Françoise Livolant

In viruses and cells, DNA is closely packed and tightly curved thanks to polyvalent cations inducing an effective attraction between its negatively charged filaments. Our understanding of this effective attraction remains very incomplete, partly because experimental data is limited to bulk measurements on large samples of mostly uncurved DNA helices. Here we use cryo electron microscopy to shed light on the interaction between highly curved helices. We find that the spacing between DNA helices in spermine-induced DNA toroidal condensates depends on their location within the torus, consistent with a mathematical model based on the competition between electrostatic interactions and the bending rigidity of DNA. We use our model to infer the characteristics of the interaction potential, and find that its equilibrium spacing strongly depends on the curvature of the filaments. In addition, the interaction is much softer than previously reported in bulk samples using different salt conditions. Beyond viruses and cells, our characterization of the interactions governing DNA-based dense structures could help develop robust designs in DNA nanotechnologies.

In Eukaryotes, DNA is wound around the histone core octamer to form the basic chromatin unit, the nucleosome. Atomic resolution structures have been obtained from crystallography and single particle cryo-electron microscopy of identical engineered particles. But native nucleosomes are dynamical entities with diverse DNA sequence and histone content, and little is known about their conformational variability, especially in the cellular context. Using cryo-electron microscopy and tomography of vitreous sections we analyse the conformation of native nucleosomes, both in vitro , using purified particles solubilised at physiologically relevant concentrations (25-50 %), and in situ , within interphase nuclei. We visualise individual nucleosomes at a level of detail that allows us to analyse the conformation of the DNA wrapped around, and measure the distance between the DNA gyres. We evidence a variety of conformations. In interphase nuclei open nucleosomes predominate, with an average inter-gyre distance larger than that of the canonical particle. In concentrated solutions, we evidence a salt–dependant transition, with high salt compact conformations resembling the canonical nucleosome, and open low salt ones, closer to nuclear nucleosomes. Although further particle characterisation and cartography are needed to understand the relationship between this conformational variability and chromatin functional states, this work opens a route to chromatin exploration in situ .

As revealed initially by X‐ray diffraction and visualized later on by cryoEM in several bacteriophages, the DNA chain confined in the capsid is densely packed and organized locally in such a way that DNA segments form an hexagonal lattice with an average inter‐helix spacing aH close to 26 – 27 Å. The 3D organization of DNA in the capsid or the path followed by the chain remains unknown. DNA is kinetically constrained and undergoes out‐of‐equilibrium conformational dynamics during packaging in the capsid. The structural analysis of full capsids also indicates that there is not a unique deterministic DNA packaging pathway. All consequences of this high confinement on DNA ordering and mobility are not yet well understood. Our aim is here to explore how confinement determines DNA organization in the full capsid and to what extent spermine and temperature may affect this ordering. CryoEM has been used to visualize DNA patterns in individual capsids, Circular Dichroism (CD) to detect possible changes of the conformation of DNA and of its supramolecular chiral organization and Small Angle X‐ray Scattering (SAXS) to detect possible variations of inter‐helix distances, while controlling the conformation of the full capsids. We have compared T5, λ, T7, and Φ29 bacteriophages. This selection let us explore the effect of extreme confinement and compare capsids of different shape (prolate or isometric icosahedron) and size, with or without an internal core. We also compared T5 strains containing the full‐length genome to the mutant T5st0 containing a shorter DNA chain, to detect whether possible effects may be increased or reduced at lower densities. Selected CryoEM views show the overall shape of the phages (Figure 1A) and highlight the local hexagonal DNA lattice seen in top view (domains framed in red in Figure 1B). In the direction normal to the capsid faces, up to 5‐6 layers define the thickness of the domain. Figure 1C presents structure factors recorded for all phages. ‐ First, we did not detect any change of DNA organization as a function of temperature between 20 to 40°C, as opposed to previous results (1, 2). ‐ Second, the presence of spermine (4+) enlarges the size of the hexagonal domains determined by the width of the diffraction peak: from 139 to 156 Å in T5, from 111 to 123 Å in λ, from 111 to 128 Å in T7, and from 96 to 110 Å in Φ29 when 100 mM sp is added in HS buffer (10 mM Tris‐HCl, 100 mM NaCl, 1 mM MgCl2, and 1 mM CaCl2). 4 mM spermine is enough to produce this effect. We interpret this increase of the domain size detected for all phages as an increase of the DNA ordering upon spermine addition in agreement with (3). The addition of spermine, by reducing the repulsive interactions between DNA strands would help the local hexagonal order to expand and slightly move the defects further apart. In this hypothesis, DNA reorganization would require very minor sliding of the DNA chain inside the capsid. The enlargement of the DNA hexagonal domains by spermine does not modify the CD spectrum of the encapsidated DNA that stays close to the typical B‐type spectrum. Our estimations of the DNA concentrations based on SAXS measurements (540 ± 20 mg/ml in T5, 525 ± 20 mg/ml in λ, 575 ± 20 in T7 and 615 ± 50 in Φ29) seem to indicate that the smallest phages (T7 and Φ29) are the most densely packed. The comparison between λ and T7 (both icosahedral, with dimensions and DNA length in the same range) suggest that the presence of an internal core in T7 facilitate DNA organization by wounding part of the chain around it in a toroidal organization, thus reducing the defect lattice required to solve the frustration arising from the confinement of the hexagonal lattice in an icosahedral capsid (4).

We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.

Highly charged polyelectrolytes can self-assemble in presence of condensing agents such as multivalent cations, amphiphilic molecules or proteins of opposite charge. Aside precipitation, the formation of soluble micro- and nano-particles has been reported in multiple systems. However a precise control of experimental conditions needed to achieve the desired structures has been so far hampered by the extreme sensitivity of the samples to formulation pathways. Herein we combine experiments and molecular modelling to investigate the detailed microscopic dynamics and the structure of self-assembled hexagonal bundles made of short dsDNA fragments complexed with small basic proteins. We suggest that inhomogeneous mixing conditions are required to form and stabilize charged self-assembled nano-aggregates in large excess of DNA. Our results should help re-interpreting puzzling behaviors reported for a large class of strongly charged polyelectrolyte systems.

The highly charged DNA chain may be either in an extended conformation, the coil, or condensed into a highly dense and ordered structure, the toroid. The transition, also called collapse of the chain, can be triggered in different ways, for example by changing the ionic conditions of the solution. We observe individual DNA molecules one by one, kept separated and confined inside a protein shell (the envelope of a bacterial virus, 80 nm in diameter). For subcritical concentrations of spermine (4+), part of the DNA is condensed and organized in a toroid and the other part of the chain remains uncondensed around. Two states coexist along the same DNA chain. These ‘hairy’ globules are imaged by cryo-electron microscopy. We describe the global conformation of the chain and the local ordering of DNA segments inside the toroid.