Nucleosome assembly and disassembly pathways in vitro.

Structural fluctuations of nucleosomes modulate the access to internal DNA in eukaryotic cells; clearly characterisation of this fundamental process is crucial to understanding gene regulation. Here we apply PhAST (Photochemical Analysis of Structural Transitions) to monitor at a base pair level, structural alterations induced all along the DNA upon histone binding or release. By offering the first reliable, detailed comparison of nucleosome assembly and disassembly in vitro, we reveal similarities and differences between the two processes. We identify multiple, sequential intermediate states characterised by specific PhAST signals whose localisation and amplitude reflect asymmetries of DNA/histone interactions with respect to the nucleosome pseudo dyad. These asymmetries involve not only the DNA extremities but also regions close to the pseudo dyad. Localisations of asymmetries develop in a consistent manner during both assembly and disassembly processes; they primarily reflect the DNA sequence effect on the efficiency of DNA-histone binding. More unexpectedly, the amplitude component of PhAST signals not only evolves as a function of intermediate states but does so differently between assembly and disassembly pathways. Our observation of differences between assembly and disassembly opens up new avenues to define the role of the DNA sequence in processes underlying the regulation of gene expression. Overall, we provide new insights into how the intrinsic properties of DNA are integrated into a holistic mechanism that controls chromatin structure.

A dynamic view of DNA structure within the nucleosome: Biological implications.

Most of eukaryotic cellular DNA is packed in nucleosome core particles (NCPs), in which the DNA (DNANCP) is wrapped around histones. The influence of this organization on the intrinsic local dynamics of DNA is largely unknown, in particular because capturing such information from experiments remains notoriously challenging. Given the importance of dynamical properties in DNA functions, we addressed this issue using CHARMM36 MD simulations of a nucleosome containing the NCP positioning 601 sequence and four related free dodecamers. Comparison between DNANCP and free DNA reveals a limited impact of the dense DNA-histone interface on correlated motions of dinucleotide constituents and on fluctuations of inter base pair parameters. A characteristic feature intimately associated with the DNANCP super-helical path is a set of structural periodicities that includes a marked alternation of regions enriched in backbone BI and BII conformers. This observation led to uncover a convincing correspondence between the sequence effect on BI/BII propensities in both DNANCP and free DNA, strengthening the idea that the histone preference for particular DNA sequences relies on those intrinsic structural properties. These results offer for the first time a detailed view of the DNA dynamical behavior within NCP. They show in particular that the DNANCP dynamics is substantial enough to preserve the ability to structurally adjust to external proteins, for instance remodelers. Also, fresh structural arguments highlight the relevance of relationships between DNA sequence and structural properties for NCP formation. Overall, our work offers a more rational framework to approach the functional, biological roles of NCP.

Structural Explorations of NCp7-Nucleic Acid Complexes Give Keys to Decipher the Binding Process.

A comprehensive view of all the structural aspects related to NCp7 is essential to understand how this protein, crucial in many steps of the HIV-1 cycle, binds and anneals nucleic acids (NAs), mainly thanks to two zinc fingers, ZF1 and ZF2. Here, we inspected the structural properties of the available experimental models of NCp7 bound to either DNA or RNA molecules, or free of ligand. Our analyses included the characterization of the relative positioning of ZF1 and ZF2, accessibility measurements and the exhaustive, quantitative mapping of the contacts between amino acids and nucleotides by a recent tessellation method, VLDM. This approach unveiled the intimate connection between NA binding process and the conformations explored by the free protein. It also provided new insights into the functional specializations of ZF1 and ZF2. The larger accessibility of ZF2 in free NCp7 and the consistency of the ZF2/NA interface in different models and conditions give ZF2 the lead of the binding process. ZF1 contributes to stabilize the complexes through various organizations of the ZF1/NA interface. This work outcome is a global binding scheme of NCp7 to DNA and RNA, and an example of how protein-NA complexes are stabilized.

Holding the Nucleosome Together: A Quantitative Description of the DNA-Histone Interface in Solution.

The nucleosome is the fundamental unit of eukaryotic genome packaging in the chromatin. In this complex, the DNA wraps around eight histone proteins to form a superhelical double helix. The resulting bending, stronger than anything observed in free DNA, raises the question of how such a distortion is stabilized by the proteic and solvent environments. In this work, the DNA-histone interface in solution was exhaustively analyzed from nucleosome structures generated by molecular dynamics. An original Voronoi tessellation technique, measuring the topology of interacting elements without any empirical or subjective adjustment, was used to characterize the interface in terms of contact area and occurrence. Our results revealed an interface more robust than previously known, combining extensive, long-lived nonelectrostatic and electrostatic interactions between DNA and both structured and unstructured histone regions. Cation accumulation makes the proximity of juxtaposed DNA gyres in the superhelix possible by shielding the strong electrostatic repulsion of the charged phosphate groups. Overall, this study provides new insights on the nucleosome cohesion, explaining how DNA distortions can be maintained in a nucleoprotein complex.