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.

Nick Induced Dynamics in Supercoiled DNA Facilitates the Protein Target Search Process.

A DNA nick, defined as a discontinuity in a double-stranded DNA molecule where the phosphodiester bond between adjacent nucleotides of one strand is absent due to enzyme action, serves as an effective mechanism to alleviate stress in supercoiled DNA. This stress release is essential for the smooth operation of transcriptional machinery. However, the underlying mechanisms and their impact on protein search dynamics, which are crucial for initiating transcription, remain unclear. Through extensive computer simulations, we unravel the molecular picture, demonstrating that intramolecular stress release due to a DNA nick is driven by a combination of writhing and twisting motions, depending on the nick's position. This stress release is quantitatively manifested as a step-like increase in the linking number. Furthermore, we elucidate that the nicked supercoiled minicircles exhibit enhanced torsional dynamics, promoting rapid conformational changes and frequent shifts in the identities of juxtaposed DNA sites on the plectoneme. The dynamics of the juxtaposition sites facilitates communication between protein and DNA, resulting in faster protein diffusion compared with native DNA with the same topology. Our findings highlight the mechanistic intricacies and underscore the importance of DNA nicks in facilitating transcription elongation by actively managing torsional stress during DNA unwinding by the RNA polymerase.

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 stability and 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.

RNA interacts with topoisomerase I to adjust DNA topology.

Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.

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.

Inhibition of topoisomerase 2 catalytic activity impacts the integrity of heterochromatin and repetitive DNA and leads to interlinks between clustered repeats.

DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an SLX4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements.

In Vivo Genomic Supercoiling Mapping Using Psora-seq.

Supercoiling is a fundamental property of DNA that governs all strand opening reactions, including DNA replication, transcription, and homologous recombination. However, traditional genomic supercoiling assays are difficult and lack sensitivity. Building on prior assays using the DNA intercalator psoralen, we developed a supercoil mapping assay that is robust and sensitive to a wide range of supercoiling while requiring only commercially available reagents and common laboratory equipment. This method, psoralen affinity purification with genomic sequencing (Psora-seq), utilizes biotinylated psoralen and streptavidin-conjugated magnetic beads to facilitate efficient pull-down of psoralen-bound DNA, followed by deep sequencing to identify and quantify supercoiling at 1 kb resolution. Psora-seq overcomes two major bottlenecks associated with existing psoralen pull-down assays, inefficient photo-binding of psoralen-bound molecules, and poor recovery of cross-linked DNA.

Methods to Quantitatively Measure Topological Changes Induced by DNA-Binding Proteins In Vivo and In Vitro.

Agarose gel electrophoresis in the presence of chloroquine (an intercalating agent) can be used to resolve and characterize the population of topoisomers present in supercoiled plasmid DNA. Here, we describe how chloroquine gel electrophoresis can capture changes in the topoisomer distribution of plasmid DNA that bears a recognition site for a given protein, if that plasmid is isolated from cells producing the protein of interest. We also describe two complementary in vitro assays, which can be used to capture transient changes in DNA supercoiling caused when the purified protein of interest engages its recognition site. These are the topoisomerase I-mediated relaxation assay (TMRA) and the ligase-mediated supercoiling assay (LMSA). Together, these in vivo and in vitro methods allow the capture and measurement of changes in DNA topology that are triggered by DNA-binding proteins, especially those that multimerize on or spread along DNA.

Nucleosomes play a dual role in regulating transcription dynamics.

Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design.

Low levels of supercoiled mitochondrial DNA are involved in heart failure induced by transverse aortic constriction in mice via an inflammatory response mediated by ZBP1.

BACKGROUND: Inflammation in the myocardium plays a critical role in cardiac remodeling and the pathophysiology of heart failure (HF). Previous studies have shown that mitochondrial DNA (mtDNA) can exist in different topological forms. However, the specific influence of the ratio of supercoiled/relaxed mtDNA on the inflammatory response in cardiomyocytes remains poorly understood. The aim of this study was to elucidate the differential effects of different mtDNA types on cardiomyocyte inflammation through regulation of ZBP1. MATERIALS AND METHODS: A mouse model of HF was established by transverse aortic constriction (TAC) or doxorubicin (Doxo) induction. Histopathological changes were assessed by HE staining. ELISA was used to measure cytokine levels (IL-1ß and IL-6). Southern blot analysis was performed to examine the different topology of mtDNA. Pearson correlation analysis was used to determine the correlation between the ratio of supercoiled/relaxed mtDNA and inflammatory cytokines. Reverse transcription quantitative PCR (RT-qPCR) was used to measure the mRNA expression levels of cytokines (IL-1ß, IL-6) and Dloop, as an mtDNA marker. RESULTS: The ratio of supercoiled to relaxed mtDNA was significantly increased in the myocardium of Doxo-induced mice, whereas no significant changes were observed in TAC-induced mice. The levels of IL-1ß and IL-6 were positively correlated with the cytoplasmic mtDNA supercoiled/relaxed circle ratio. Different mtDNA topology has different effects on inflammatory pathways. Low supercoiled mtDNA primarily activates the NF-κB (Ser536) pathway via ZBP1, whereas high supercoiled mtDNA significantly affects the STAT1 and STAT2 pathways. The RIPK3-NF-κB pathway, as a downstream target of ZBP1, mediates the inflammatory response induced by low supercoiled mtDNA. Knockdown of TLR9 enhances the expression of ZBP1, p-NF-κB, and RIPK3 in cardiomyocytes treated with low supercoiled mtDNA, indicating the involvement of TLR9 in the anti-inflammatory role of ZBP1 in low supercoiled mtDNA-induced inflammation. CONCLUSION: Different ratios of supercoiled to relaxed mtDNA influence the inflammatory response of cardiomyocytes and contribute to HF through the involvement of ZBP1. ZBP1, together with its downstream inflammatory mechanisms, mediates the inflammatory response induced by a low ratio of supercoiled mtDNA.

Compaction, Relaxation, and Linearization of Megadalton-Sized DNA Plasmids: DNA Structures Probed by CD-MS.

High purity plasmid DNA is a raw material for recombinant protein production as well as an active ingredient in DNA vaccines. There are four primary plasmid structures that can be observed in a typical plasmid formulation: supercoiled, relaxed (circular), linearized, and condensed. Determining what structures are present in a sample is important, as the structure can affect activity; the supercoiled structure has the highest activity, and >90% supercoiled is desired for industry standards. Recently, charge detection mass spectrometry (CD-MS) was used to distinguish two of the structures, supercoiled and condensed, by measuring the charge deposited on the ions by positive mode electrospray. Here, CD-MS is used to probe the structures of DNA plasmids during compaction with polycations, and through enzymatic treatment to relax and linearize plasmids. We find that all four structural types for plasmid DNA have unique charging profiles that can be distinguished using CD-MS. The extent of mechanical shearing of the DNA plasmids during electrospray is strongly influenced by the structural type.

DNA topology: A central dynamic coordinator in chromatin regulation.

Double helical DNA winds around nucleosomes, forming a beads-on-a-string array that further contributes to the formation of high-order chromatin structures. The regulatory components of the chromatin, interacting intricately with DNA, often exploit the topological tension inherent in the DNA molecule. Recent findings shed light on, and simultaneously complicate, the multifaceted roles of DNA topology (also known as DNA supercoiling) in various aspects of chromatin regulation. Different studies may emphasize the dynamics of DNA topological tension across different scales, interacting with diverse chromatin factors such as nucleosomes, nucleic acid motors that propel DNA-tracking processes, and DNA topoisomerases. In this review, we consolidate recent studies and establish connections between distinct scientific discoveries, advancing our current understanding of chromatin regulation mediated by the supercoiling tension of the double helix. Additionally, we explore the implications of DNA topology and DNA topoisomerases in human diseases, along with their potential applications in therapeutic interventions.

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.

Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Structural basis of DNA crossover capture by Escherichia coli DNA gyrase.

DNA supercoiling must be precisely regulated by topoisomerases to prevent DNA entanglement. The interaction of type IIA DNA topoisomerases with two DNA molecules, enabling the transport of one duplex through the transient double-stranded break of the other, remains elusive owing to structures derived solely from single linear duplex DNAs lacking topological constraints. Using cryo-electron microscopy, we solved the structure of Escherichia coli DNA gyrase bound to a negatively supercoiled minicircle DNA. We show how DNA gyrase captures a DNA crossover, revealing both conserved molecular grooves that accommodate the DNA helices. Together with molecular tweezer experiments, the structure shows that the DNA crossover is of positive chirality, reconciling the binding step of gyrase-mediated DNA relaxation and supercoiling in a single structure.

Bridging DNA contacts allow Dps from E. coli to condense DNA.

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Selective hydrophobic interaction chromatography for high purity of supercoiled DNA plasmids.

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.

The acidic C-terminal tail of DNA Gyrase of Salmonella enterica serovar Typhi controls DNA relaxation in an acidic environment.

The intracellular bacteria, Salmonella Typhi adapts to acidic conditions in the host cell by resetting the chromosomal DNA topology majorly controlled by DNA Gyrase, a Type II topoisomerase. DNA Gyrase forms a heterodimer A2B2 complex, which manages the DNA supercoiling and relaxation in the cell. DNA relaxation forms a part of the regulatory mechanism to activate the transcription of genes required to survive under hostile conditions. Acid-induced stress attenuates the supercoiling activity of the DNA Gyrase, resulting in DNA relaxation. Salmonella DNA becomes relaxed as the bacteria adapt to the acidified intracellular environment. Despite comprehensive studies on DNA Gyrase, the mechanism to control supercoiling activity needs to be better understood. A loss in supercoiling activity in E. coli was observed upon deletion of the non-conserved acidic C-tail of Gyrase A subunit. Salmonella Gyrase also contains an acidic tail at the C-terminus of Gyrase A, where its deletion resulted in reduced supercoiling activity compared to wild-type Gyrase. Interestingly, we also found that wild-type Gyrase compromises supercoiling activity at acidic pH 2-3, thereby causing DNA relaxation. The absence of a C-tail displayed DNA supercoiling to some extent between pH 2-9. Hence, the C-tail of Gyrase A might be one of the controlling factors that cause DNA relaxation in Salmonella at acidic pH conditions. We propose that the presence of the C-tail of GyraseA causes acid-mediated inhibition of the negative supercoiling activity of Gyrase, resulting in relaxed DNA that attracts DNA-binding proteins for controlling the transcriptional response.

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.

Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments.

DNA stores our genetic information and is ubiquitous in applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and can change local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model with a straight-forward numerical implementation of how the global constraints introduced by DNA topology modulate binding. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Our model correctly describes bulk experiments using plasmids with different starting topologies, different intercalators, and over a broad range of intercalator and DNA concentrations. We demonstrate and quantitatively model supercoiling-dependent binding in a single-molecule assay, where we directly observe the different intercalator densities going from supercoiled to nicked DNA. The single-molecule assay provides direct access to binding kinetics and DNA supercoil dynamics. Our model has broad implications for the detection and quantification of DNA, including the use of psoralen for UV-induced DNA crosslinking to quantify torsional tension in vivo, and for the modulation of DNA binding in cellular contexts.