showing Histone proteins and DNA in different organizational states. We observe that the Histone Octamer undergoes conformational changes that distort the overall nucleosome structure. As such, rearrangements in the Histone core α-helices and DNA induce strain that distorts and moves DNA at SHL 2. Distortion of the nucleosome structure detaches Histone α-helices from the DNA, leading to their rearrangement and DNA translocation. Biochemical assays show that cross-linked Histone Octamers are immobilized on DNA, indicating that structural changes in the Octamer move DNA. This intrinsic plasticity of the nucleosome is exploited by chromatin remodelers and might be used by other chromatin machineries.
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Structural rearrangements of the Histone Octamer translocate DNA
Nature Publishing Group, 2018Co-Authors: Silvija Bilokapic, Mike Strauss, Mario HalicAbstract:Nucleosomes are dynamic and can move along DNA in an uncatalyzed manner but little is known about the mechanisms of Histone Octamer translocation. Here the authors present cryo-EM structures of nucleosomes in differently organized Histone Octamer and DNA states and show how Histone Octamers translocate DNA
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Histone Octamer rearranges to adapt to DNA unwrapping.
Nature structural & molecular biology, 2017Co-Authors: Silvija Bilokapic, Mike Strauss, Mario HalicAbstract:Nucleosomes, the basic units of chromatin, package and regulate expression of eukaryotic genomes. Although the structure of the intact nucleosome is well characterized, little is known about structures of partially unwrapped, transient intermediates. In this study, we present nine cryo-EM structures of distinct conformations of nucleosome and subnucleosome particles. These structures show that initial DNA breathing induces conformational changes in the Histone Octamer, particularly in Histone H3, that propagate through the nucleosome and prevent symmetrical DNA opening. Rearrangements in the H2A-H2B dimer strengthen interaction with the unwrapping DNA and promote nucleosome stability. In agreement with this, cross-linked H2A-H2B that cannot accommodate unwrapping of the DNA is not stably maintained in the nucleosome. H2A-H2B release and DNA unwrapping occur simultaneously, indicating that DNA is essential in stabilizing the dimer in the nucleosome. Our structures reveal intrinsic nucleosomal plasticity that is required for nucleosome stability and might be exploited by extrinsic protein factors.
Horace R. Drew - One of the best experts on this subject based on the ideXlab platform.
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DNA recognition and nucleosome organization.
Biopolymers, 1997Co-Authors: Andrew Travers, Horace R. DrewAbstract:The affinity of a DNA sequence for the Histone Octamer in a core nucleosome depends on the intrinsic flexibility of the DNA. This parameter can be affected both by the sequence-dependent conformational preferences of individual base steps and by the nature and location of the exocyclic groups of the DNA bases. By adopting highly preferred conformations particular types of base step can influence the rotational positioning of the DNA on the surface of the Histone Octamer. The asymmetry of the next higher order of chromatin structure is determined in part by the asymmetric binding of the globular domain of Histone H5 to the core nucleosome.
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Reconstitution of short-spaced chromatin from the Histone Octamer and either HMG-14,17 or Histone H1.
Journal of molecular biology, 1993Co-Authors: Horace R. DrewAbstract:Abstract Two new chromatin-assembly reactions are described. The first involves the addition of phosphorylated HMG-14,17 to the Histone Octamer plus DNA in high concentrations of salt and yields a repeating particle size or spacing of about 165 base-pairs. The second involves the addition of Histone H1 to the acetylated Histone Octamer plus poly(glutamate) in low concentrations of salt, followed by the addition of DNA; and it yields a spacing of about 170 base-pairs. Plots of band size versus band number in gels, often used to determine nucleosome repeat-length, yield slopes of 138 base-pairs for the Histone Octamer alone, or 155 base-pairs with HMG-14,17 or 160 base-pairs with Histone H1, and intercepts of 10, 25 and 20 base-pairs, respectively, in the three cases. Attempts were made to combine the spacing activities of HMG-14,17 and Histone H1 within a single assembly reaction, to provide an even longer spacing of about 190 base-pairs (as observed in cell extracts to which H1 has been added), but our present methods did not allow this. The two assembly reactions described here will be of use for structural studies of chromatin having defined length and sequence, and potentially of practical use for the regular, ordered condensation of very long DNA.
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Can one measure the free energy of binding of the Histone Octamer to different DNA sequences by salt-dependent reconstitution?
Journal of molecular biology, 1991Co-Authors: Horace R. DrewAbstract:Abstract I explain why many recently reported measurements for the “free energy” of positioning of the Histone Octamer on different DNA sequences are likely to be in error: i.e. because Histone Octamers do not equilibrate between different DNA molecules at low salt, but only at high salt. Thus, the reported “free energies” refer to an equilibrium at high salt, under nearly dissociating conditions between protein and DNA, and they are likely to be much too small on an absolute scale. There are many other lines of evidence to suggest that the preferences of the Histone Octamer for different DNA sequences are rather strong and of importance in biological systems.
Andrew Travers - One of the best experts on this subject based on the ideXlab platform.
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Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the Histone Octamer.
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Manu Shubhdarshan Shukla, Dimitar Angelov, Jan Bednar, Andrew Travers, Sajad Hussain Syed, Fabien Montel, Cendrine Faivre-moskalenko, Stefan DimitrovAbstract:Chromatin remodelers are sophisticated nano-machines that are able to alter Histone-DNA interactions and to mobilize nucleosomes. Neither the mechanism of their action nor the conformation of the remodeled nucleosomes are, however, yet well understood. We have studied the mechanism of Remodels Structure of Chromatin (RSC)-nucleosome mobilization by using high-resolution microscopy and biochemical techniques. Atomic force microscopy and electron cryomicroscopy (EC-M) analyses show that two types of products are generated during the RSC remodeling: (i) stable non-mobilized particles, termed remosomes that contain about 180 bp of DNA associated with the Histone Octamer and, (ii) mobilized particles located at the end of DNA. EC-M reveals that individual remosomes exhibit a distinct, variable, highly-irregular DNA trajectory. The use of the unique "one pot assays" for studying the accessibility of nucleosomal DNA towards restriction enzymes, DNase I footprinting and ExoIII mapping demonstrate that the Histone-DNA interactions within the remosomes are strongly perturbed, particularly in the vicinity of the nucleosome dyad. The data suggest a two-step mechanism of RSC-nucleosome remodeling consisting of an initial formation of a remosome followed by mobilization. In agreement with this model, we show experimentally that the remosomes are intermediate products generated during the first step of the remodeling reaction that are further efficiently mobilized by RSC.
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Relative affinities of DNA sequences for the Histone Octamer depend strongly upon both the temperature and Octamer concentration.
Biochemistry, 2005Co-Authors: Andrew TraversAbstract:Using a novel competition assay to determine the relative strength of different Histone Octamer-binding sites, we have compared three natural and two synthetic sites. We show that the relative affinities of these sites for the Histone Octamer depend upon both the temperature and Octamer concentration. In particular, under certain conditions, a natural Octamer-binding site from a yeast promoter outcompetes a synthetic sequence of comparable affinity to the strongest previously described positioning sequence. Under other conditions, this synthetic sequence is the preferred Octamer ligand. We infer that sequence selection by the Histone Octamer depends strongly upon both the sequence-dependent anisotropy of DNA bending and on DNA deformability and that these parameters may contribute differently to nucleosome formation. These findings indicate that previous studies designed to identify strong Octamer-binding sites may fail to select some natural strong binding sites.
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The exocyclic groups of DNA modulate the affinity and positioning of the Histone Octamer.
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Memmo Buttinelli, Andrew Minnock, Gianna Panetta, Michael J. Waring, Andrew TraversAbstract:To investigate the nature of the chemical determinants in DNA required for nonspecific binding and bending by proteins we have created a novel DNA in which inosine–5-methylcytosine and 2,6-diaminopurine–uracil base pairs are substituted for normal base pairs in a defined DNA sequence. This procedure completely switches the patterns of the base pair H bonding and attachment of exocyclic groups. We show that this DNA binds a Histone Octamer more tightly than normal DNA but, surprisingly, does not alter the orientation of the sequence on the surface of the protein. However, in general, the addition or removal of DNA exocyclic groups reduces or increases, respectively, the affinity for the Histone Octamer. The average incremental change in binding energy for a single exocyclic group is ≈40 J/mol. The orientation of the DNA in core nucleosomes also is sensitive to the number and nature of the exocyclic groups present. Notably, substitution with the naturally occurring cytosine analogue, 5-methylcytosine, shifts the preferred rotational position by 3 bp, whereas incorporating 2,6-diaminopurine shifts it 2 bp in the opposite direction. These manipulations potentially would alter the accessibility of a protein recognition sequence on the surface of the Histone Octamer. We propose that exocyclic groups impose steric constraints on protein-induced DNA wrapping and are also important in determining the orientation of DNA on a protein surface. In addition, we consider the implications of the selection of A-T and G-C base pairs in natural DNA.
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DNA recognition and nucleosome organization.
Biopolymers, 1997Co-Authors: Andrew Travers, Horace R. DrewAbstract:The affinity of a DNA sequence for the Histone Octamer in a core nucleosome depends on the intrinsic flexibility of the DNA. This parameter can be affected both by the sequence-dependent conformational preferences of individual base steps and by the nature and location of the exocyclic groups of the DNA bases. By adopting highly preferred conformations particular types of base step can influence the rotational positioning of the DNA on the surface of the Histone Octamer. The asymmetry of the next higher order of chromatin structure is determined in part by the asymmetric binding of the globular domain of Histone H5 to the core nucleosome.
Evangelos N. Moudrianakis - One of the best experts on this subject based on the ideXlab platform.
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Histone Octamer function in vivo: mutations in the dimer–tetramer interfaces disrupt both gene activation and repression
The EMBO journal, 1997Co-Authors: Maria Soledad Santisteban, Evangelos N. Moudrianakis, Gina Arents, M. Mitchell SmithAbstract:Within the core Histone Octamer each Histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, we made site-directed amino acid substitutions at each of these tyrosine residues. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA. These results demonstrate the critical role of Histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression.
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Histone Octamer function in vivo mutations in the dimer tetramer interfaces disrupt both gene activation and repression
The EMBO Journal, 1997Co-Authors: Maria Soledad Santisteban, Evangelos N. Moudrianakis, Gina Arents, M. Mitchell SmithAbstract:Within the core Histone Octamer each Histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, we made site-directed amino acid substitutions at each of these tyrosine residues. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA. These results demonstrate the critical role of Histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression.
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Topography of the Histone Octamer surface: repeating structural motifs utilized in the docking of nucleosomal DNA.
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Gina Arents, Evangelos N. MoudrianakisAbstract:The Histone Octamer core of the nucleosome is a protein superhelix of four spirally arrayed Histone dimers. The cylindrical face of this superhelix is marked by intradimer and interdimer pseudodyad axes, which derive from the nature of the Histone fold. The Histone fold appears as the result of a tandem, parallel duplication of the "helix-strand-helix" motif. This motif, by its occurrence in the four dimers, gives rise to repetitive structural elements--i.e., the "parallel beta bridges" and the "paired ends of helix I" motifs. A preponderance of positive charges on the surface of the Octamer appears as a left-handed spiral situated at the expected path of the DNA. We have matched a subset of DNA pseudodyads with the Octamer pseudodyads and thus have built a model of the nucleosome. In it, the two DNA strands coincide with the path of the Histone-positive charges, and the central 12 turns of the double helix contact the surface of the Octamer at the repetitive structural motifs. The properties of these complementary contacts appear to explain the preference of Histones for double-helical DNA and to suggest a possible basis for allosteric regulation of nucleosome function.
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Enhanced stability of Histone Octamers from plant nucleosomes: role of H2A and H2B Histones.
Biochemistry, 1992Co-Authors: Charles P. Moehs, Andreas D. Baxevanis, Evangelos N. Moudrianakis, Steven SpikerAbstract:Gel filtration and sedimentation studies have previously established that the vertebrate animal core Histone Octamer is in equilibrium with an (H3-H4)2 tetramer and an H2A-H2B dimer [Eickbush, T. H., & Moudrianakis, E. N. (1978) Biochemistry 17, 4955-4964; Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346]. We have investigated the core Histone Octamer of wheat (Triticum aestivum L.) and have found it to be much more stable than its vertebrate animal counterpart. When vertebrate animal Histone Octamers are subjected to gel filtration in 2 M NaCl, a trailing peak of H2A-H2B dimer can be clearly resolved from the main Octamer peak. When the plant Octamer is subjected to the identical procedure, there is no trailing peak of H2A-H2B dimer, but rather a single peak containing the Octamer. A sampling across the Octamer peak from leading to trailing edge shows no change in the ratio of H2A-H2B to (H3-H4)2. Surprisingly, the plant Octamer shows the same stability at 0.6 M NaCl, a salt concentration in which the vertebrate animal Octamer dissociates into dimers and tetramers. Equilibrium sedimentation data indicate that the assembly potential of the wheat Histones in 2 M NaCl is very high at all protein concentrations above 0.1 mg mL-1. In order to disrupt the forces stabilizing the plant Histone Octamer at high Histone concentrations, the concentration of NaCl must be lowered to approximately 0.3 M.(ABSTRACT TRUNCATED AT 250 WORDS)
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The nucleosomal core Histone Octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Gina Arents, Rufus W. Burlingame, Bi-cheng Wang, Warner E. Love, Evangelos N. MoudrianakisAbstract:Abstract The structure of the Octameric Histone core of the nucleosome has been determined by x-ray crystallography to a resolution of 3.1 A. The Histone Octamer is a tripartite assembly in which a centrally located (H3-H4)2 tetramer is flanked by two H2A-H2B dimers. It has a complex outer surface; depending on the perspective, the structure appears as a wedge or as a flat disk. The disk represents the planar projection of a left-handed proteinaceous superhelix with approximately 28 A pitch. The diameter of the particle is 65 A and the length is 60 A at its maximum and approximately 10 A at its minimum extension; these dimensions are in agreement with those reported earlier by Klug et al. [Klug, A., Rhodes, D., Smith, J., Finch, J. T. & Thomas, J. O. (1980) Nature (London) 287, 509-516]. The folded Histone chains are elongated rather than globular and are assembled in a characteristic "handshake" motif. The individual polypeptides share a common central structural element of the helix-loop-helix type, which we name the Histone fold.