Negative DNA Supercoiling Research Articles

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Structural insights into the transient closed conformation and pH dependent ATPase activity of S.Typhi GyraseB N- terminal domain

DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.

Effects of DNA Topology on Transcription from rRNA Promoters in Bacillus subtilis.

The expression of rRNA is one of the most energetically demanding cellular processes and, as such, it must be stringently controlled. Here, we report that DNA topology, i.e., the level of DNA supercoiling, plays a role in the regulation of Bacillus subtilis σA-dependent rRNA promoters in a growth phase-dependent manner. The more negative DNA supercoiling in exponential phase stimulates transcription from rRNA promoters, and DNA relaxation in stationary phase contributes to cessation of their activity. Novobiocin treatment of B. subtilis cells relaxes DNA and decreases rRNA promoter activity despite an increase in the GTP level, a known positive regulator of B. subtilis rRNA promoters. Comparative analyses of steps during transcription initiation then reveal differences between rRNA promoters and a control promoter, Pveg, whose activity is less affected by changes in supercoiling. Additional data then show that DNA relaxation decreases transcription also from promoters dependent on alternative sigma factors σB, σD, σE, σF, and σH with the exception of σN where the trend is the opposite. To summarize, this study identifies DNA topology as a factor important (i) for the expression of rRNA in B. subtilis in response to nutrient availability in the environment, and (ii) for transcription activities of B. subtilis RNAP holoenzymes containing alternative sigma factors.

Selection for ancient periodic motifs that do not impart DNA bending.

A ~10–11 bp periodicity in dinucleotides imparting DNA bending, with shorter periods found in organisms with positively-supercoiled DNA and longer periods found in organisms with negatively-supercoiled DNA, was previously suggested to assist in DNA compaction. However, when measured with more robust methods, variation in the observed periods between organisms with different growth temperatures is not consistent with that hypothesis. We demonstrate that dinucleotide periodicity does not arise solely by mutational biases but is under selection. We found variation between genomes in both the period and the suite of dinucleotides that are periodic. Whereas organisms with similar growth temperatures have highly variable periods, differences in periods increase with phylogenetic distance between organisms. In addition, while the suites of dinucleotides under selection for periodicity become more dissimilar among more distantly-related organisms, there is a core set of dinucleotides that are strongly periodic among genomes in all domains of life. Notably, this core set of periodic motifs are not involved in DNA bending. These data indicate that dinucleotide periodicity is an ancient genomic architecture which may play a role in shaping the evolution of genes and genomes.

Functional interactions between gyrase subunits are optimized in a species-specific manner

DNA gyrase is a bacterial DNA topoisomerase that catalyzes ATP-dependent negative DNA supercoiling and DNA decatenation. The enzyme is a heterotetramer comprising two GyrA and two GyrB subunits. Its overall architecture is conserved, but species-specific elements in the two subunits are thought to optimize subunit interaction and enzyme function. Toward understanding the roles of these different elements, we compared the activities of Bacillus subtilis, Escherichia coli, and Mycobacterium tuberculosis gyrases and of heterologous enzymes reconstituted from subunits of two different species. We show that B. subtilis and E. coli gyrases are proficient DNA-stimulated ATPases and efficiently supercoil and decatenate DNA. In contrast, M. tuberculosis gyrase hydrolyzes ATP only slowly and is a poor supercoiling enzyme and decatenase. The heterologous enzymes are generally less active than their homologous counterparts. The only exception is a gyrase reconstituted from mycobacterial GyrA and B. subtilis GyrB, which exceeds the activity of M. tuberculosis gyrase and reaches the activity of the B. subtilis gyrase, indicating that the activities of enzymes containing mycobacterial GyrB are limited by ATP hydrolysis. The activity pattern of heterologous gyrases is in agreement with structural features present: B. subtilis gyrase is a minimal enzyme, and its subunits can functionally interact with subunits from other bacteria. In contrast, the specific insertions in E. coli and mycobacterial gyrase subunits appear to prevent efficient functional interactions with heterologous subunits. Understanding the molecular details of gyrase adaptations to the specific physiological requirements of the respective organism might aid in the development of species-specific gyrase inhibitors.

Significance of the chromosomal positions of the genes encoding DNA gyrase in Salmonella enterica Serovar Typhimurium

Changing positions of genes on a chromosome is an informative way of learning about why an existing chromosome structure and gene order was selected by evolution in bacteria. In Salmonella gyrA and gyrB – genes encoding DNA gyrase – enzyme that introduces negative supercoils in DNA, are located almost at opposite poles of a chromosome. However, in many other bacteria they are arranged in a gyrBA operon. In order to investigate the significance of this fact, gyrBA operon was made in Salmonella by bringing gyrA open reading frame and its ribosome binding site directly downstream of gyrB and under a control of its regulatory regions, the original copy of gyrA was deleted. The gyrBA strain obtained as a result exhibits no differences from the wild type in growth and morphology, however, the ability to supercoil DNA is altered between gyrBA and the WT. This is specifically important at conditions mimicking environment inside a macrophage in terms of Mg2+ concentration, as it may suggest alteration of gyrBA survival inside a macrophage. An attempt to make strain with gyrAB operon was not successful, as it was not possible to delete the original gyrB, suggesting particular importance of its position.

Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends onMUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

New Insights into Structural and Functional Roles of Indole-3-acetic acid (IAA): Changes in DNA Topology and Gene Expression in Bacteria.

Indole-3-acetic acid (IAA) is a major plant hormone that affects many cellular processes in plants, bacteria, yeast, and human cells through still unknown mechanisms. In this study, we demonstrated that the IAA-treatment of two unrelated bacteria, the Ensifer meliloti 1021 and Escherichia coli, harboring two different host range plasmids, influences the supercoiled state of the two plasmid DNAs in vivo. Results obtained from in vitro assays show that IAA interacts with DNA, leading to DNA conformational changes commonly induced by intercalating agents. We provide evidence that IAA inhibits the activity of the type IA topoisomerase, which regulates the DNA topological state in bacteria, through the relaxation of the negative supercoiled DNA. In addition, we demonstrate that the treatment of E. meliloti cells with IAA induces the expression of some genes, including the ones related to nitrogen fixation. In contrast, these genes were significantly repressed by the treatment with novobiocin, which reduces the DNA supercoiling in bacterial cells. Taking into account the overall results reported, we hypothesize that the IAA action and the DNA structure/function might be correlated and involved in the regulation of gene expression. This work points out that checking whether IAA influences the DNA topology under physiological conditions could be a useful strategy to clarify the mechanism of action of this hormone, not only in plants but also in other unrelated organisms.

Transcriptional Response of Streptomyces coelicolor to Rapid Chromosome Relaxation or Long-Term Supercoiling Imbalance.

Negative DNA supercoiling allows chromosome condensation and facilitates DNA unwinding, which is required for the occurrence of DNA transaction processes, i.e., DNA replication, transcription and recombination. In bacteria, changes in chromosome supercoiling impact global gene expression; however, the limited studies on the global transcriptional response have focused mostly on pathogenic species and have reported various fractions of affected genes. Furthermore, the transcriptional response to long-term supercoiling imbalance is still poorly understood. Here, we address the transcriptional response to both novobiocin-induced rapid chromosome relaxation or long-term topological imbalance, both increased and decreased supercoiling, in environmental antibiotic-producing bacteria belonging to the Streptomyces genus. During the Streptomyces complex developmental cycle, multiple copies of GC-rich linear chromosomes present in hyphal cells undergo profound topological changes, from being loosely condensed in vegetative hyphae, to being highly compacted in spores. Moreover, changes in chromosomal supercoiling have been suggested to be associated with the control of antibiotic production and environmental stress response. Remarkably, in S. coelicolor, a model Streptomyces species, topoisomerase I (TopA) is solely responsible for the removal of negative DNA supercoils. Using a S. coelicolor strain in which topA transcription is under the control of an inducible promoter, we identified genes involved in the transcriptional response to long-term supercoiling imbalance. The affected genes are preferentially organized in several clusters, and a supercoiling-hypersensitive cluster (SHC) was found to be located in the core of the S. coelicolor chromosome. The transcripts affected by long-term topological imbalance encompassed genes encoding nucleoid-associated proteins, DNA repair proteins and transcriptional regulators, including multiple developmental regulators. Moreover, using a gyrase inhibitor, we identified those genes that were directly affected by novobiocin, and found this was correlated with increased AT content in their promoter regions. In contrast to the genes affected by long-term supercoiling changes, among the novobiocin-sensitive genes, a significant fraction encoded for proteins associated with membrane transport or secondary metabolite synthesis. Collectively, our results show that long-term supercoiling imbalance globally regulates gene transcription and has the potential to impact development, secondary metabolism and DNA repair, amongst others.

Crystal structure of the 65-kilodalton amino-terminal fragment of DNA topoisomerase I from the gram-positive model organism Streptococcus mutans

Herein we report the first structure of topoisomerase I determined from the gram-positive bacterium, S. mutans. Bacterial topoisomerase I is an ATP-independent type 1A topoisomerase that uses the inherent torsional strain within hyper-negatively supercoiled DNA as an energy source for its critical function of DNA relaxation. Interest in the enzyme has gained momentum as it has proven to be essential in various bacterial organisms. In order to aid in further biochemical characterization, the apo 65-kDa amino-terminal fragment of DNA topoisomerase I from the gram-positive model organism Streptococcus mutans was crystalized and a three-dimensional structure was determined to 2.06 Å resolution via x-ray crystallography. The overall structure illustrates the four classic major domains that create the traditional topoisomerase I “lock” formation comprised of a sizable toroidal aperture atop what is considered to be a highly dynamic body. A catalytic tyrosine residue resides at the interface between two domains and is known to form a 5’ phosphotyrosine DNA-enzyme intermediate during transient single-stranded cleavage required for enzymatic relaxation of hyper negative DNA supercoils. Surrounding the catalytic tyrosine residue is the remainder of the highly conserved active site. Within 5 Å from the catalytic center, only one dissimilar residue is observed between topoisomerase I from S. mutans and the gram-negative model organism E. coli. Immediately adjacent to the conserved active site, however, S. mutans topoisomerase I displays a somewhat unique nine residue loop extension not present in any bacterial topoisomerase I structures previously determined other than that of an extremophile.

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35⁻38 Amino Acids of GyrA.

Prokaryotes have an essential gene—gyrase—that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35–38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to β-pinwheel elements is presented.

The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a.

Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by Lachnospiraceae bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with Streptococcus thermophilus DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.

Formation of G-quadruplex structure in supercoiled DNA under molecularly crowded conditions.

G-quadruplex is a secondary structure of nucleic acids that plays crucial roles in many significant biological processes. Potential G-quadruplex-forming sequences exist widely in various regions of the genome such as telomeres and gene promoters. In spite of the fact that G-quadruplex can be readily assembled from a single-stranded segment of DNA, its formation from duplex DNA is very difficult under physiological conditions because Watson–Crick interactions in guanine rich segments need to be weakened first. It is demonstrated in our studies that intrastrand G-quadruplex generated from a perfectly matched guanine-rich duplex in a circular DNA as a result of significant quadruplex stabilization and duplex destabilization created by the combined actions of negative DNA supercoiling and molecular crowding conditions.

Molecular characterization of pea DNA gyrase-A reveals dual localization of protein in plastid and mitochondria

Gyrases are a class of Topoisomerase II that facilitate ATP dependent negative supercoiling of DNA during transcription and replication. They aid in strand breakage during chromosome condensation and take part in separation of intertwined DNA during mitosis. The present study reports gyrase-A (PsGryA) CDS clone from Pisum sativum (L.) comprising of 2817 bp that encodes for a peptide of 939 amino acids. The estimated molecular weight of PsGryA is approximately 104 kDa with pI as 6.99. The protein sequence showed maximum similarity with GyrA of Medicago truncatula. The PsGyrA polypeptide has approximately 42% overall sequence identity with Escherichia coli gyrase-A. The three-dimensional structure of PsGyrA generated based on the X-ray structure of E. coli gyrase-A revealed a putative active site region with amino acid (AA) sequence AAMRYTE located between 208th and 214th AA with a tyrosine residue at position 212. In silico and In vivo immunolocalization studies establishes the dual targeting of PsGyrA to both chloroplast and mitochondria.

Gyrase containing a single C-terminal domain catalyzes negative supercoiling of DNA by decreasing the linking number in steps of two.

The topological state of DNA in vivo is regulated by topoisomerases. Gyrase is a bacterial topoisomerase that introduces negative supercoils into DNA at the expense of ATP hydrolysis. According to the strand-passage mechanism, a double-strand of the DNA substrate is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. The correct orientation of these DNA segments for strand passage is achieved by wrapping of the DNA around gyrase, which involves the C-terminal domains (CTDs) of both GyrA subunits in the A2B2 heterotetramer. Gyrase lacking both CTDs cannot introduce negative supercoils into DNA. Here, we analyze the requirements for the two CTDs in individual steps in the supercoiling reaction. Gyrase that contains a single CTD binds, distorts, and cleaves DNA similarly to wildtype gyrase. It also shows wildtype-like DNA-dependent ATPase activity, and undergoes DNA-induced movement of the CTD as well as N-gate narrowing. Most importantly, the enzyme still introduces negative supercoils into DNA in an ATP-dependent reaction, with a velocity similar to wildtype gyrase, and decreases the linking number of the DNA in steps of two. One CTD is thus sufficient to support DNA supercoiling.

Why Two? On the Role of (A-)Symmetry in Negative Supercoiling of DNA by Gyrase.

Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce negative supercoils into DNA by converting a positive DNA node into a negative node through strand passage: First, it cleaves both DNA strands of a double-stranded DNA, termed the G-segment, and then it passes a second segment of the same DNA molecule, termed the T-segment, through the gap created. As a two-fold symmetric enzyme, gyrase contains two copies of all elements that are key for the supercoiling reaction: The GyrB subunits provide two active sites for ATP binding and hydrolysis. The GyrA subunits contain two C-terminal domains (CTDs) for DNA binding and wrapping to stabilize the positive DNA node, and two catalytic tyrosines for DNA cleavage. While the presence of two catalytic tyrosines has been ascribed to the necessity of cleaving both strands of the G-segment to enable strand passage, the role of the two ATP hydrolysis events and of the two CTDs has been less clear. This review summarizes recent results on the role of these duplicate elements for individual steps of the supercoiling reaction, and discusses the implications for the mechanism of DNA supercoiling.

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress.

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4-5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic-membrane-located inhibitor of proton-driven F1 F0 ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin-resistant (NovR ) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with NovR gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug-treated bacteria. The Salmonella cytosol reaches pH 5-6 in response to an external pH of 4-5: the ATP-dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP-dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid-mediated impairment of the negative supercoiling activity of gyrase.

Direct Observation and Quantification of Protein Dynamics on Negatively-Supercoiled DNA

Direct Observation and Quantification of Protein Dynamics on Negatively-Supercoiled DNA

Interaction between Sso7d and DNA studied by single-molecule technique

Each organism has its own set of chromatin proteins to protect the stable structure of DNA and thus maintain the stability of genes. Sso7d is a small nonspecific DNA-binding protein from the hyperthermophilic archaea Sulfolobus solfataricus. This protein has high thermal and acid stability. It stabilizes dsDNA and constrains negative DNA supercoils. Besides, the Sso7d binds in a minor groove of DNA and causes a sharp kink in DNA. By observing the interaction between chromatin protein and DNA structure, we can understand the function and mechanism of chromatin protein. Sulfolobus solfataricus can survive at high temperature. To understand why the DNA of Sulfolobus solfataricus retains activity at high temperature, we investigate the interaction between Sso7d and DNA by atomic force microscope (AFM) and magnetic tweezers. Atomic force microscope and magnetic tweezers are advanced single molecule experimental tools that can be used to observe the interaction between individual molecules. The experimental result of AFM reveals the process of interaction between Sso7d and DNA. The DNA structure changes at a different concentration of Sso7d and depends on reaction time. At a relatively low concentration of Sso7d, DNA strand forms a kink structure. When the concentration of Sso7d is increased, DNA loops appear. Finally, DNA becomes a dense nuclear structure at a high concentration of Sso7d. If the time of the interaction between Sso7d and DNA is increased, DNA structure tends to be more compact. These results indicate that high concentration of Sso7d is important for the compact structure of DNA. We design an experiment to find out the formation of the looped structure on DNA. Moreover, we measure the angle of kinked DNA and compared it with previous result. Through the experiment of magnetic tweezers, we measure the forces of unfolding the double-stranded DNA complexed with Sso7d at different concentrations. The experimental results show that the binding between Sso7d and DNA increases the force of unfolding the double-stranded DNA. The binding energy between Sso7d and dsDNA is 3.1kBT which is calculated from experimental data. It indicates that DNA base pairs are more stable when chromatin protein Sso7d exists. These results can explain the survival of Sulfolobus in high temperature environment.

The Use of Psoralen Photobinding to Study Transcription-Induced Supercoiling.

Proteins manipulating intracellular DNA necessarily impart torsional stress, which redistributes across the DNA. Overtwisting and undertwisting of the double helix result in the manifestation of positive and negative DNA supercoiling. A growing body of evidence indicates that DNA topology is an important player in the key regulatory steps of genome function, highlighting the need for biochemical techniques to detect dynamic changes in the DNA structure. Psoralen binding to DNA in vivo is proportional to the level of supercoiling, providing an excellent probe for the topological state of nuclear DNA. Here we describe a psoralen-based methodology to detect transcription-induced DNA supercoiling genome-wide. The DNA samples generated with this approach can be hybridized to microarray platforms or high-throughput sequenced to provide a topological snapshot of the whole genome.