X-ray structure of the nucleosome core particle

Two monoclinic crystal forms (P2(1),C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2(1) crystal form has been solved to 15 A resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2(1) asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

X-Ray Structure of the Nucleosome Core Particle

Abstract

Two monoclinic crystal forms (P2₁,C2) of chicken erythrocyte nucleosomes have been under study in this laboratory. The x-ray structure of the P2₁ crystal form has been solved to 15 Å resolution. The B-DNA superhelix has a relatively uniform curvature, with only several local distortions observed in the superhelix. The individual histone domains have been localized and specific contacts between each histone and the DNA can be observed. Histone contacts to the inner surface of the DNA superhelix occur predominantly at the minor groove sites. Most of the histone core is contained within the inner surface of the superhelical DNA, except for part of H2A which extends between the DNA gyres near the terminus of the DNA. No part of H2A blocks the DNA terminus or would prevent a smooth exit of the DNA into the linker region. A similar extension of a portion of histone H4 between the DNA gyres occurs close to the dyad axis. Both unique nucleosomes in the P2₁ asymmetric unit demonstrate good dyad symmetry and are similar to each other throughout the histone core and DNA regions.     

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.     

Superhelix dimensions of a 1868 base pair plasmid determined by scanning force microscopy in air and in aqueous solution.

We have used scanning force microscopy (SFM) to study the conformation of a 1868 base pair plasmid (p1868) in its open circular form and at a superhelical density of sigma= -0.034. The samples were deposited on a mica surface in the presence of MgCl2. DNA images were obtained both in air and in aqueous solutions, and the dimensions of the DNA superhelix were analysed. Evaluation of the whole plasmid yielded average superhelix dimensions of 27 +/- 9 nm (outer superhelix diameter D), 107 +/- 51 nm (superhelix pitch P), and 54 +/-8 degrees (superhelix pitch angle alpha). We also analysed compact superhelical regions within the plasmid separately, and determined values of D = 9.2 +/- 3.3 nm, P = 42 +/- 13 nm and alpha= 63 +/- 20 degrees for samples scanned in air or rehydrated in water. These results indicate relatively large conformation changes between superhelical and more open regions of the plasmid. In addition to the analysis of the DNA superhelix dimensions, we have followed the deposition process of open circular p1868 to mica in real time. These experiments show that it is possible to image DNA samples by SFM without prior drying, and that the surface bound DNA molecules retain some ability to change their position on the surface.     

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.     

Structural flexibility of the nucleosome core particle at atomic resolution studied by molecular dynamics simulation

Comparative explicit solvent molecular dynamics (MD) simulations have been performed on a complete nucleosome core particle with and without N-terminal histone tails for more than 20 ns. Main purpose of the simulations was to study the dynamics of mobile elements such as histone N-terminal tails and how packing and DNA-bending influences the fine structure and dynamics of DNA. Except for the tails, histone and DNA molecules stayed on average close to the crystallographic start structure supporting the quality of the current force field approach. Despite the packing strain, no increase of transitions to noncanonical nucleic acid backbone conformations compared to regular B-DNA was observed. The pattern of kinks and bends along the DNA remained close to the experiment overall. In addition to the local dynamics, the simulations allowed the analysis of the superhelical mobility indicating a limited relative mobility of DNA segments separated by one superhelical turn (mean relative displacement of approximately +/-0.2 nm, mainly along the superhelical axis). An even higher rigidity was found for relative motions (distance fluctuations) of segments separated by half a superhelical turn (approximately +/-0.1 nm). The N-terminal tails underwent dramatic conformational rearrangements on the nanosecond time scale toward partially and transiently wrapped states around the DNA. Many of the histone tail changes corresponded to coupled association and folding events from fully solvent-exposed states toward complexes with the major and minor grooves of DNA. The simulations indicate that the rapid conformational changes of the tails can modulate the DNA accessibility within a few nanoseconds.     

Mediation of the A/B-DNA helix transition by G-tracts in the crystal structure of duplex CATGGGCCCATG

The crystal structure of the DNA dodecamer duplex CATGGGCCCATG lies on a structural continuum along the transition between A- and B-DNA. The dodecamer possesses the normal vector plot and inclination values typical of B-DNA, but has the crystal packing, helical twist, groove width, sugar pucker, slide and x-displacement values typical of A-DNA. The structure shows highly ordered water structures, such as a double spine of water molecules against each side of the major groove, stabilizing the GC base pairs in an A-like conformation. The different hydration of GC and AT base pairs provides a physical basis for solvent-dependent facilitation of the A↔B helix transition by GC base pairs. Crystal structures of CATGGGCCCATG and other A/B-DNA intermediates support a ‘slide first, roll later’ mechanism for the B→A helix transition. In the distribution of helical parameters in protein–DNA crystal structures, GpG base steps show A-like properties, reflecting their innate predisposition for the A conformation.     

Low-angle neutron scattering from chromatin subunit particles.

Monomer chromatin particles containing 140 base pairs of DNA and eight histone molecules have been studied by neutron scattering. From measurements in various H2O/D2O mixtures, radii of gyration and the average scattering density of the particle were determined. The radius of gyration under conditions when scattering from the DNA dominates is 50A, and when scattering from the protein dominates, 30A. Consequently the core of the particle is largely occupied by the histones while the outer shell consists of DNA together with some of the histone.     

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.     

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.     

Theoretical model for the equilibrium behavior of DNA superhelices

A statistical-mechanical model for superhelical DNA is presented. The partition function for a DNA superhelix is written by using a combinatorial approach in order to allow for the known relation between the number of superhelical twists and the states of the base pairs in the double helix. While the theory allows any factors which might contribute to the free energy of superhelical twisting to be included in the statistical weights of the superhelical twists, only the reduction in configurational entropy is considered in this paper. Similarities between an imperfectly matched DNA double helix and a DNA superhelix are used in the derivation of expressions for the entropy of superhelical DNA. Although the partition function is presented in a general form, permitting many equilibrium properties of DNA superhelices to be treated, the application considered in this paper is the calculation of helix–coil transition curves. Several experimentally observed features of such transitions are predicted. For example, the curves are bimodal, with an early and a late transition relative to that of a nicked molecule. The results are very sensitive to the volume within which two parts of the double helix must meet when forming a superhelical twist. The free energy of superhelix formation is calculated, and the results are compared with those obtained from the data of Bauer and Vinograd for ethidium bromide intercalation. In the present model, the free energy increases less sharply with an increase in the number of superhelical twists than observed experimentally, indicating that factors other than configurational entropy probably make important contributions to the free energy of superhelix formation.     

Spatial organization of histones and DNA in the nucleosome core particle: A model

We present here an attempt to build up a space-filling model of the nucleosome core particle based on the chemical crosslinking data of Mirzabekov and coworkers (23). It is shown that the models proposed earlier are inconsistent with the results of these authors. The main characteristics of our model are as follows: a) the DNA superhelix contains at least 90 base pairs (bp) per turn; b) the particle has a dyad axis of symmetry; c) the histone octamer may be regarded as consisting of two heterotypic tetramers. The possible shape and function of core histones are discussed in the light of the model.     

Structural characterization of separated H DNA conformers

Polypyrimidine/polypurine DNA sequences in plasmids can adopt protonated triplex-containing structures (H DNA) in response to negative superhelical stress and low pH. A d(TC)17-d(GA)17 insert adopts two isomeric protonated structures, which differ in degree of helical unwinding. The variant forms of individual topoisomers were separated by agarose gel electrophoresis and their reactivities to permanganate and acid-induced depurination were compared. Depurination patterns of the individual conformers indicate that in the more mobile form (H-y5) the 5'-half of the d(GA)n strand participates in a triplex while in the other (H-y3) the 3'-half forms the triplex. The H-y5 form is more stable than the H-y3 form at low negative superhelix densities. Because of the difference in helical unwinding, the H-y5 form becomes relatively less stable as the superhelix density increases. Topological models of the two forms show that providing there is no linkage at the tips of the triple helical segments one more positive twist is localized in the H-y5 form than in the H-y3 form. The foldback in the pyrimidine strand of the H-y5 form is less accessible to solvent than that of the H-y3 form as assessed by its lower reactivity to permanganate. Consideration of a pyrimidine loop model (Harvey, S. C., Luo, J., & Lavery, R. (1988) Nucleic Acids Res. 16, 11795-11809) suggests that the unique stability of the H-y5 form results from Watson-Crick base pairs between residues of the d(TC)n loop and the d(GA)n strand as it exits the triplex.     

S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA.

The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.     

Sequence-dependent bending of DNA and phasing of nucleosomes

Conformational analysis has revealed anisotropic flexibility of the B-DNA double helix: it bends most easily into the grooves, being the most rigid when bent in a perpendicular direction. This result implies that DNA in a nucleosome is curved by means of relatively sharp bends ("mini-kinks") which are directed into the major and minor grooves alternatively and separated by 5-6 base pairs. The "mini-kink" model proved to be in keeping with the x-ray structure of the B-DNA dodecamer resolved later, which exhibits two "annealed kinks", also directed into the grooves. The anisotropy of B DNA is sequence-dependent: the pyrimidine-purine dimers (YR) favor bending into the minor groove, and the purine-pyrimidine dinucleotides (RY), into the minor one. The RR and YY dimers appear to be the most rigid dinucleotides. Thus, a DNA fragment consisting of the interchanging oligopurine and oligopyrimidine blocks 5-6 base pairs long should manifest a spectacular curvature in solution. Similarly, a nucleotide sequence containing the RY and YR dimers separated by a half-pitch of the double helix is the most suitable for wrapping around the nucleosomal core. Analysis of the numerous examples demonstrating the specific alignment of nucleosomes on DNA confirms this concept. So, the sequence-dependent "mechanical" properties of the double helix influence the spatial arrangement of DNA in chromatin.     

The three-dimensional structure of supercoiled deoxyribonucleic acid in solution. Evidence obtained from the angular distribution of scattered light

1. The size and shape of superhelical double-stranded circular DNA from bacteriophage ØX174 were investigated by light-scattering. The molecular weight of the DNA is 3.17×10⁶ and the root-mean-square radius is 103.5nm. 2. The light-scattering envelopes of various theoretical three-dimensional models for such DNA molecules were calculated by repetitive computational techniques, and the results were compared with the experimental findings. 3. It is concluded that the structure of supercoiled DNA containing −12 superhelical turns in buffer of I0.2 corresponds best to one of the more compact models for superhelix structure such as the branched model, and the commonly employed straight interwound superhelix model is incompatible with the experimental results, at the superhelix density found.     

The extended and eccentric E-DNA structure induced by cytosine methylation or bromination

Cytosine methylation or bromination of the DNA sequence d(GGCGCC)2 is shown here to induce a novel extended and eccentric double helix, which we call E-DNA. Like B-DNA, E-DNA has a long helical rise and bases perpendicular to the helix axis. However, the 3'-endo sugar conformation gives the characteristic deep major groove and shallow minor groove of A-DNA. Also, if allowed to crystallize for a period of time longer than that yielding E-DNA, the methylated sequence forms standard A-DNA, suggesting that E-DNA is a kinetically trapped intermediate in the transition to A-DNA. Thus, the structures presented here chart a crystallographic pathway from B-DNA to A-DNA through the E-DNA intermediate in a single sequence. The E-DNA surface is highly accessible to solvent, with waters in the major groove sitting on exposed faces of the stacked nucleotides. We suggest that the geometry of the waters and the stacked base pairs would promote the spontaneous deamination of 5-methylcytosine in the transition mutation of dm5C-dG to dT-dA base pairs.     

Dynamics of Forced Nucleosome Unraveling and Role of Nonuniform Histone-DNA Interactions

A coarse-grained model of the nucleosome is introduced to investigate the dynamics of force-induced unwrapping of DNA from histone octamers. In this model, the DNA is treated as a charged, discrete worm-like chain, and the octamer is treated as a rigid cylinder carrying a positively charged superhelical groove that accommodates 1.7 turns of DNA. The groove charges are parameterized to reproduce the nonuniform histone/DNA interaction free energy profile and the loading rate-dependent unwrapping forces, both obtained from single-molecule experiments. Brownian dynamics simulations of the model under constant loading conditions reveal that nucleosome unraveling occurs in three distinct stages. At small extensions, the flanking DNA exhibits rapid unwrapping-rewrapping (breathing) dynamics and the octamer flips ～180° and moves toward the pulling axis. At intermediate extensions, the outer turn of DNA unwraps gradually and the octamer swivels about the taut linkers and flips a further ～90° to orient its superhelical axis almost parallel to the pulling axis. At large extensions, a portion of the inner turn unwraps abruptly with a notable rip in the force-extension plot and a >90° flip of the octamer. The remaining inner turn unwraps reversibly to leave a small portion of DNA attached to the octamer despite extended pulling. Our simulations further reveal that the nonuniform histone/DNA interactions in canonical nucleosomes serve to: stabilize the inner turn against unraveling while enhancing the breathing dynamics of the nucleosome and prevent dissociation of the octamer from the DNA while facilitating its mobility along the DNA. Thus, the modulation of the histone/DNA interactions could constitute one possible mechanism for regulating the accessibility of the nucleosome-wound DNA sequences.     

A new particle of chromatin obtained by proteolysis of histones with clostripain

The proteolysis of the chromatin core particle by the arginine-specific endopeptidase clostripain yields a new nucleoprotein particle containing an unaltered DNA fragment of about 145 base pairs in length and a protein octameric core made up of twice the four histone fragments H2A, H2B, H3 and H4. This composition is suggested by the molecular weight of about 180 kd determined for the new particle by small angle neutron scattering. The histone fragments differ by about 2-3 kd each from the initial histones H2A, H2B, H3 and H4 and they correspond to the cleavage of the N-terminal part of the sequence (20-30 residues). A preliminary investigation by thermal denaturation, circular dichroism and small angle neutron scattering (measurement of a radius of gyration by the H2O-D2O contrast variation technique) indicates that the spatial organization of the new chromatin particle closely resembles that of the initial core particle.