Positive Supercoiling Research Articles

A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions.

DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but does not affect the rate of ATP-induced sliding clamp formation. These findings not only underscore the versatility of our protocol but also opens new avenues for exploring the intricate dynamics of protein-DNA interactions under the influences of supercoiling.

Type II topoisomerases shape multi-scale 3D chromatin folding in regions of positive supercoils.

Type II topoisomerases (TOP2s) resolve torsional stress accumulated during various cellular processes and are enriched at chromatin loop anchors and topologically associated domain (TAD) boundaries, where, when trapped, can lead to genomic instability promoting the formation of oncogenic fusions. Whether TOP2s relieve topological constraints at these positions and/or participate in 3D chromosome folding remains unclear. Here, we combine 3D genomics, imaging, and GapRUN, a method for the genome-wide profiling of positive supercoiling, to assess the role of TOP2s in shaping chromosome organization in human cells. Acute TOP2 depletion led to the emergence of new, large-scale contacts at the boundaries between active, positively supercoiled, and lamina-associated domains. TOP2-dependent changes at the higher-order chromatin folding were accompanied by remodeling of chromatin-nuclear lamina interactions and of gene expression, while at the chromatin loop level, TOP2 depletion predominantly remodeled transcriptionally anchored, positively supercoiled loops. We propose that TOP2s act as a fine regulator of chromosome folding at multiple scales.

Human Smc5/6 recognises transcription-generated positive DNA supercoils

Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon integration of the DNA into the chromosome. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further demonstrate that the transcriptional process, per se, and particularly the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. More specifically, we find that in vivo Smc5/6 binds to positively supercoiled DNA. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localizes at few but highly transcribed chromosome loci, not only unveil a previously unforeseen role of Smc5/6 in DNA topology management during transcription but highlight the significance of sensing DNA topology as an antiviral defense mechanism.

Innovative Tools for DNA Topology Probing in Human Cells Reveal a Build-Up of Positive Supercoils Following Replication Stress at Telomeres and at the FRA3B Fragile Site.

Linear unconstrained DNA cannot harbor supercoils since these supercoils can diffuse and be eliminated by free rotation of the DNA strands at the end of the molecule. Mammalian telomeres, despite constituting the ends of linear chromosomes, can hold supercoils and be subjected to topological stress. While negative supercoiling was previously observed, thus proving the existence of telomeric topological constraints, positive supercoils were never probed due to the lack of an appropriate tool. Indeed, the few tools available currently could only investigate unwound (Trioxsalen) or overwound (GapR) DNA topology (variations in twist) but not the variations in writhe (supercoils and plectonemes). To address this question, we have designed innovative tools aimed at analyzing both positive and negative DNA writhe in cells. Using them, we could observe the build-up of positive supercoils following replication stress and inhibition of Topoisomerase 2 on telomeres. TRF2 depletion caused both telomere relaxation and an increase in positive supercoils while the inhibition of Histone Deacetylase I and II by TSA only caused telomere relaxation. Moving outside telomeres, we also observed a build-up of positive supercoils on the FRA3B fragile site following replication stress, suggesting a topological model of DNA fragility for this site.

Highly sensitive mapping of in vitro type II topoisomerase DNA cleavage sites with SHAN-seq.

Type II topoisomerases (topos) are a ubiquitous and essential class of enzymes that form transient enzyme-bound double-stranded breaks on DNA called cleavage complexes. The location and frequency of these cleavage complexes on DNA is important for cellular function, genomic stabilityand a number of clinically important anticancer and antibacterial drugs, e.g. quinolones. We developed a simple high-accuracy end-sequencing (SHAN-seq) method to sensitively map type II topo cleavage complexes on DNA in vitro. Using SHAN-seq, we detected Escherichia coli gyrase and topoisomerase IV cleavage complexes at hundreds of sites on supercoiled pBR322 DNA, approximately one site every ten bp, with frequencies that varied by two-to-three orders of magnitude. These sites included previously identified sites and 20-50-fold more new sites. We show that the location and frequency of cleavage complexes at these sites are enzyme-specific and vary substantially in the presence of the quinolone, ciprofloxacin, but not with DNA supercoil chirality, i.e. negative versus positive supercoiling. SHAN-seq's exquisite sensitivity provides an unprecedented single-nucleotide resolution view of the distribution of gyrase and topoisomerase IV cleavage complexes on DNA. Moreover, the discovery that these enzymes can cleave DNA at orders of magnitude more sites than the relatively few previously known sites resolves the apparent paradox of how these enzymes resolve topological problems throughout the genome.

Phylogenetic distribution of DNA topoisomerase VI and its distinction from SPO11.

DNA topoisomerases (topos) are major targets for antimicrobial and chemotherapeutic drugs due to their fundamental roles in regulating DNA topology. Type II topos are essential for chromosome segregation and relaxing positive DNA supercoils, and are exemplified by topo II in eukaryotes, topo IV and DNA gyrase in bacteria, and topo VI in archaea. Topo VI occurs ubiquitously in plants and sporadically in bacteria, algae, and other protists and is highly homologous to Spo11, which initiates eukaryotic homologous recombination. This homology makes the two complexes difficult to distinguish by sequence and leads to discrepancies such as the identity of the putative topo VI in malarial Plasmodium species. A lack of understanding of the role and distribution of topo VI outside of archaea hampers its pursuit as a potential drug target, and the present study addresses this with an up-to-date and extensive phylogenetic analysis. We show that the A and B subunits of topo VI and Spo11 can be distinguished using phylogenetics and structural modelling, and that topo VI is not present in Plasmodium nor other members of the phylum Apicomplexa. These findings provide insights into the evolutionary relationships between topo VI and Spo11, and their adoption alongside other type II topos.

Depletion of CpG dinucleotides in bacterial genomes may represent an adaptation to high temperatures.

Dinucleotide biases have been widely investigated in the genomes of eukaryotes and viruses, but not in bacteria. We assembled a dataset of bacterial genomes (>15000), which are representative of the genetic diversity in the kingdom Eubacteria, and we analyzed dinucleotide biases in relation to different traits. We found that TpA dinucleotides are the most depleted and that CpG dinucleotides show the widest dispersion. The abundances of both dinucleotides vary with genomic G+C content and show a very strong phylogenetic signal. After accounting for G+C content and phylogenetic inertia, we analyzed different bacterial lifestyle traits. We found that temperature preferences associate with the abundance of CpG dinucleotides, with thermophiles/hyperthemophiles being particularly depleted. Conversely, the TpA dinucleotide displays a bias that only depends on genomic G+C composition. Using predictions of intrinsic cyclizability we also show that CpG depletion may associate with higher DNA bendability in both thermophiles/hyperthermophiles and mesophiles, and that the former are predicted to have significantly more flexible genomes than the latter. We suggest that higher bendability is advantageous at high temperatures because it facilitates DNA positive supercoiling and that, through modulation of DNA mechanical properties, local or global CpG depletion controls genome organization, most likely not only in bacteria.

Bacterial chromatin proteins, transcription, and DNA topology: Inseparable partners in the control of gene expression.

DNA in bacterial chromosomes is organized into higher-order structures by DNA-binding proteins called nucleoid-associated proteins (NAPs) or bacterial chromatin proteins (BCPs). BCPs often bind to or near DNA loci transcribed by RNA polymerase (RNAP) and can either increase or decrease gene expression. To understand the mechanisms by which BCPs alter transcription, one must consider both steric effects and the topological forces that arise when DNA deviates from its fully relaxed double-helical structure. Transcribing RNAP creates DNA negative (-) supercoils upstream and positive (+) supercoils downstream whenever RNAP and DNA are unable to rotate freely. This (-) and (+) supercoiling generates topological forces that resist forward translocation of DNA through RNAP unless the supercoiling is constrained by BCPs or relieved by topoisomerases. BCPs also may enhance topological stress and overall can either inhibit or aid transcription. Here, we review current understanding of how RNAP, BCPs, and DNA topology interplay to control gene expression.

Transcription-driven DNA supercoiling counteracts H-NS-mediated gene silencing in bacterial chromatin

In all living cells, genomic DNA is compacted through interactions with dedicated proteins and/or the formation of plectonemic coils. In bacteria, DNA compaction is achieved dynamically, coordinated with dense and constantly changing transcriptional activity. H-NS, a major bacterial nucleoid structuring protein, is of special interest due to its interplay with RNA polymerase. H-NS:DNA nucleoprotein filaments inhibit transcription initiation by RNA polymerase. However, the discovery that genes silenced by H-NS can be activated by transcription originating from neighboring regions has suggested that elongating RNA polymerases can disassemble H-NS:DNA filaments. In this study, we present evidence that transcription-induced counter-silencing does not require transcription to reach the silenced gene; rather, it exerts its effect at a distance. Counter-silencing is suppressed by introducing a DNA gyrase binding site within the intervening segment, suggesting that the long-range effect results from transcription-driven positive DNA supercoils diffusing toward the silenced gene. We propose a model wherein H-NS:DNA complexes form in vivo on negatively supercoiled DNA, with H-NS bridging the two arms of the plectoneme. Rotational diffusion of positive supercoils generated by neighboring transcription will cause the H-NS-bound negatively-supercoiled plectoneme to “unroll” disrupting the H-NS bridges and releasing H-NS.

Loop-extruding Smc5/6 organizes transcription-induced positive DNA supercoils

The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.

Flexible structural arrangement and DNA-binding properties of protein p6 from Bacillus subtillis phage φ29.

The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein-DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.

How Do Thermophiles Organize Their Genomes?

All cells must maintain the structural and functional integrity of the genome under a wide range of environments. High temperatures pose a formidable challenge to cells by denaturing the DNA double helix, causing chemical damage to DNA, and increasing the random thermal motion of chromosomes. Thermophiles, predominantly classified as bacteria or archaea, exhibit an exceptional capacity to mitigate these detrimental effects and prosper under extreme thermal conditions, with some species tolerating temperatures higher than 100°C. Their genomes are mainly characterized by the presence of reverse gyrase, a unique topoisomerase that introduces positive supercoils into DNA. This enzyme has been suggested to maintain the genome integrity of thermophiles by limiting DNA melting and mediating DNA repair. Previous studies provided significant insights into the mechanisms by which NAPs, histones, SMC superfamily proteins, and polyamines affect the 3D genomes of thermophiles across different scales. Here, I discuss current knowledge of the genome organization in thermophiles and pertinent research questions for future investigations.

ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

The function of the mitogen-activated protein kinase signaling pathway is required for the activation of immediate early genes (IEGs), including EGR1 and FOS, for cell growth and proliferation. Recent studies have identified topoisomerase II (TOP2) as one of the important regulators of the transcriptional activation of IEGs. However, the mechanism underlying transcriptional regulation involving TOP2 in IEG activation has remained unknown. Here, we demonstrate that ERK2, but not ERK1, is important for IEG transcriptional activation and report a critical ELK1 binding sequence for ERK2 function at the EGR1 gene. Our data indicate that both ERK1 and ERK2 extensively phosphorylate the C-terminal domain of TOP2B at mutual and distinctive residues. Although both ERK1 and ERK2 enhance the catalytic rate of TOP2B required to relax positive DNA supercoiling, ERK2 delays TOP2B catalysis of negative DNA supercoiling. In addition, ERK1 may relax DNA supercoiling by itself. ERK2 catalytic inhibition or knock-down interferes with transcription and deregulates TOP2B in IEGs. Furthermore, we present the first cryo-EM structure of the human cell-purified TOP2B and etoposide together with the EGR1 transcriptional start site (–30 to +20) that has the strongest affinity to TOP2B within –423 to +332. The structure shows TOP2B-mediated breakage and dramatic bending of the DNA. Transcription is activated by etoposide, while it is inhibited by ICRF193 at EGR1 and FOS, suggesting that TOP2B-mediated DNA break to favor transcriptional activation. Taken together, this study suggests that activated ERK2 phosphorylates TOP2B to regulate TOP2-DNA interactions and favor transcriptional activation in IEGs. We propose that TOP2B association, catalysis, and dissociation on its substrate DNA are important processes for regulating transcription and that ERK2-mediated TOP2B phosphorylation may be key for the catalysis and dissociation steps.

Differential roles of positive and negative supercoiling in organizing the E. coli genome.

This study aims to explore whether and how positive and negative supercoiling contribute to the three-dimensional (3D) organization of the bacterial genome. We used recently published Escherichia coli GapR ChIP-seq and TopoI ChIP-seq (also called EcTopoI-seq) data, which marks positive and negative supercoiling sites, respectively, to study how supercoiling correlates with the spatial contact maps obtained from chromosome conformation capture sequencing (Hi-C and 5C). We find that supercoiled chromosomal loci have overall higher Hi-C contact frequencies than sites that are not supercoiled. Surprisingly, positive supercoiling corresponds to higher spatial contact than negative supercoiling. Additionally, positive, but not negative, supercoiling could be identified from Hi-C data with high accuracy. We further find that the majority of positive and negative supercoils coincide with highly active transcription units, with a minor group likelyassociated with replication and other genomic processes. Our results show that both positive and negative supercoiling enhance spatial contact, with positive supercoiling playing a larger role in bringing genomic loci closer in space. Based on our results, we propose new physical models of how the E. coli chromosome is organized by positive and negative supercoils.

To Break or Not to Break: The Role of TOP2B in Transcription.

Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This can inhibit transcriptional elongation and other processes essential to transcription. In addition, chromatin remodeling associated with gene activation can generate or be hindered by excess DNA torsional stress in gene regulatory regions. These topological challenges are solved by DNA topoisomerases via a strand-passage reaction which involves transiently breaking and re-joining of one (type I topoisomerases) or both (type II topoisomerases) strands of the phosphodiester backbone. This review will focus on one of the two mammalian type II DNA topoisomerase enzymes, DNA topoisomerase II beta (TOP2B), that have been implicated in correct execution of developmental transcriptional programs and in signal-induced transcription, including transcriptional activation by nuclear hormone ligands. Surprisingly, several lines of evidence indicate that TOP2B-mediated protein-free DNA double-strand breaks are involved in signal-induced transcription. We discuss the possible significance and origins of these DSBs along with a network of protein interaction data supporting a variety of roles for TOP2B in transcriptional regulation.

Impact of R-loops on oncogene-induced replication stress in cancer cells.

Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability and cancer development. It is now well established that transcription can interfere with genome replication in different ways, such as head-on collisions between polymerases, accumulation of positive DNA supercoils or formation of R-loops. These structures form during transcription when nascent RNA reanneals with DNA behind the RNA polymerase, forming a stable DNA:RNA hybrid. In this review, we discuss how these different cotranscriptional processes disrupt the progression of replication forks and how they contribute to genetic instability in cancer cells.

Identification of potential DNA gyrase inhibitors: virtual screening, extra-precision docking and molecular dynamics simulation study

DNA gyrase brings negative supercoils into DNA and loosens up certain positive supercoils that collect during replication and transcription and is a notable antibacterial target. To fight against the menace of antibiotic-resistant bacterial infections, we have employed various computational tools like high throughput virtual screening (HTVS), standard precision (SP) docking, extra precision (XP) docking, and molecular dynamics (MD) simulation studies to identify some potential DNA gyrase inhibitors. A focused library of 5968 anti-bacterial compounds was screened using the HTVS docking protocol of the glide module of Maestro. The top 200 docked compounds were further filtered using SP and XP docking protocols, and their free binding energies were calculated using MM-GBSA studies. The binding and stability of the top two compounds which showed better docking scores than the co-crystallized ligand (Clorobiocin) of DNA gyrase (PDB ID: 1KZN) were further probed by MD simulation of 100 ns using GROMACS. MD simulation study suggested that the compounds AM1 and AM5 form a stable complex with DNA gyrase with a good number of hydrogen bonds. XP docking study showed that interaction with the crucial amino acids for compounds AM1 and AM5 was like the co-crystallized ligand. These compounds were also predicted to be drug-like molecules with good water solubility and excellent absorption profiles. Based on the above studies, herein we report compounds AM1 (1R,3S)-1-(2-((3-(ammoniomethyl)phenyl)amino)-2-oxoethyl)-3-carbamoylpiperidin-1-ium and AM5 (1'S,2 s,4R)-4-ammonio-6-ethyl-1'-methylspiro[chromane-2,4'-piperidin]-1'-ium as potential DNA gyrase inhibitors which can be further developed as a potential lead against the menace of antibiotic resistance.

Sen1 and Rrm3 ensure permissive topological conditions for replication termination

Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and transmission electron microscopy, we find that Rrm3hPif1 and Sen1hSenataxin helicases assist termination at TERs; Sen1 specifically acts at telomeres. rrm3 and sen1 genetically interact and fail to terminate replication, exhibiting fragility at termination zones (TERs) and telomeres. sen1rrm3 accumulates RNA-DNA hybrids and X-shaped gapped or reversed converging forks at TERs; sen1, but not rrm3, builds up RNA polymerase II (RNPII) at TERs and telomeres. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of Top1 and Top2 when forks encounter transcription head on or codirectionally, respectively, thus preventing the slowing down of DNA and RNA polymerases. Hence Rrm3 and Sen1 are indispensable to generate permissive topological conditions for replication termination.

Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a β-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length andis dispensable for DNA supercoiling, the β-bulge loop is required for supercoiling activity. It has previously been shown that the β-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a β-bulge loop as a minimal latch is reported. It is shown that the β-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

Single-nucleotide resolution detection of Topo IV cleavage activity in the Escherichia coli genome with Topo-Seq.

Topoisomerase IV (Topo IV) is the main decatenation enzyme in Escherichia coli; it removes catenation links that are formed during DNA replication. Topo IV binding and cleavage sites were previously identified in the E. coli genome with ChIP-Seq and NorfIP. Here, we used a more sensitive, single-nucleotide resolution Topo-Seq procedure to identify Topo IV cleavage sites (TCSs) genome-wide. We detected thousands of TCSs scattered in the bacterial genome. The determined cleavage motif of Topo IV contained previously known cleavage determinants (-4G/+8C, -2A/+6 T, -1 T/+5A) and additional, not observed previously, positions -7C/+11G and -6C/+10G. TCSs were depleted in the Ter macrodomain except for two exceptionally strong non-canonical cleavage sites located in 33 and 38 bp from the XerC-box of the dif-site. Topo IV cleavage activity was increased in Left and Right macrodomains flanking the Ter macrodomain and was especially high in the 50-60 kb region containing the oriC origin of replication. Topo IV enrichment was also increased downstream of highly active transcription units, indicating that the enzyme is involved in relaxation of transcription-induced positive supercoiling.