Explaining the striking difference in twist-stretch coupling between DNA and RNA: A comparative molecular dynamics analysis

Double stranded helical DNA and RNA are flexible molecules that can undergo global conformational fluctuations. Their bending, twisting and stretching deformabilities are of similar magnitude. However, recent single-molecule experiments revealed a striking qualitative difference indicating an opposite sign for the twist-stretch couplings of dsDNA and dsRNA [Lipfert et al. 2014. Proc. Natl. Acad. Sci. U.S.A. 111, 15408] that is not explained by existing models. Employing unconstrained Molecular Dynamics (MD) simulations we are able to reproduce the qualitatively different twist-stretch coupling for dsDNA and dsRNA in semi-quantitative agreement with experiment. Similar results are also found in simulations that include an external torque to induce over-or unwinding of DNA and RNA. Detailed analysis of the helical deformations coupled to twist indicate that the interplay of helical rise, base pair inclination and displacement from the helix axis upon twist changes are responsible for the different twist-stretch correlations. Overwinding of RNA results in more compact conformations with a narrower major groove and consequently reduced helical extension. Overwinding of DNA decreases the size of the minor groove and the resulting positive base pair inclination leads to a slender and more extended helical structure

Explaining the striking difference in twist-stretch coupling between DNA and RNA: A comparative molecular dynamics analysis

Double stranded helical DNA and RNA are flexible molecules that can undergo global conformational fluctuations. Their bending, twisting and stretching deformabilities are of similar magnitude. However, recent single-molecule experiments revealed a striking qualitative difference indicating an opposite sign for the twist-stretch couplings of dsDNA and dsRNA [Lipfert et al. 2014. Proc. Natl. Acad. Sci. U.S.A. 111, 15408] that is not explained by existing models. Employing unconstrained Molecular Dynamics (MD) simulations we are able to reproduce the qualitatively different twist-stretch coupling for dsDNA and dsRNA in semi-quantitative agreement with experiment. Similar results are also found in simulations that include an external torque to induce over-or unwinding of DNA and RNA. Detailed analysis of the helical deformations coupled to twist indicate that the interplay of helical rise, base pair inclination and displacement from the helix axis upon twist changes are responsible for the different twist-stretch correlations. Overwinding of RNA results in more compact conformations with a narrower major groove and consequently reduced helical extension. Overwinding of DNA decreases the size of the minor groove and the resulting positive base pair inclination leads to a slender and more extended helical structure

Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease

Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

Nucleosomes are the basic units of chromatin and critical for storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks, in which post-translational modifications of histones play a key role. However, the mode of action and the structural implications at the single-molecule level of nucleosomes is still poorly understood. Here we apply a high-throughput atomic force microscopy imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants three additional methyl groups at lysine 36 of histone H3 (H3K36me3), phosphorylation of H3 histones at serine 10 (H3S10phos), and acetylation of H4 histones at lysines 5, 8, 12, and 16 (H4K5/8/12/16ac). Our data set of more than 25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5-bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild-type nucleosomes and additionally unwrap stochastically from both sides, similar to centromere protein A (CENP-A) nucleosomes and in contrast to the highly anticooperative unwrapping of wild-type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild-type nucleosomes and also retain the same level of anticooperativity. Our findings help to put the mode of action of these modifications into context. Although H3K36me3 likely acts partially by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament's torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca2+ ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament

Applying torque to the Escherichia coli flagellar motor using magnetic tweezers

The bacterial flagellar motor of Escherichia coli is a nanoscale rotary engine essential for bacterial propulsion. Studies on the power output of single motors rely on the measurement of motor torque and rotation under external load. Here, we investigate the use of magnetic tweezers, which in principle allow the application and active control of a calibrated load torque, to study single flagellar motors in Escherichia coli. We manipulate the external load on the motor by adjusting the magnetic field experienced by a magnetic bead linked to the motor, and we probe the motor's response. A simple model describes the average motor speed over the entire range of applied fields. We extract the motor torque at stall and find it to be similar to the motor torque at drag-limited speed. In addition, use of the magnetic tweezers allows us to force motor rotation in both forward and backward directions. We monitor the motor's performance before and after periods of forced rotation and observe no destructive effects on the motor. Our experiments show how magnetic tweezers can provide active and fast control of the external load while also exposing remaining challenges in calibration. Through their non-invasive character and straightforward parallelization, magnetic tweezers provide an attractive platform to study nanoscale rotary motors at the single-motor level

High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes

Nucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ∼10 000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but in contrast to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Notably, centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes. Finally, our results reconcile previous conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability and to facilitate future high-throughput AFM studies that involve heterogeneous nucleoprotein complexes

DNA fluctuations reveal the size and dynamics of topological domains

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein-induced topological domains in DNA, quantitative and time-resolved approaches are required. Here, we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real time and at the single-molecule level. Our approach is based on quantifying the extension fluctuations—in addition to the mean extension—of supercoiled DNA in magnetic tweezers (MT). Using a combination of high-speed MT experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime, the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how the transient (partial) dissociation of DNA-bridging proteins results in the dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our results to further the understanding and optimization of magnetic tweezer measurements and to enable quantification of the dynamics and reaction pathways of DNA processing enzymes in the context of physiologically relevant forces and supercoiling densities

The free energy landscape of retroviral integration

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo