Ralf Everaers

Single-base resolution mapping of H1–nucleosome interactions and 3D organization of the nucleosome

Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution •OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121–127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.

Stress Relaxation in Entangled Polymer Melts

We present an extensive set of simulation results for the stress relaxation in equilibrium and step-strained bead-spring polymer melts. The data allow us to explore the chain dynamics and the shear relaxation modulus, G(t), into the plateau regime for chains with Z=40 entanglements and into the terminal relaxation regime for Z=10. Using the known (Rouse) mobility of unentangled chains and the melt entanglement length determined via the primitive path analysis of the microscopic topological state of our systems, we have performed parameter-free tests of several different tube models. We find excellent agreement for the Likhtman-McLeish theory using the double reptation approximation for constraint release, if we remove the contribution of high-frequency modes to contour length fluctuations of the primitive chain.

From crystal and NMR structures, footprints and cryo-electron-micrographs to large and soft structures: nanoscale modeling of the nucleosomal stem

The interaction of histone H1 with linker DNA results in the formation of the nucleosomal stem structure, with considerable influence on chromatin organization. In a recent paper [Syed,S.H., Goutte-Gattat,D., Becker,N., Meyer,S., Shukla,M.S., Hayes,J.J., Everaers,R., Angelov,D., Bednar,J. and Dimitrov,S. (2010) Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome. Proc. Natl Acad. Sci. USA, 107, 9620–9625], we published results of biochemical footprinting and cryo-electron-micrographs of reconstituted mono-, di- and tri-nucleosomes, for H1 variants with different lengths of the cationic C-terminus. Here, we present a detailed account of the analysis of the experimental data and we include thermal fluctuations into our nano-scale model of the stem structure. By combining (i) crystal and NMR structures of the nucleosome core particle and H1, (ii) the known nano-scale structure and elasticity of DNA, (iii) footprinting information on the location of protected sites on the DNA backbone and (iv) cryo-electron micrographs of reconstituted tri-nucleosomes, we arrive at a description of a polymorphic, hierarchically organized stem with a typical length of 20u2009±u20092 base pairs. A comparison to linker conformations inferred for poly-601 fibers with different linker lengths suggests, that intra-stem interactions stabilize and facilitate the formation of dense chromatin fibers.

The radial distribution function of worm-like chains

Abstract.Thermal conformations of semiflexible macromolecules are generically described by the worm-like chain model. The end-to-end distance distribution, a fundamental quantity of the model, is not yet known in closed form. We provide a solution to the practical problem of choosing an appropriate approximation. First, a comprehensive review of existing approximations and exact limiting results is given. We then propose an explicit expression which interpolates between all relevant limiting cases. We show that it accurately reproduces, at no computational cost, high-precision Monte Carlo data, covering a wide range from stiff to flexible chains and from looped to fully stretched configurations. Using this result we quantify the enhancement of short worm-like loop formation by (protein) bridges.

A Unified Poland-Scheraga Model of Oligo- and Polynucleotide DNA Melting: Salt Effects and Predictive Power

Key biological and nano-technological processes require the partial or complete association and dissociation of complementary DNA strands. We present a variant of the Poland-Scheraga model for DNA melting where we introduce a local, sequence-dependent salt correction of the nearest-neighbor parameters. Furthermore, our formulation accounts for capping and interfacial energies of helical and coiled chain sections. We show that the model reproduces experimental data for melting temperatures over the full experimental range of strand length, strand concentration, and ionic strength of the solution. In particular, we reproduce a phenomenological relation by Frank-Kamenetskii for very long chains using a parameterization based on melting curves for short oligomers. However, we also show that the parameters of the Poland-Scheraga model are still not known with sufficient precision to quantitatively predict the fine structure of melting curves. This formulation of the Poland-Scheraga model opens the possibility to overcome this limitation by optimizing parameters with respect to an extended base of experimental data for short-, medium-, and long-chain melting. We argue that the often-discarded melting data for longer oligomers exhibiting non-two-state transitions could play a particularly important role.

Temperature Dependence of the DNA Double Helix at the Nanoscale: Structure, Elasticity, and Fluctuations

Biological organisms exist over a broad temperature range of -15°C to +120°C, where many molecular processes involving DNA depend on the nanoscale properties of the double helix. Here, we present results of extensive molecular dynamics simulations of DNA oligomers at different temperatures. We show that internal basepair conformations are strongly temperature-dependent, particularly in the stretch and opening degrees of freedom whose harmonic fluctuations can be considered the initial steps of the DNA melting pathway. The basepair step elasticity contains a weaker, but detectable, entropic contribution in the roll, tilt, and rise degrees of freedom. To extend the validity of our results to the temperature interval beyond the standard melting transition relevant to extremophiles, we estimate the effects of superhelical stress on the stability of the basepair steps, as computed from the Benham model. We predict that although the average twist decreases with temperature in vitro, the stabilizing external torque in vivo results in an increase of ∼1°/bp (or a superhelical density of Δσ ≃ +0.03) in the interval 0-100°C. In the final step, we show that the experimentally observed apparent bending persistence length of torsionally unconstrained DNA can be calculated from a hybrid model that accounts for the softening of the double helix and the presence of transient denaturation bubbles. Although the latter dominate the behavior close to the melting transition, the inclusion of helix softening is important around standard physiological temperatures.

DNA nanomechanics: How proteins deform the double helix

It is a standard exercise in mechanical engineering to infer the external forces and torques on a body from a given static shape and known elastic properties. Here we apply this kind of analysis to distorted double-helical DNA in complexes with proteins: We extract the local mean forces and torques acting on each base pair of bound DNA from high-resolution complex structures. Our analysis relies on known elastic potentials and a careful choice of coordinates for the well-established rigid base-pair model of DNA. The results are robust with respect to parameter and conformation uncertainty. They reveal the complex nanomechanical patterns of interaction between proteins and DNA. Being nontrivially and nonlocally related to observed DNA conformations, base-pair forces and torques provide a new view on DNA-protein binding that complements structural analysis.

Prediction of RNA multiloop and pseudoknot conformations from a lattice-based, coarse-grain tertiary structure model

We present a semiquantitative lattice model of RNA folding, which is able to reproduce complex folded structures such as multiloops and pseudoknots without relying on the frequently employed ad hoc generalization of the Jacobson-Stockmayer loop entropy. We derive the model parameters from the Turner description of simple secondary structural elements and pay particular attention to the unification of mismatch and coaxial stacking parameters as well as of border and nonlocal loop parameters, resulting in a reduced, unified parameter set for simple loops of arbitrary type and size. For elementary structures, the predictive power of the model is comparable to the standard secondary structure approaches, from which its parameters are derived. For complex structures, our approach offers a systematic treatment of generic effects of chain connectivity as well as of excluded volume or attractive interactions between and within all elements of the secondary structure. We reproduce the native structures of tRNA multiloops and of viral frameshift signal pseudoknots.

Genome wide application of DNA melting analysis

Correspondences between functional and thermodynamic melting properties in a genome are being increasingly employed for abxa0initio gene finding and for the interpretation of the evolution of genomes. Here we present the first systematic genome wide comparison between biologically coding domains and thermodynamically stable regions. In particular, we develop statistical methods to estimate the reliability of the resulting predictions. Not surprisingly, we find that the success of the approach depends on the difference in GC content between the coding and the non-coding parts of the genome and on the percentage of coding base-pairs in the sequence. These prerequisites vary strongly between species, where we observe no systematic differences between eukaryotes and prokaryotes. We find a number of organisms in which the strong correlation of coding domains and thermodynamically stable regions allows us to identify putative exons or genes to complement existing approaches. In contrast to previous investigations along these lines we have not employed the Poland-Scheraga (PS) model of DNA melting but use the earlier Zimm-Bragg (ZB) model. The Ising-like form of the ZB model can be viewed as an approximation to the PS model, with averaged loop entropies included into the cooperative factor [Formula: see text]. This results in a speed-up by a factor of 20-100 compared to the Fixman-Freire algorithm for the solution of the PS model. We show that for genomic sequences the resulting systematic errors are negligible compared to the parameterization uncertainty of the models. We argue that for limited computing resources, available CPU power is better invested in broadening the statistical base for genomic investigations than in marginal improvements of the description of the physical melting behavior.

Computer simulations of randomly branching polymers: annealed versus quenched branching structures

We present computer simulations of three systems of randomly branching polymers in d = 3 dimensions: ideal trees and self-avoiding trees with annealed and quenched connectivities. In all cases, we performed a detailed analysis of trees connectivities, spatial conformations and statistical properties of linear paths on trees, and compare the results to the corresponding predictions of Flory theory. We confirm that, overall, the theory predicts correctly that trees with quenched ideal connectivity exhibit less overall swelling in good solvent than corresponding trees with annealed connectivity even though they are more strongly stretched on the path level. At the same time, we emphasize the inadequacy of the Flory theory in predicting the behaviour of other, and equally relevant, observables like contact probabilities between tree nodes. We show, then, that contact probabilities can be aptly characterized by introducing a novel critical exponent, , which accounts for how they decay as a function of the node-to-node path distance on the tree.