Positive Supercoiling Research Articles

Dissecting the in vivo dynamics of transcription locking due to positive supercoiling buildup

Positive supercoiling buildup (PSB) is a pervasive phenomenon in the transcriptional programs of Escherichia coli. After finding a range of Gyrase concentrations where the inverse of the transcription rate of a chromosome-integrated gene changes linearly with the inverse of Gyrase concentration, we apply a LineWeaver-Burk plot to dissect the expected in vivo transcription rate in absence of PSB. We validate the estimation by time-lapse microscopy of single-RNA production kinetics of the same gene when single-copy plasmid-borne, shown to be impervious to Gyrase inhibition. Next, we estimate the fraction of time in locked states and number of transcription events prior to locking, which we validate by measurements under Gyrase inhibition. Replacing the gene of interest by one with slower transcription rate decreases the fraction of time in locked states due to PSB. Finally, we combine data from both constructs to infer a range of possible transcription initiation locking kinetics in a chromosomal location, obtainable by tuning the transcription rate. We validate with measurements of transcription activity at different induction levels. This strategy for dissecting transcription initiation locking kinetics due to PSB can contribute to resolve the transcriptional programs of E. coli and in the engineering of synthetic genetic circuits.

Histone-DNA Interactions in the Archaeon Methanocaldococcus Jannaschii

The requirement for DNA compaction is universal across all domains of life. Although the histone proteins of Archaea and Eukaryotes have low sequence homology, the core histone fold is well-conserved and structures are virtually identical. However, small structural differences mean that Archaeal nucleosomes might assemble into a continuous solenoid-like fiber: the ‘hypernucleosome’. Given that M. jannaschii thrives at 80oC and its genomic DNA is positively supercoiled (the opposite to mesophiles like us), the mechanism of DNA packaging may show interesting adaptations and diversity that will inform us about the biophysics of chromatin in general. Here, we have used magnetic tweezers to control the supercoiling of DNA while quantifying the kinetics and energetics of DNA-histone binding, wrapping and compaction. Using this technique, we have investigated the properties of M. jannaschii chromatin with its most abundant histone protein “A3”. We propose a model for the force-extension properties of Archaeal chromatin, analogous to that for Eukaryotic chromatin, and demonstrate that the force-extension properties are dependent upon protein concentration. We find that, under moderate load, histone binding confers stability to DNA by preventing base-pair unstacking when negative supercoiling is applied, and the chromatin fiber shows a sharp buckling transition between both positive and negative supercoiling regimes. At high load (>5pN), the chromatin fiber is disrupted and the force-extension diagram returns to the worm-like chain, typical of naked DNA. Our model suggests a mechanism by which DNA processing enzymes may shunt or displace nucleosomes, obviating the need for active chromatin remodeling enzymes.

Coarse-Grained Modeling of DNA Plectoneme Formation in the Presence of Base-Pair Mismatches

Defects in double stranded DNA can arise from damaged bases or mismatched base pairs. In vivo, defects in DNA must be rapidly and efficiently repaired since they affect a variety of cell processes. Here we use molecular dynamics to study the effect of mismatched bps on DNA supercoiling. Under external torsional stress, an elastic rod with a defect would buckle at the defect; provided the defect reduces the local bending stiffness. In genomic DNA however, the entropic cost associated with plectoneme localization could make pinning unfavorable. Magnetic tweezers-based studies of DNA supercoiling have shown that in DNA harboring a single mismatch, the plectoneme always localizes at the mismatch. These studies were conducted at relatively high salt concentrations (>0.5M NaCl). Theoretical studies have predicted that under physiological salt concentrations of ∼0.2M NaCl, plectoneme localization becomes probabilistic. However, both experimental and theoretical approaches are currently limited to positively supercoiled DNA. We develop a simulation framework using the oxDNA model to study the effect of mismatches on both positively and negatively supercoiled DNA in physiologically relevant salt concentrations. We study both positive and negative supercoiling in a 610bp long DNA with 0,2,4 and 6 mismatches at its center. We find that for a positively supercoiled DNA, the oxDNA framework can reproduce the experimentally observed plectoneme pinning at high force (2 pN) and high salt concentrations (1M NaCl). Under physiological salt concentrations (0.2M NaCl), we find that the plectoneme localization at the mismatch becomes probabilistic. To determine the effects of entropy on plectoneme localization directly, we simulate a 1010bp DNA under identical conditions. The utility of the simulation approach is highlighted by the ability to quantify the effect of mismatches on the motion of plectonemes along the DNA molecule.

Positive Supercoiling Ahead of RNA Polymerase Aids Exit from Protein-Mediated Loops

Positive Supercoiling Ahead of RNA Polymerase Aids Exit from Protein-Mediated Loops

Mechanistic insight into the role of Poly(ADP-ribosyl)ation in DNA topology modulation and response to DNA damage.

Genotoxic stress generates single- and double-strand DNA breaks either through direct damage by reactive oxygen species or as intermediates of DNA repair. Failure to detect and repair DNA strand breaks leads to deleterious consequences such as chromosomal aberrations, genomic instability and cell death. DNA strand breaks disrupt the superhelical state of cellular DNA, which further disturbs the chromatin architecture and gene activity regulation. Proteins from the poly(ADP-ribose) polymerase (PARP) family, such as PARP1 and PARP2, use NAD+ as a substrate to catalyse the synthesis of polymeric chains consisting of ADP-ribose units covalently attached to an acceptor molecule. PARP1 and PARP2 are regarded as DNA damage sensors that, upon activation by strand breaks, poly(ADP-ribosyl)ate themselves and nuclear acceptor proteins. Noteworthy, the regularly branched structure of poly(ADP-ribose) polymer suggests that the mechanism of its synthesis may involve circular movement of PARP1 around the DNA helix, with a branching point in PAR corresponding to one complete 360° turn. We propose that PARP1 stays bound to a DNA strand break end, but rotates around the helix displaced by the growing poly(ADP-ribose) chain, and that this rotation could introduce positive supercoils into damaged chromosomal DNA. This topology modulation would enable nucleosome displacement and chromatin decondensation around the lesion site, facilitating the access of DNA repair proteins or transcription factors. PARP1-mediated DNA supercoiling can be transmitted over long distances, resulting in changes in the high-order chromatin structures. The available structures of PARP1 are consistent with the strand break-induced PAR synthesis as a driving force for PARP1 rotation around the DNA axis.

Constitutively active Artemis nuclease recognizes structures containing single-stranded DNA configurations

The Artemis nuclease recognizes and endonucleolytically cleaves at single-stranded to double-stranded DNA (ss/dsDNA) boundaries. It is also a key enzyme in the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. Previously, a truncated form, Artemis-413, was developed that is constitutively active both in vitro and in vivo. Here, we use this constitutively active form of Artemis to detect DNA structures with ss/dsDNA boundaries that arise under topological stress. Topoisomerases prevent abnormal levels of torsional stress through modulation of positive and negative supercoiling. We show that overexpression of Artemis-413 in yeast cells carrying genetic mutations that ablate topoisomerase activity have an increased frequency of DNA double-strand breaks (DSBs). Based on the biochemical activity of Artemis, this suggests an increase in ss/dsDNA-containing structures upon increased torsional stress, with DSBs arising due to Artemis cutting at these ss/dsDNA structures. Camptothecin targets topoisomerase IB (Top1), and cells treated with camptothecin show increased DSBs. We find that expression of Artemis-413 in camptothecin-treated cells leads to a reduction in DSBs, the opposite of what we find with topoisomerase genetic mutations. This contrast between outcomes not only confirms that topoisomerase mutation and topoisomerase poisoning have distinct effects on cells, but also demonstrates the usefulness of Artemis-413 to study changes in DNA structure.

Closing the DNA replication cycle: from simple circular molecules to supercoiled and knotted DNA catenanes.

Due to helical structure of DNA, massive amounts of positive supercoils are constantly introduced ahead of each replication fork. Positive supercoiling inhibits progression of replication forks but various mechanisms evolved that permit very efficient relaxation of that positive supercoiling. Some of these mechanisms lead to interesting topological situations where DNA supercoiling, catenation and knotting coexist and influence each other in DNA molecules being replicated. Here, we first review fundamental aspects of DNA supercoiling, catenation and knotting when these qualitatively different topological states do not coexist in the same circular DNA but also when they are present at the same time in replicating DNA molecules. We also review differences between eukaryotic and prokaryotic cellular strategies that permit relaxation of positive supercoiling arising ahead of the replication forks. We end our review by discussing very recent studies giving a long-sought answer to the question of how slow DNA topoisomerases capable of relaxing just a few positive supercoils per second can counteract the introduction of hundreds of positive supercoils per second ahead of advancing replication forks.

PICH and TOP3A cooperate to induce positive DNA supercoiling.

All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.

Twist to disentangle.

Intertwining of DNA molecules frequently results in the formation of ‘ultrafine bridges’ between sister chromatids that need to be resolved during segregation of the chromatids into daughter cells. Although it has been established that these DNA bridges are coated by the helicase PICH, it has remained unknown how PICH assists in their resolution. A study now reveals that PICH directs the formation of positive DNA supercoiling in the presence of type I topoisomerases to promote the subsequent disentanglement of these DNA helices by type II topoisomerases. Remarkably, PICH might be able to reconfigure DNA topology by extruding loops of DNA while it moves along the double helix.

Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures

Temperature shifts trigger genome-wide changes in Escherichia coli’s gene expression. We studied if chromosome integration impacts on a gene’s sensitivity to these shifts, by comparing the single-RNA production kinetics of a PLacO3O1 promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated PLacO3O1 has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.

Transcription Machine Studied in a Nutshell on T7 RNA Polymerase Mechanochemistry, Fidelity Control, and Bursting Activity

RNA polymerase (RNAP) is an essential enzyme that transcribes DNA into messenger RNA to further express proteins. Bacteriophage T7 RNAP is widely utilized in lab gene expression system and synthetic circuit as it transcribes self-sufficiently, without transcription factors, and with high promoter activity. We have investigated T7 RNAP on both its mechano-chemical couplings and fidelity control during transcription, combining physical modeling with structural dynamics simulations. In particular, we have implemented extensive atomistic molecular dynamics (MD) simulations and constructed the Markov state model (MSM) to investigate product release and translocation process of T7 RNAP, as it couples Brownian thermal motions with essential structural elements to assist PPi release, to facilitate the RNAP translocation, and to prevent backtracking. Meanwhile, we systematically quantified the nucleotide selectivity of this non-proofreading RNAP at multiple kinetic checkpoints prior to the full nucleotide incorporation. Essential selection against the non-cognate nucleotide species appears to happen at early checkpoints at the pre-insertion and right after, with a critical amino acid serving as a paw for selective ratcheting RNAP along the DNA. In addition, we also model the complete transcription process along with RNAP traffic, as positive supercoiling is accumulated in front of a leading RNAP to inhibit the transcription, and then being resolved gradually to allow transcriptional bursting.

DNA Shape Shifting as a Gene Therapy Tool

DNA supercoiling affects DNA metabolism yet much about how it does so is unknown. Using 336 bp DNA minicircles covering a range of positive to negative supercoiling, we unveiled the first three-dimensional structures of supercoiled DNA using cryo-electron tomography. With supercoiling, DNA can form far more bent and contorted shapes than predicted. We sought to understand how the interplay of DNA sequence and supercoiling drives the formation of these shapes using coarse-grained simulations and biochemical probing. Base pair disruptions indicate regions of high bending, as localized denaturation (from base flipping) creates flexible hinges. At the same time, sharp bending at the apices of highly writhed DNA circles leads to broken base pairs. Probing with nuclease Bal-31 revealed exposed bases as a function of supercoiling. Bal31 cleaved all the negatively supercoiled 336 bp minicircles but the rate increased beyond a distinct negative supercoiling threshold. This threshold shifted to more negative supercoiling for 672 bp minicircles with inherently less curvature, demonstrating the relationship between bending and base accessibility. A sharp positive supercoiling threshold was required for Bal-31 cleavage to even occur. We mapped Bal-31 cleavage sites and, using coarse-grained simulations, determined the DNA register of our cryo-electron micrograph images. Our data reveal two hotspots of Bal-31 cleavage located ∼180° apart along the DNA circumference, suggesting that exposed bases are predominantly located at superhelical apices and that a particular three-dimensional conformation is dominant. The relative probability of Bal-31 cleaving at either site varied as a function of supercoiling. Together these data reveal the interplay among sequence, supercoiling, and shape, resulting in conformational changes that should profoundly influence DNA interactions with proteins. Understanding these changes could facilitate the design of supercoiling-dependent DNA nanostructures for gene therapy.

Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

DNA Replication Arrest at Protein and Transcription Barriers is Governed by DNA Supercoiling

The unscheduled arrest of DNA replication forks is a frequent and mutagenic phenomenonaffecting all growing cells. DNA-bound proteins and elongating transcription complexes are thetwo major triggers of unscheduled fork arrest, and it is generally thought that these effectsoperate by physical occlusion. We show here that, instead, replication fork arrest results from theaccumulation of superhelical stress between the fork and barrier, thousands of base pairs away.Reducing positive supercoiling prevented fork arrest, allowing fork progression through both astable protein complex and a highly transcribed site oriented toward oncoming replication forks.Reducing positive supercoiling improved fork progression globally and decreased the occurrenceof spontaneous fork stalling, indicating that spontaneous fork blockages are frequent and alsosupercoiling-mediated. These results transform our understanding of how DNA-bound obstaclesimpede replication forks — by restricting the passage of inhibitory supercoiling instead of bydirect collision.

Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

Increase of positive supercoiling in a hyperthermophilic archaeon after UV irradiation.

Diverse DNA repair mechanisms are essential to all living organisms. Some of the most widespread repair systems allow recovery of genome integrity in the face of UV radiation. Here, we show that the hyperthermophilic archaeon Thermococcus nautili possesses a remarkable ability to recovery from extreme chromosomal damage. Immediately following UV irradiation, chromosomal DNA of T. nautili is fragmented beyond recognition. However, the extensive UV-induced double-stranded breaks (DSB) are repaired over the course of several hours, allowing restoration of growth. DSBs also disrupted plasmid DNA in this species. Similar to the chromosome, plasmid integrity was restored during an outgrowth period. Intriguingly, the topology of recovered pTN1 plasmids differed from control strain by being more positively supercoiled. As reverse gyrase (RG) is the only enzyme capable of inducing positive supercoiling, our results suggest the activation of RG activity by UV-induced stress. We suggest simple UV stress could be used to study archaeal DNA repair and responses to DSB.

Single-molecule imaging of DNA gyrase activity in living Escherichia coli.

Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ∼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ∼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ∼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.

A Bacterial Chromosome Structuring Protein Binds Overtwisted DNA to Stimulate Type II Topoisomerases and Enable DNA Replication

When DNA is unwound during replication, it becomes overtwisted and forms positive supercoils in front of the translocating DNA polymerase. Unless removed or dissipated, this superhelical tension can impede replication elongation. Topoisomerases, including gyrase and topoisomerase IV in bacteria, are required to relax positive supercoils ahead of DNA polymerase but may not be sufficient for replication. Here, we find that GapR, a chromosome structuring protein in Caulobacter crescentus, is required to complete DNA replication. GapR associates invivo with positively supercoiled chromosomal DNA, and our biochemical and structural studies demonstrate that GapR forms a dimer-of-dimers that fully encircles overtwisted DNA. Further, we show that GapR stimulates gyrase and topo IV to relax positive supercoils, thereby enabling DNA replication. Analogous chromosome structuring proteins that locate to the overtwisted DNA in front of replication forks may be present in other organisms, similarly helping to recruit and stimulate topoisomerases during DNA replication.

How does supercoiling regulation on a battery of RNA polymerases impact on bacterial transcription bursting?

Transcription plays an essential role in gene expression. The transcription bursting in bacteria has been suggested to be regulated by positive supercoiling accumulation in front of a transcribing RNA polymerase (RNAP) together with gyrase binding on DNA to release the supercoiling. In this work, we study the supercoiling regulation in the case of a battery of RNAPs working together on DNA by constructing a multi-state quantitative model, which allows gradual and stepwise supercoiling accumulation and release in the RNAP transcription. We solved for transcription characteristics under the multi-state bursting model for a single RNAP transcription, and then simulated for a battery of RNAPs on DNA with T7 and Escherichia coli RNAP types of traffic, respectively, probing both the average and fluctuation impacts of the supercoiling regulation. Our studies show that due to the supercoiling accumulation and release, the number of RNAP molecules loaded onto the DNA vary significantly along time in the traffic condition. Though multiple RNAPs in transcription promote the mRNA production, they also enhance the supercoiling accumulation to suppress the production. In particular, the fluctuations of the mRNA transcripts become highly pronounced for a battery of RNAPs transcribing together under the supercoiling regulation, especially for a long process of transcription elongation. In such an elongation process, though a single RNAP can work at a high duty ratio, multiple RNAPs are hardly able to do so. Our multi-state model thus provides a systematical characterization of the quantitative features of the bacterial transcription bursting; it also supports improved physical examinations on top of this general modeling framework.

DNA Topoisomerase I differentially modulates R-loops across the human genome

BackgroundCo-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.ResultsHere, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.ConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation.