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.

Single-molecule visualization of twin-supercoiled domains generated during transcription.

Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.

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.

Detection of Z-DNA Structures in Supercoiled Genome.

Z-DNAs are nucleic acid secondary structures that form within a special pattern of nucleotides and are promoted by DNA supercoiling. Through Z-DNA formation, DNA encodes information by dynamic changes in its secondary structure. A growing body of evidence indicates that Z-DNA formation can play a role in gene regulation; it can affect chromatin architecture and demonstrates its association with genomic instability, genetic diseases, and genome evolution. Many functional roles of Z-DNA are yet to be discovered highlighting the need for techniques to detect genome-wide folding of DNA into this structure. Here, we describe an approach to convert linear genome into supercoiled genome sponsoring Z-DNA formation. Applying permanganate-based methodology and high-throughput sequencing to supercoiled genome allows genome-wide detection of single-stranded DNA. Single-stranded DNA is characteristic of the junctions between the classical B-form of DNA and Z-DNA. Consequently, analysis of single-stranded DNA map provides snapshots of the Z-DNA conformation over the whole genome.

Enhanced binding of an HU homologue under increased DNA supercoiling preserves chromosome organisation and sustains Streptomyces hyphal growth.

Bacterial chromosome topology is controlled by topoisomerases and nucleoid-associated proteins (NAPs). While topoisomerases regulate DNA supercoiling, NAPs introduce bends or coat DNA upon its binding, affecting DNA loop formation. Streptomyces, hyphal, multigenomic bacteria known for producing numerous clinically important compounds, use the highly processive topoisomerase I (TopA) to remove excessive negative DNA supercoils. Elongated vegetative Streptomyces cells contain multiple copies of their linear chromosome, which remain relaxed and relatively evenly distributed. Here, we explored how TopA cooperates with HupA, an HU homologue that is the most abundant Streptomyces NAP. We verified that HupA has an increased affinity for supercoiled DNA in vivo and in vitro. Analysis of mutant strains demonstrated that HupA elimination is detrimental under high DNA supercoiling conditions. The absence of HupA, combined with decreased TopA levels, disrupted chromosome distribution in hyphal cells, eventually inhibiting hyphal growth. We concluded that increased HupA binding to DNA under elevated chromosome supercoiling conditions is critical for the preservation of chromosome organisation.

Supercoiling-mediated feedback rapidly couples and tunes transcription.

Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. However, a theoretical framework is needed to integrate biophysical regulation with biochemical transcriptional regulation. To investigate the role of supercoiling-mediated feedback within multi-gene systems, we model transcriptional regulation under the influence of supercoiling-mediated polymerase dynamics, allowing us to identify patterns of expression that result from physical inter-gene coupling. We find that gene syntax-the relative ordering and orientation of genes-defines the expression profiles, variance, burst dynamics, and inter-gene correlation of two-gene systems. Furthermore, supercoiling can enhance or weaken biochemical regulation. Our results suggest that supercoiling couples behavior between neighboring genes, providing a regulatory mechanism that tunes transcriptional variance in engineered gene networks and explains the behavior of co-localized native circuits.

What is a supercoiling-sensitive gene? Insights from topoisomerase I inhibition in the Gram-negative bacterium Dickeya dadantii.

DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by DNA gyrase inhibition were categorised as 'supercoiling-sensitive'. Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after DNA gyrase or topoisomerase I inhibition.

Alteration of DNA supercoiling serves as a trigger of short-term cold shock repressed genes of E. coli.

Cold shock adaptability is a key survival skill of gut bacteria of warm-blooded animals. Escherichia coli cold shock responses are controlled by a complex multi-gene, timely-ordered transcriptional program. We investigated its underlying mechanisms. Having identified short-term, cold shock repressed genes, we show that their responsiveness is unrelated to their transcription factors or global regulators, while their single-cell protein numbers' variability increases after cold shock. We hypothesized that some cold shock repressed genes could be triggered by high propensity for transcription locking due to changes in DNA supercoiling (likely due to DNA relaxation caused by an overall reduction in negative supercoiling). Concomitantly, we found that nearly half of cold shock repressed genes are also highly responsive to gyrase inhibition (albeit most genes responsive to gyrase inhibition are not cold shock responsive). Further, their response strengths to cold shock and gyrase inhibition correlate. Meanwhile, under cold shock, nucleoid density increases, and gyrases and nucleoid become more colocalized. Moreover, the cellular energy decreases, which may hinder positive supercoils resolution. Overall, we conclude that sensitivity to diminished negative supercoiling is a core feature of E. coli's short-term, cold shock transcriptional program, and could be used to regulate the temperature sensitivity of synthetic circuits.

Quantitative contribution of the spacer length in the supercoiling-sensitivity of bacterial promoters.

DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the -10 and -35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the 'spacer' between them. Here, we develop a thermodynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions of the relative activation of promoters containing a short versus long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in Escherichia coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the model's predictions. Altogether, this study shows an example of mechanical constraints associated to promoter binding by RNA Polymerase underpinning a basal and global regulatory mechanism.

Spatiotemporal Coupling of DNA Supercoiling and Genomic Sequence Organization-A Timing Chain for the Bacterial Growth Cycle?

In this article we describe the bacterial growth cycle as a closed, self-reproducing, or autopoietic circuit, reestablishing the physiological state of stationary cells initially inoculated in the growth medium. In batch culture, this process of self-reproduction is associated with the gradual decline in available metabolic energy and corresponding change in the physiological state of the population as a function of "travelled distance" along the autopoietic path. We argue that this directional alteration of cell physiology is both reflected in and supported by sequential gene expression along the chromosomal OriC-Ter axis. We propose that during the E. coli growth cycle, the spatiotemporal order of gene expression is established by coupling the temporal gradient of supercoiling energy to the spatial gradient of DNA thermodynamic stability along the chromosomal OriC-Ter axis.

Psoralen mapping reveals a bacterial genome supercoiling landscape dominated by transcription.

DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction - an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.

Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation.

Many cellular processes occur out of equilibrium. This includes site-specific unwinding in supercoiled DNA, which may play an important role in gene regulation. Here, we use the Convex Lens-induced Confinement (CLiC) single-molecule microscopy platform to study these processes with high-throughput and without artificial constraints on molecular structures or interactions. We use two model DNA plasmid systems, pFLIP-FUSE and pUC19, to study the dynamics of supercoiling-induced secondary structural transitions after perturbations away from equilibrium. We find that structural transitions can be slow, leading to long-lived structural states whose kinetics depend on the duration and direction of perturbation. Our findings highlight the importance of out-of-equilibrium studies when characterizing the complex structural dynamics of DNA and understanding the mechanisms of gene regulation.

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops.

Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates.

MYC assembles and stimulates topoisomerases 1 and 2 in a "topoisome".

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

The bacterial promoter spacer modulates promoter strength and timing by length, TG-motifs and DNA supercoiling sensitivity.

Transcription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals an extension to the -10 region that enhances RNAP binding but damps promoter activity. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.

DNA Supercoiling Drives a Transition between Collective Modes of Gene Synthesis.

Transcription of genes can be affected by both biochemical and mechanical factors. Recent experiments suggested that the mechanical stress associated with transcription-induced DNA supercoiling is responsible for the transition from cooperative to antagonistic group dynamics of RNA polymerases (RNAPs) upon promoter repression. To underpin the mechanism behind this drastic transition, we developed a continuum deterministic model for transcription under torsion. In our model, the speed of an RNAP is affected by the local DNA supercoiling, as well as two global factors: (i) the number of RNAPs on the gene affecting the torsional stress experienced by individual RNAPs and (ii) transcription factors blocking the diffusion of DNA supercoils. Our minimal model can successfully reproduce the experimental findings and helps elucidate the interplay of mechanical and biological factors in the collective dynamics of molecular machines involved in gene expression.

Negative DNA supercoiling makes protein-mediated looping deterministic and ergodic within the bacterial doubling time.

Protein-mediated DNA looping is fundamental to gene regulation and such loops occur stochastically in purified systems. Additional proteins increase the probability of looping, but these probabilities maintain a broad distribution. For example, the probability of lac repressor-mediated looping in individual molecules ranged 0-100%, and individual molecules exhibited representative behavior only in observations lasting an hour or more. Titrating with HU protein progressively compacted the DNA without narrowing the 0-100% distribution. Increased negative supercoiling produced an ensemble of molecules in which all individual molecules more closely resembled the average. Furthermore, in only 12 min of observation, well within the doubling time of the bacterium, most molecules exhibited the looping probability of the ensemble. DNA supercoiling, an inherent feature of all genomes, appears to impose time-constrained, emergent behavior on otherwise random molecular activity.

The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase.

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.