Long-Distance Cooperative and Antagonistic RNA Polymerase Dynamics via DNA Supercoiling.

Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.

Synergistic Coordination of Chromatin Torsional Mechanics and Topoisomerase Activity.

DNA replication in eukaryotes generates DNA supercoiling, which may intertwine (braid) daughter chromatin fibers to form precatenanes, posing topological challenges during chromosome segregation. The mechanisms that limit precatenane formation remain unclear. By making direct torque measurements, we demonstrate that the intrinsic mechanical properties of chromatin play a fundamental role in dictating precatenane formation and regulating chromatin topology. Whereas a single chromatin fiber is torsionally soft, a braided fiber is torsionally stiff, indicating that supercoiling on chromatin substrates is preferentially directed in front of the fork during replication. We further show that topoisomerase II relaxation displays a strong preference for a single chromatin fiber over a braided fiber. These results suggest a synergistic coordination-the mechanical properties of chromatin inherently suppress precatenane formation during replication elongation by driving DNA supercoiling ahead of the fork, where supercoiling is more efficiently removed by topoisomerase II. VIDEO ABSTRACT.

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.

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.

DNA supercoiling restricts the transcriptional bursting of neighboring eukaryotic genes.

DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes.

Multiscale Dynamic Structuring of Bacterial Chromosomes.

Since the nucleoid was isolated from bacteria in the 1970s, two fundamental questions emerged and are still in the spotlight: how bacteria organize their chromosomes to fit inside the cell and how nucleoid organization enables essential biological processes. During the last decades, knowledge of bacterial chromosome organization has advanced considerably, and today, such chromosomes are considered to be highly organized and dynamic structures that are shaped by multiple factors in a multiscale manner. Here we review not only the classical well-known factors involved in chromosome organization but also novel components that have recently been shown to dynamically shape the 3D structuring of the bacterial genome. We focus on the different functional elements that control short-range organization and describe how they collaborate in the establishment of the higher-order folding and disposition of the chromosome. Recent advances have opened new avenues for a deeper understanding of the principles and mechanisms of chromosome organization in bacteria.

In vivo assembly of bacterial partition condensates on circular supercoiled and linear DNA.

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2 Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Finally, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

Variable DNA topology is an epigenetic generator of physiological heterogeneity in bacterial populations.

Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This pattern of diversity reflects, in part, the burst-like nature of transcription. Transcription bursting has many causes and a failure to remove the supercoils that accumulate in DNA during transcription elongation is an important contributor. Positive supercoiling of the DNA ahead of the transcription elongation complex can result in RNA polymerase stalling if this DNA topological roadblock is not removed. The relaxation of these positive supercoils is performed by the ATP-dependent type II topoisomerases DNA gyrase and topoisomerase IV. Interference with the action of these topoisomerases involving, inter alia, topoisomerase poisons, fluctuations in the [ATP]/[ADP] ratio, and/or the intervention of nucleoid-associated proteins with GapR-like or YejK-like activities, may have consequences for the smooth operation of the transcriptional machinery. Antibiotic-tolerant (but not resistant) persister cells are among the phenotypic outliers that may emerge. However, interference with type II topoisomerase activity can have much broader consequences, making it an important epigenetic driver of physiological diversity in the bacterial population.

Distinct subunit architecture and assembly pattern of DNA gyrase from mycobacteria.

DNA gyrase, the sole negative supercoiling type II topoisomerase, is composed of two subunits, GyrA and GyrB, encoded by the gyrA and gyrB genes, respectively, that form a quaternary complex of A2 B2 . In this study, we have investigated the assembly of mycobacterial DNA gyrase from its individual subunits, a step prerequisite for its activity. Using analytical size-exclusion chromatography, we show that GyrA from Mycobacterium tuberculosis and Mycobacterium smegmatis forms tetramers (A4 ) in solution unlike in Escherichia coli and other bacteria where GyrA exists as a dimer. GyrB, however, persists as a monomer, resembling the pattern found in E. coli. GyrB in both mycobacterial species interacts with GyrA and triggers the dissociation of the GyrA tetramer to facilitate the formation of catalytically active A2 B2 . Despite oligomerisation, the GyrA tetramer retained its DNA binding ability, and DNA binding had no effect on GyrA's oligomeric state in both species. Moreover, the presence of DNA facilitated the assembly of holoenzyme in the case of M. smegmatis by stabilising the GyrA2 B2 tetramer but with little effect in M. tuberculosis. Thus, in addition to the distinct organisation and regulation of the gyr locus in mycobacteria, the enzyme assembly also follows a different pattern.

Keeping intracellular DNA untangled: A new role for condensin?

The DNA-passage activity of topoisomerase II accidentally produces DNA knots and interlinks within and between chromatin fibers. Fortunately, these unwanted DNA entanglements are actively removed by some mechanism. Here we present an outline on DNA knot formation and discuss recent studies that have investigated how intracellular DNA knots are removed. First, although topoisomerase II is able to minimize DNA entanglements in vitro to below equilibrium values, it is unclear whether such capacity performs equally in vivo in chromatinized DNA. Second, DNA supercoiling could bias topoisomerase II to untangle the DNA. However, experimental evidence indicates that transcriptional supercoiling of intracellular DNA boosts knot formation. Last, cohesin and condensin could tighten DNA entanglements via DNA loop extrusion (LE) and force their dissolution by topoisomerase II. Recent observations indicate that condensin activity promotes the removal of DNA knots during interphase and mitosis. This activity might facilitate the spatial organization and dynamics of chromatin.

Reactions of deoxyribonucleotide bases with sulfooxymethyl or halomethyl polycyclic aromatic hydrocarbons induce unwinding of DNA supercoils.

Torsional stress in double-stranded DNA enables and regulates facets of chromosomal metabolism, replication, and transcription and requires regulatory enzymatic systems including topoisomerases and histone methyltransferases. As such, this machinery may be subject to deleterious effects from reactive mutagens, including ones from carcinogenic polycyclic aromatic hydrocarbon (PAH) adduct formation with DNA. Supercoiled plasmid DNA was investigated for its torsional responses to adducts formed in vitro from PAH benzylic carbocation reactive intermediates created spontaneously by release of leaving groups. PAH sulfate esters were found to (1) unwind DNA in a concentration dependent manner, and (2) provide maximum unwinding in a pattern consistent with known carcinogenicities of the parent PAHs, that is, 6-methylbenzo[a]pyrene > 7,12-methylbenz[a]anthracene > 3-methylcholanthrene > 9-methylanthracene > 7-methylbenz[a]anthracene > 1-methylpyrene. Supercoil unwinding was demonstrated to be dependent on the presence of sulfate or chloride leaving groups such that reactive carbocations were generated in situ by hydrolysis. In silico modeling of intercalative complex topology showed PAH benzylic carbocation reactive functional groups in alignment with target nucleophiles on guanine bases in a 5'-dCdG-3' pocket in agreement with known formation of nucleotide adducts. Inhibitory or modulatory effects on PAH-induced supercoil unwinding were seen with ascorbic acid and an experimental antineoplastic agent Antineoplaston A10 in agreement with their known anticarcinogenic properties. In summary, the reactive PAH intermediates studied here undoubtedly participate in well-known mutational mechanisms such as frameshifts and apurinic site generation. However, they are also capable of random disruption of chromosomal supercoiling in a manner consistent with the known carcinogenicities of the parent compounds, and this mechanism may represent an additional detrimental motif worthy of further study for a more complete understanding of chemical carcinogenicity.

Synthetic lethal mutants in Escherichia coli define pathways necessary for survival with RNase H deficiency.

Ribonucleotides frequently contaminate DNA and, if not removed, cause genomic instability. Consequently, all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids (RDHs). Escherichia coli lacking RNase HI (rnhA) and RNase HII (rnhB) enzymes, the ∆rnhA ∆rnhB double mutant, accumulates RDHs in its DNA. These RDHs can convert into RNA-containing DNA lesions (R-lesions) of unclear nature that compromise genomic stability. The ∆rnhAB double mutant has severe phenotypes, like growth inhibition, replication stress, sensitivity to ultraviolet radiation, SOS induction, increased chromosomal fragmentation, and defects in nucleoid organization. In this study, we found that RNase HI deficiency also alters wild-type levels of DNA supercoiling. Despite these severe chromosomal complications, ∆rnhAB double mutant survives, suggesting that dedicated pathways operate to avoid or repair R-lesions. To identify these pathways, we systematically searched for mutants synthetic lethal (colethal) with the rnhAB defect using an unbiased color screen and a candidate gene approach. We identified both novel and previously reported rnhAB-colethal and -coinhibited mutants, characterized them, and sorted them into avoidance or repair pathways. These mutants operate in various parts of nucleic acid metabolism, including replication fork progression, R-loop prevention and removal, nucleoid organization, tRNA modification, recombinational repair, and chromosome-dimer resolution, demonstrating the pleiotropic nature of RNase H deficiency. IMPORTANCE Ribonucleotides (rNs) are structurally very similar to deoxyribonucleotides. Consequently, rN contamination of DNA is common and pervasive across all domains of life. Failure to remove rNs from DNA has severe consequences, and all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids. RNase H deficiency leads to complications in bacteria, yeast, and mouse, and diseases like progressive external ophthalmoplegia (mitochondrial defects in RNASEH1) and Aicardi-Goutières syndrome (defects in RNASEH2) in humans. Escherichia coli ∆rnhAB mutant, deficient in RNases H, has severe chromosomal complications. Despite substantial problems, nearly half of the mutant population survives. We have identified novel and previously confirmed pathways in various parts of nucleic acid metabolism that ensure survival with RNase H deficiency.

DNA topoisomerases: Advances in understanding of cellular roles and multi-protein complexes via structure-function analysis.

DNA topoisomerases, capable of manipulating DNA topology, are ubiquitous and indispensable for cellular survival due to the numerous roles they play during DNA metabolism. As we review here, current structural approaches have revealed unprecedented insights into the complex DNA-topoisomerase interaction and strand passage mechanism, helping to advance our understanding of their activities in vivo. This has been complemented by single-molecule techniques, which have facilitated the detailed dissection of the various topoisomerase reactions. Recent work has also revealed the importance of topoisomerase interactions with accessory proteins and other DNA-associated proteins, supporting the idea that they often function as part of multi-enzyme assemblies in vivo. In addition, novel topoisomerases have been identified and explored, such as topo VIII and Mini-A. These new findings are advancing our understanding of DNA-related processes and the vital functions topos fulfil, demonstrating their indispensability in virtually every aspect of DNA metabolism.

Physiologic and Transcriptomic Effects Triggered by Overexpression of Wild Type and Mutant DNA Topoisomerase I in Streptococcus pneumoniae.

Topoisomerase I (TopoI) in Streptococcus pneumoniae, encoded by topA, is a suitable target for drug development. Seconeolitsine (SCN) is a new antibiotic that specifically blocks this enzyme. We obtained the topARA mutant, which encodes an enzyme less active than the wild type (topAWT) and more resistant to SCN inhibition. Likely due to the essentiality of TopoI, we were unable to replace the topAWT allele by the mutant topARA version. We compared the in vivo activity of TopoIRA and TopoIWT using regulated overexpression strains, whose genes were either under the control of a moderately (PZn) or a highly active promoter (PMal). Overproduction of TopoIRA impaired growth, increased SCN resistance and, in the presence of the gyrase inhibitor novobiocin (NOV), caused lower relaxation than TopoIWT. Differential transcriptomes were observed when the topAWT and topARA expression levels were increased about 5-fold. However, higher increases (10-15 times), produced a similar transcriptome, affecting about 52% of the genome, and correlating with a high DNA relaxation level with most responsive genes locating in topological domains. These results confirmed that TopoI is indeed the target of SCN in S. pneumoniae and show the important role of TopoI in global transcription, supporting its suitability as an antibiotic target.

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.

What's on the Other Side of the Gate: A Structural Perspective on DNA Gate Opening of Type IA and IIA DNA Topoisomerases.

DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and catalyse diverse topological reactions by cutting and re-joining DNA ends. Type IA and IIA topoisomerases, which work by strand passage mechanisms, share catalytic domains for DNA binding and cleavage. Structural information has accumulated over the past decades, shedding light on the mechanisms of DNA cleavage and re-ligation. However, the structural rearrangements required for DNA-gate opening and strand transfer remain elusive, in particular for the type IA topoisomerases. In this review, we compare the structural similarities between the type IIA and type IA topoisomerases. The conformational changes that lead to the opening of the DNA-gate and strand passage, as well as allosteric regulation, are discussed, with a focus on the remaining questions about the mechanism of type IA topoisomerases.

StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae.

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO4-inducible PZn promoter (strain ΔstaRPZnstaR) or of the maltose-inducible PMal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRPZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = -0.049) than when StaR was overproduced (σ = -0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction.

Consequences of producing DNA gyrase from a synthetic gyrBA operon in Salmonella enterica serovar Typhimurium.

DNA gyrase is an essential type II topoisomerase that is composed of two subunits, GyrA and GyrB, and has an A2 B2 structure. Although the A and B subunits are required in equal proportions to form DNA gyrase, the gyrA and gyrB genes that encode them in Salmonella (and in many other bacteria) are at separate locations on the chromosome, are under separate transcriptional control, and are present in different copy numbers in rapidly growing bacteria. In wild-type Salmonella, gyrA is near the chromosome's replication terminus, while gyrB is near the origin. We generated a synthetic gyrBA operon at the oriC-proximal location of gyrB to test the significance of the gyrase gene position for Salmonella physiology. Although the strain producing gyrase from an operon had a modest alteration to its DNA supercoiling set points, most housekeeping functions were unaffected. However, its SPI-2 virulence genes were expressed at a reduced level and its survival was reduced in macrophage. Our data reveal that the horizontally acquired SPI-2 genes have a greater sensitivity to disturbance of DNA topology than the core genome and we discuss its significance in the context of Salmonella genome evolution and the gyrA and gyrB gene arrangements found in other bacteria.

A Genome-Wide Evolutionary Simulation of the Transcription-Supercoiling Coupling.

DNA supercoiling, the level of under- or overwinding of the DNA polymer around itself, is widely recognized as an ancestral regulation mechanism of gene expression in bacteria. Higher levels of negative supercoiling facilitate the opening of the DNA double helix at gene promoters and thereby increase gene transcription rates. Different levels of supercoiling have been measured in bacteria exposed to different environments, leading to the hypothesis that variations in supercoiling could be a response to changes in the environment. Moreover, DNA transcription has been shown to generate local variations in the supercoiling level and, therefore, to impact the transcription rate of neighboring genes. In this work, we study the coupled dynamics of DNA supercoiling and transcription at the genome scale. We implement a genome-wide model of gene expression based on the transcription-supercoiling coupling. We show that, in this model, a simple change in global DNA supercoiling is sufficient to trigger differentiated responses in gene expression levels via the transcription-supercoiling coupling. Then, studying our model in the light of evolution, we demonstrate that this non-linear response to different environments, mediated by the transcription-supercoiling coupling, can serve as the basis for the evolution of specialized phenotypes.

Supercoiling DNA optically.

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.