Topological Barriers Research Articles

Transcribing RNA polymerases: Dynamics of twin supercoiled domains

Gene transcription by an RNA polymerase (RNAP) enzyme requires that double-stranded DNA be locally and transiently opened, which results in an increase of DNA supercoiling downstream of the RNAP and a decrease of supercoiling upstream of it. When the DNA is initially torsionally relaxed and the RNAP experiences sufficiently large rotational drag, these variations lead to positively supercoiled plectonemes ahead of the RNAPs and negatively supercoiled ones behind it, a feature known as “twin supercoiled domain” (TSD). This work aims at deciphering into some more detail the torsional dynamics of circular DNA molecules being transcribed by RNAP enzymes. To this end, we performed Brownian dynamics simulations with a specially designed coarse-grained model. Depending on the superhelical density of the DNA molecule and the ratio of RNAP’s twist injection rate and rotational relaxation speed, simulations reveal a rich panel of behaviors, which sometimes differ markedly from the crude TSD picture. In particular, for sufficiently slow rotational relaxation speed, positively supercoiled plectonemes never form ahead of an RNAP that transcribes a DNA molecule with physiological negative supercoiling. Rather, negatively supercoiled plectonemes form almost periodically at the upstream side of the RNAP and grow up to a certain length before detaching from the RNAP and destabilizing rapidly. The extent to which topological barriers hinder the dynamics of TSDs is also discussed.

Variation of Structure and Cellular Functions of Type IA Topoisomerases across the Tree of Life.

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.

Topological magnetic structure generation using VAE-GAN hybrid model and discriminator-driven latent sampling

Recently, deep generative models using machine intelligence are widely utilized to investigate scientific systems by generating scientific data. In this study, we experiment with a hybrid model of a variational autoencoder (VAE) and a generative adversarial network (GAN) to generate a variety of plausible two-dimensional magnetic topological structure data. Due to the topological properties in the system, numerous and diverse metastable magnetic structures exist, and energy and topological barriers separate them. Thus, generating a variety of plausible spin structures avoiding those barrier states is a challenging problem. The VAE-GAN hybrid model can present an effective approach to this problem because it brings the advantages of both VAE’s diversity and GAN’s fidelity. It allows one to perform various applications including searching a desired sample from a variety of valid samples. Additionally, we perform a discriminator-driven latent sampling (DDLS) using our hybrid model to improve the quality of generated samples. We confirm that DDLS generates various plausible data with large coverage, following the topological rules of the target system.

AlphaFold Blindness to Topological Barriers Affects Its Ability to Correctly Predict Proteins' Topology.

AlphaFold is a groundbreaking deep learning tool for protein structure prediction. It achieved remarkable accuracy in modeling many 3D structures while taking as the user input only the known amino acid sequence of proteins in question. Intriguingly though, in the early steps of each individual structure prediction procedure, AlphaFold does not respect topological barriers that, in real proteins, result from the reciprocal impermeability of polypeptide chains. This study aims to investigate how this failure to respect topological barriers affects AlphaFold predictions with respect to the topology of protein chains. We focus on such classes of proteins that, during their natural folding, reproducibly form the same knot type on their linear polypeptide chain, as revealed by their crystallographic analysis. We use partially artificial test constructs in which the mutual non-permeability of polypeptide chains should not permit the formation of complex composite knots during natural protein folding. We find that despite the formal impossibility that the protein folding process could produce such knots, AlphaFold predicts these proteins to form complex composite knots. Our study underscores the necessity for cautious interpretation and further validation of topological features in protein structures predicted by AlphaFold.

Assessing in vivo the impact of gene context on transcription through DNA supercoiling.

Gene context can have significant impact on gene expression but is currently not integrated in quantitative models of gene regulation despite known biophysical principles and quantitative in vitro measurements. Conceptually, the simplest gene context consists of a single gene framed by two topological barriers, known as the twin transcriptional-loop model, which illustrates the interplay between transcription and DNA supercoiling. In vivo, DNA supercoiling is additionally modulated by topoisomerases, whose modus operandi remains to be quantified. Here, we bridge the gap between theory and in vivo properties by realizing in Escherichia coli the twin transcriptional-loop model and by measuring how gene expression varies with promoters and distances to the topological barriers. We find that gene expression depends on the distance to the upstream barrier but not to the downstream barrier, with a promoter-dependent intensity. We rationalize these findings with a first-principle biophysical model of DNA transcription. Our results are explained if TopoI and gyrase both act specifically, respectively upstream and downstream of the gene, with antagonistic effects of TopoI, which can repress initiation while facilitating elongation. Altogether, our work sets the foundations for a systematic and quantitative description of the impact of gene context on gene regulation.

Molecular Dynamics of Topological Barriers on the Crystallization Behavior of Ring Polyethylene Melts with Trefoil Knots

Topological barriers in ring polyethylene (PE) melts of trefoil knots inhibit crystallization due to self-entanglement, as confirmed by united atom molecular dynamics (UAMD) simulations. In this study, we clarified the decrease in the topological barriers of self-entangled knots with increasing polymer chain length (N) and the localization of the knotted segments in the noncrystallized region. In the UAMD simulations, isothermal crystallization processes were performed at T = 300 K for the trefoil knots using a crystallization time tIC = 200 ns. Here, the trefoil knot gives the simplest nontrivial topology with nonzero crossing number. Crystallization was not observed in trefoil PE knots for N ≤ 120; however, crystallization did take place in the ring PE melt of the trivial knot with trivial topology and zero minimal crossing number. For N = 140, suppression of the growth of a crystalline phase (i.e., the polycrystalline phase) was observed in the trefoil PE melts, whereas such suppression was not detected in the trivial PE melts. In contrast, no significant differences in the crystallization behaviors of the trefoil and trivial PE melts were observed at N = 200. These results indicated that the topological barriers decreased with increasing N. Furthermore, to investigate the relationship between the chain conformation and degree of local crystallization, we developed a new mathematical method for searching the knotted segment of a trefoil knot in a crystallized trefoil PE melt. We found that trefoil knots with large subloops (“large-leaves”) exhibited localization of the knotted segment. In addition, the large leaves of the localized knots dominated in the crystallized region, and the knotted segments of the localized knots were located mainly in the noncrystallized region.

Interaction between magnon and skyrmion: Toward quantum magnonics

In recent years, magnon and spin texture are attracting great interest in condensed matter physics and magnetism. Magnonics is aiming to use magnon as information carriers to realize functions for storage, transmission, and processing. Magnetic skyrmion is representative spin texture due to its topologically nontrivial properties. Since skyrmions are topologically protected, their transformation to other spin configurations requires overcoming additional topological energy barriers. Therefore, skyrmions are more stable than other trivial spin textures. In addition, the characters of nanoscale size, quasiparticle properties, and various excitation modes make them a potential candidate for spintronic application. Magnon and skyrmion, as two fundamental excitations, can coexist in magnetic systems and interplay with each other through direct exchange interactions. In this review, we provide an overview of recent theoretical and experimental studies on magnon–skyrmion interactions. We mainly focus on three kinds of magnon–skyrmion interactions: (i) magnon scattering by skyrmion, (ii) skyrmion motion driven by magnon, and (iii) coupling between magnon and skyrmion modes. The first two kinds of interactions could be clearly explained by the wave-particle interaction model on the classical level. Alternatively, the last kind of interaction could be understood by the coupled harmonic oscillator model on the quantum level, which indicates fast energy exchange and hybrid magnon states. The exploration focused on quantum phenomena of magnon has led to the emerging field of quantum magnonics and promoted applications of magnon in quantum information storage and processing. In the end, we give a perspective on the exploration of magnon–skyrmion interaction in quantum magnonics.

Models of topological barriers and molecular motors of bacterial DNA

ABSTRACT Bacterial genomes are partitioned into kilobases long domains that are topologically independent from each other, meaning that change of DNA superhelicity in one domain does not propagate to neighbours. This is made possible by proteins like the LacI repressor, which behave like topological barriers and block the diffusion of torsion along the DNA. Other proteins, like DNA gyrases and RNA polymerases, called molecular motors, use the energy released by the hydrolysis of ATP to apply forces and/or torques to the DNA and modify its superhelicity. Here, we report on simulation work aimed at enlightening the interplay between DNA supercoiling, topological barriers, and molecular motors. To this end, we developed a coarse-grained Hamiltonian model of topological barriers and a model of molecular motors and investigated their properties through Brownian dynamics simulations. We discuss their influence on the contact map of a model nucleoid and the steady state values of twist and writhe in the DNA. These coarse-grained models, which are able to predict the dynamics of plectonemes depending on the position of topological barriers and molecular motors, should prove helpful to back up experimental efforts, like the development of Chromosome Conformation Capture techniques, and decipher the organisational mechanisms of bacterial chromosomes.

Topologically switchable and gated transcription machinery.

Topological barriers control in nature the transcription machinery, thereby perturbing gene expression. Here we introduce synthetically designed DNA templates that include built-in topological barriers for switchable, triggered-controlled transcription of RNA aptamers. This is exemplified with the design of transcription templates that include reversible and switchable topological barriers consisting of a Sr2+-ion-stabilized G-quadruplex and its separation by kryptofix [2.2.2], KP, for the switchable transcription of the malachite green (MG) RNA aptamer, the T-A·T triplex barrier being separated by a fuel-strand for the cyclic triggered transcription of the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI)-binding aptamer, and the use of a photoactivated cis/trans azobenzene-modified nucleic acid barrier for the switchable “ON”/“OFF” transcription of the MG RNA aptamer. By applying a mixture of topologically triggered templates consisting of the photoresponsive barrier and the T-A·T triplex barrier, the gated transcription of the MG aptamer or the DFHBI-binding aptamer is demonstrated. In addition, a Sr2+-ion/KP topologically triggered DNA tetrahedra promoter-transcription scaffold, for the replication of the MG RNA aptamer, and T7 RNA polymerase are integrated into DNA-based carboxymethyl cellulose hydrogel microcapsules acting as cell-like assemblies. The switchable, reversible transcription of the MG RNA aptamer in a cell-like containment is introduced.

Revealing Topological Barriers against Knot Untying in Thermal and Mechanical Protein Unfolding by Molecular Dynamics Simulations.

The knot is one of the most remarkable topological features identified in an increasing number of proteins with important functions. However, little is known about how the knot is formed during protein folding, and untied or maintained in protein unfolding. By means of all-atom molecular dynamics simulation, here we employ methyltransferase YbeA as the knotted protein model to analyze changes of the knotted conformation coupled with protein unfolding under thermal and mechanical denaturing conditions. Our results show that the trefoil knot in YbeA is occasionally untied via knot loosening rather than sliding under enhanced thermal fluctuations. Through correlating protein unfolding with changes in the knot position and size, several aspects of barriers that jointly suppress knot untying are revealed. In particular, protein unfolding is always prior to knot untying and starts preferentially from separation of two α-helices (α1 and α5), which protect the hydrophobic core consisting of β-sheets (β1–β4) from exposure to water. These β-sheets form a loop through which α5 is threaded to form the knot. Hydrophobic and hydrogen bonding interactions inside the core stabilize the loop against loosening. In addition, residues at N-terminal of α5 define a rigid turning to impede α5 from sliding out of the loop. Site mutations are designed to specifically eliminate these barriers, and easier knot untying is achieved under the same denaturing conditions. These results provide new molecular level insights into the folding/unfolding of knotted proteins.

Topological barriers to defect nucleation generate large mechanical forces in an ordered fluid

Common fluids cannot sustain static mechanical stresses at the macroscopic scale because they lack molecular order. Conversely, crystalline solids exhibit long-range order and mechanical strength at the macroscopic scale. Combining the properties of fluids and solids, liquid crystal films respond to mechanical confinement by both flowing and generating static forces. The elastic response, however, is very weak for film thicknesses exceeding 10 nm. In this study, the mechanical strength of a fluid film was enhanced by introducing topological defects in a cholesteric liquid crystal, producing unique viscoelastic and optomechanical properties. The cholesteric was confined under strong planar anchoring conditions between two curved surfaces with sphere-sphere contact geometry similar to that of large colloidal particles, creating concentric dislocation loops. During surface retraction, the loops shrank and periodically disappeared at the surface contact point, where the cholesteric helix underwent discontinuous twist transitions, producing weak oscillatory surface forces. On the other hand, new loop nucleation was frustrated by a topological barrier during fluid compression, creating a metastable state. This generated exceptionally large forces with a range exceeding 100 nm as well as extended blueshifts of the photonic bandgap. The metastable cholesteric helix eventually collapsed under a high compressive load, triggering a stick-slip-like cascade of defect nucleation and twist reconstruction events. These findings were explained using a simple theoretical model and suggest a general approach to enhance the mechanical strength of one-dimensional periodic materials, particularly cholesteric colloid mixtures.

Structure and Connectivity of Hydrothermal Vent Communities Along the Mid-Ocean Ridges in the West Indian Ocean: A Review

To date, 13 biologically active hydrothermal vent (HTV) fields have been described on the West Indian Ocean ridges. Knowledge of benthic communities of these vent ecosystems serves as scientific bases for assessing the resilience of these ecosystems under the global effort to strike an elegant balance between future deep-sea mining and biodiversity conservation. This review aims to summarize our up-to-date knowledge of the benthic community structure and connectivity of these Indian vents and to identify knowledge gaps and key research questions to be prioritized in order to assess the resilience of these communities. The HTVs in the West Indian Ocean are home to many unique invertebrate species such as the remarkable scaly-foot snail. While distinct in composition, the macrofaunal communities of the Indian HTVs share many characteristics with those of other HTVs, including high endemism, strong zonation at the local scale, and a simple food web structure. Furthermore, Indian vent benthic communities are mosaic compositions of Atlantic, Pacific, and Antarctic HTV fauna possibly owning to multiple waves of past colonization. Phylogeographic studies have shed new light into these migratory routes. Current animal connectivity across vent fields appears to be highly influenced by distance and topological barriers. However, contrasting differences in gene flow have been documented across species. Thus, a better understanding of the reproductive biology of the Indian vent animals and the structure of their population at the local scale is crucial for conservation purposes. In addition, increased effort should be given to characterizing the vents’ missing diversity (at both the meio and micro-scale) and elucidating the functional ecology of these vents.

The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin.

Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation.

Mechanisms of damage tolerance and repair during DNA replication.

Accurate duplication of chromosomal DNA is essential for the transmission of genetic information. The DNA replication fork encounters template lesions, physical barriers, transcriptional machinery, and topological barriers that challenge the faithful completion of the replication process. The flexibility of replisomes coupled with tolerance and repair mechanisms counteract these replication fork obstacles. The cell possesses several universal mechanisms that may be activated in response to various replication fork impediments, but it has also evolved ways to counter specific obstacles. In this review, we will discuss these general and specific strategies to counteract different forms of replication associated damage to maintain genomic stability.

The ZATT-TOP2A-PICH Axis Drives Extensive Replication Fork Reversal to Promote Genome Stability

Replication fork reversal is a global response to replication stress in mammalian cells, but precisely how it occurs remains poorly understood. Here, we show that, upon replication stress, DNA topoisomerase IIalpha (TOP2A) is recruited to stalled forks in a manner dependent on the SNF2-family DNA translocases HLTF, ZRANB3, and SMARCAL1. This is accompanied by an increase in TOP2A SUMOylation mediated by the SUMO E3 ligase ZATT and followed by recruitment of a SUMO-targeted DNA translocase, PICH. Disruption of the ZATT-TOP2A-PICH axis results in accumulation of partially reversed forks and enhanced genome instability. These results suggest that fork reversal occurs via a sequential two-step process. First, HLTF, ZRANB3, and SMARCAL1 initiate limited fork reversal, creating superhelical strain in the newly replicated sister chromatids. Second, TOP2A drives extensive fork reversal by resolving the resulting topological barriers and via its role in recruiting PICH to stalled forks.

Mechanism of Type IA Topoisomerases.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5′-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

Requirements for DNA-Bridging Proteins to Act as Topological Barriers of the Bacterial Genome

Bacterial genomes have been shown to be partitioned into several-kilobase-long chromosomal domains that are topologically independent from each other, meaning that change of DNA superhelicity in one domain does not propagate to neighbors. Both in vivo and in vitro experiments have been performed to question the nature of the topological barriers at play, leading to several predictions on possible molecular actors. Here, we address the question of topological barriers using polymer models of supercoiled DNA chains that are constrained such as to mimic the action of predicted molecular actors. More specifically, we determine under which conditions DNA-bridging proteins may act as topological barriers. To this end, we developed a coarse-grained bead-and-spring model and investigated its properties through Brownian dynamics simulations. As a result, we find that DNA-bridging proteins must exert rather strong constraints on their binding sites; they must block the diffusion of the excess of twist through the two binding sites on the DNA molecule and, simultaneously, prevent the rotation of one DNA segment relative to the other one. Importantly, not all DNA-bridging proteins satisfy this second condition. For example, single bridges formed by proteins that bind DNA nonspecifically, like H-NS dimers, are expected to fail with this respect. Our findings might also explain, in the case of specific DNA-bridging proteins like LacI, why multiple bridges are required to create stable independent topological domains. Strikingly, when the relative rotation of the DNA segments is not prevented, relaxation results in complex intrication of the two domains. Moreover, although the value of the torsional stress in each domain may vary, their differential is preserved. Our work also predicts that nucleoid-associated proteins known to wrap DNA must form higher protein-DNA complexes to efficiently work as topological barriers.

Eden growth models for flat clathrin lattices with vacancies

Clathrin-mediated endocytosis is one of the major pathways by which cells internalise cargo molecules. Recently it has been shown that clathrin triskelia can first assemble as flat lattices before the membrane starts to bend. However, for fully assembled clathrin lattices high energetic and topological barriers exist for the flat-to-curved transition. Here we explore the possibility that flat clathrin lattices grow with vacancies that are not visible in traditional imaging techniques but would lower these barriers. We identify the Eden model for cluster growth as the most appropriate modeling framework and systematically derive the four possible variants that result from the specific architecture of the clathrin triskelion. Our computer simulations show that the different models lead to clear differences in the statistical distributions of cluster shapes and densities. Experimental results from electron microscopy and correlative light microscopy provide first indications for the model variants with a moderate level of lattice vacancies.

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks.

During replication, the topology of DNA changes continuously in response to well-known activities of DNA helicases, polymerases, and topoisomerases. However, replisomes do not always progress at a constant speed and can slow-down and even stall at precise sites. The way these changes in the rate of replisome progression affect DNA topology is not yet well understood. The interplay of DNA topology and replication in several cases where progression of replication forks reacts differently to changes in DNA topology ahead is discussed here. It is proposed, there are at least two types of replication fork barriers: those that behave also as topological barriers and those that do not. Two-Dimensional (2D) agarose gel electrophoresis is the method of choice to distinguish between these two different types of replication fork barriers.

Fis protein forms DNA topological barriers to confine transcription-coupled DNA supercoiling in Escherichiacoli.

Previously, we demonstrated that transcription-coupled DNA supercoiling (TCDS) potently activated or inhibited nearby promoters in Escherichiacoli even in the presence of all four DNA topoisomerases, suggesting that DNA topoisomerases are not the only factors regulating TCDS. A different mechanism exists to confine this localized DNA supercoiling. Using an invivo system containing the TCDS-activated leu-500 promoter (Pleu-500 ), we find that the nucleoid-associated Fis protein potently inhibits the TCDS-mediated activation of Pleu-500 . We also find that deletion of the fis gene significantly enhances TCDS-mediated inhibition of transcription of three genes purH, yieP, and yrdA divergently coupled to different rrn operons in the early log phase. These results suggest that Fis protein forms DNA topological barriers upon binding to its recognition sites, blocks TCDS diffusion, and potently inhibits the TCDS-activated Pleu-500 .