Effect Of DNA Supercoiling Research Articles

The effects of DNA supercoiling on G-quadruplex formation

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson–Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation.

Effects of a Mutation in the gyrA Gene on the Virulence of Uropathogenic Escherichia coli

Fluoroquinolones are among the drugs most extensively used for the treatment of bacterial infections in human and veterinary medicine. Resistance to quinolones can be chromosome or plasmid mediated. The chromosomal mechanism of resistance is associated with mutations in the DNA gyrase- and topoisomerase IV-encoding genes and mutations in regulatory genes affecting different efflux systems, among others. We studied the role of the acquisition of a mutation in the gyrA gene in the virulence and protein expression of uropathogenic Escherichia coli (UPEC). The HC14366M strain carrying a mutation in the gyrA gene (S83L) was found to lose the capacity to cause cystitis and pyelonephritis mainly due to a decrease in the expression of the fimA, papA, papB, and ompA genes. The levels of expression of the fimA, papB, and ompA genes were recovered on complementing the strain with a plasmid containing the gyrA wild-type gene. However, only a slight recovery was observed in the colonization of the bladder in the GyrA complement strain compared to the mutant strain in a murine model of ascending urinary tract infection. In conclusion, a mutation in the gyrA gene of uropathogenic E. coli reduced the virulence of the bacteria, likely in association with the effect of DNA supercoiling on the expression of several virulence factors and proteins, thereby decreasing their capacity to cause cystitis and pyelonephritis.

The effect of DNA supercoiling on nucleosome structure and stability

Nucleosomes have to open to allow access to DNA in transcription, replication, and DNA damage repair. Changes in DNA torsional strain (e.g. during transcription elongation) influence the accessibility of nucleosomal DNA. Here we investigated the effect of DNA supercoiling-induced torsional strain on nucleosome structure and stability by scanning force microscopy (SFM) and fluorescence correlation spectroscopy (FCS). Nucleosomes were reconstituted onto 2.7 kb DNA plasmids with varying superhelical densities. The SFM results show a clear dependence of the amount of DNA wrapped around the nucleosome core on the strength and type of supercoiling. Negative supercoiling led to smaller nucleosome opening angles as compared to relaxed or positively supercoiled DNA. FCS experiments show that nucleosomes reconstituted on negatively superhelical DNA are more resistant to salt-induced destabilization, as seen by reduced H2A–H2B dimer eviction from the nucleosome. Our results show that changes in DNA topology, e.g. during transcription elongation, affect the accessibility of nucleosomal DNA.

DNA Unwinding by PcrA Helicase and RepD using TIRF and Magnetic Tweezers

Bacterial plasmids of the pT181 family are replicated by an asymmetric rolling-circle mechanism. We have studied plasmid replication that is initiated when the protein, RepD, binds to the origin of replication (OriD) making a single strand nick and forming a covalent complex with the DNA. This exposes a short length of single-stranded DNA (ssDNA) allowing PcrA helicase to bind and start unwinding the plasmid. While the DNA is being unwound, DNA Polymerase III travels behind PcrA and synthesizes a new, complementary, strand. The other strand becomes decorated with single-stranded DNA binding protein (SSB) and is later replicated by a different mechanism. In earlier studies, we monitored individual PcrA unwinding reactions using TIRF microscopy to image the accumulation of fluorescently labelled SSB on the nascent ssDNA strands (Fili et al., (2010) Nucl Acids Res. 38:4448-4457). We are now using a custom-built magnetic tweezers device to observe initiation (nicking) by RepD and translocation by PcrA. Our initial studies have focussed on the effect of DNA supercoiling upon RepD DNA-nicking kinetics and on DNA unwinding by PcrA. The initiation site, oriD, consists of three inverted complementary repeats that are predicted to form secondary DNA structures (hairpins). When the circular plasmid is damage-free (e.g. no single strand breaks) DNA gyrase converts the relaxed open circle into a compact, negatively supercoiled, form which favors extrusion of secondary structure motifs. Using magnetic tweezers to artificially supercoil a length of DNA containing the OriD sequence, we found that RepD nicking is highly sensitive to DNA supercoiling and negative supercoiling serves as a gate-keeper to ensure plasmids are damage-free before DNA replication is initiated.

The Effects of Tension and Supercoiling on Protein-Mediated DNA Looping

Protein-mediated DNA looping is widely found as a topological regulatory mechanism in DNA transcription and other genetic process in vivo. The loop formation probability is mainly regulated by the loop size, protein concentration, supercoiling level of the genomic DNA, and local tension generated from the action of motor protein and enzymes. The supercoiling level varies at different times in cell cycle and likely at different locations in the genome, and a change of the local negative supercoiling level has been known to affect gene expression and regulation. However, little is known about the dynamics of how supercoiling and enzyme-generated tension control gene regulation by affecting DNA looping probability and stability.We present single molecule measurements to study the effects of supercoiling and tension on a loop induced by the lambda repressor (CI protein) binding to two regions of specific sites in bacteriophage lambda DNA. The loop helps keeping the concentration of the repressor at the appropriate level to maintain the quiescent state, while guaranteeing efficient switching to virulence if necessary. Though even the lowest force prevents the wild type 2317 bp-long loop formation in vitro, the formation and breakdown of a 1051bp-long loop was observed under <1pN tension in unwound DNA at physiological CI concentrations. Furthermore, negative supercoiling stabilized the loop against increased tension, in agreement with our previous observation on a 400bp-long loop. The effect of DNA supercoiling on the formation and breakdown of different size loops is important for the quantitative understanding of the physiological role that DNA supercoiling and tension may have on loop-based gene regulation. Since the genome supercoiling level depends on the energy level and health status of a cell, our investigation sheds light on the dependence of some regulatory mechanisms on these two factors.

Atomic Force Microscopy Studies on the Effect of DNA Supercoiling on Nucleosome Structure

The genome of eukaryotes is folded into chromatin. Nucleosomes are its basic unit. The compaction of DNA in nucleosomes impedes nuclear processes that require DNA as a template such as transcription, replication, and DNA damage repair. It has been proposed that changes in DNA topology influence the accessibility of nucleosomal DNA. Such changes in DNA topology occur e.g. during transcription when reverse torsional tensions are being built up within the DNA during elongation in front of and behind the polymerase complex. This leads to positive or negative supercoiling of the DNA strand possibly affecting chromatin structure. Here we investigated the influence of different topological states of DNA on nucleosomal structure by qualitative analysis of atomic force microscopy (AFM) images. Recombinant histone octamers were reconstituted via salt dialysis on PUC 18 plasmids of superhelical densities σ = −0.046, −0.02, 0, +0.03 and observed by AFM. The obtained images were edited and the nucleosomal DNA opening angle (α) analysed by a half automated technique. The results show a clear dependence between the size of α and the strength and type of superhelical density of the DNA. With decreasing negative torsional strain alpha is found to be increasing whereas an increase in positive torsional strain leads to further increase of the opening angle.

Effect of DNA Supercoiling on DNA Decatenation and Unknotting Followed By Brownian Dynamics Simulations

Replication of circular DNA proceeds through a stage of multiply interlinked catenanes that have to be rapidly spatially separated. In addition, knotting of circular DNA has also to be avoided. In bacteria, topology simplification requires participation of two type II topoisomerases: gyrase and topo IV. Several simulation approaches were applied to explain the very efficient topology simplification in that system. Mainly two strategies were explored: in the first one, the system follows its free energy gradient influenced by supercoiling, and in the second one, specific geometrical rules are defined for the selection of strand passages (hooking, chirality). The Monte-Carlo methods usually used to estimate the efficiency of these strategies do not allow to follow DNA topology simplification dynamically, to evaluate its speed, for example. To overcome this limitation, we simulated DNA unknotting and decatenation by Brownian dynamics, which allows for a natural integration of the strategies mentioned above. By following the topological state of the simulated DNA chains (see figure), we show that the combination of supercoiling and local geometrical selection rules provides an important drive for unknotting and decatenation, especially at low topological complexity.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Supercoil-Accelerated DNA Threading Intercalation

The effect of DNA supercoiling on a sterically very demanding threading intercalation process is investigated here. We find that the threading rate of a dimeric ruthenium complex into a negatively supercoiled plasmid at low binding density is 2 orders of magnitude higher than into the cleaved linear form. Further saturation is on the other hand kinetically hampered in comparison to the relaxed DNA. We also observe that threading kinetics correlates with the inhibition of luciferase expression from the plasmid construct. The results show how the target torsional strain can function as a control of DNA threading kinetics and gene expression efficiency.

Effects of DNA supercoiling and topoisomerases on the expression of genes coding for F1651, a P-like fimbriae

Pathogenic Escherichia coli 4787 (O115:KV165) causes septicemia in pigs and expresses the fimbriae F165(1) encoded by the foo operon that belongs to the P fimbrial family. fooI and fooB, encoding specific foo regulators, are divergently transcribed; their intergenic region is responsible for the regulation of foo expression. The role of global and local supercoiling (transcription-induced supercoiling within the intergenic region) on the regulation of foo expression was investigated. Expression of fooB was significantly altered when global negative supercoiling was reduced by a mutation that decreases DNA gyrase activity. Deletion of the topA gene, encoding for topoisomerase I that relaxes local negative supercoiling, further reduced fooB expression. This suggests that both global and local supercoiling can significantly affect fooB expression. Moreover, FooI, a positive regulator of fooB expression, has no effect on fooB expression in the topA null mutant. This study showed that divergent transcription from a strong promoter can significantly enhance fooB expression and compensate for the absence of FooI in a wild-type strain.

Effect of DNA Supercoiling on the Geometry of Holliday Junctions

Unusual DNA conformations including cruciforms play an important role in gene regulation and various DNA transactions. Cruciforms are also the models for Holliday junctions, the transient DNA conformations critically involved in DNA homologous and site-specific recombination, repair, and replication. Although the conformations of immobile Holliday junctions in linear DNA molecules have been analyzed with the use of various techniques, the role of DNA supercoiling has not been studied systematically. We utilized atomic force microscopy (AFM) to visualize cruciform geometry in plasmid DNA with different superhelical densities at various ionic conditions. Both folded and unfolded conformations of the cruciform were identified, and the data showed that DNA supercoiling shifts the equilibrium between folded and unfolded conformations of the cruciform toward the folded one. In topoisomers with low superhelical density, the population of the folded conformation is 50-80%, depending upon the ionic strength of the buffer and a type of cation added, whereas in the sample with high superhelical density, this population is as high as 98-100%. The time-lapse studies in aqueous solutions allowed us to observe the conformational transition of the cruciform directly. The time-dependent dynamics of the cruciform correlates with the structural changes revealed by the ensemble-averaged analysis of dry samples. Altogether, the data obtained show directly that DNA supercoiling is the major factor determining the Holliday junction conformation.

The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons.

Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling). The effect of this transcription-induced DNA supercoiling on these promoters depends on their intrinsic properties. As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose. Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co-ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions. As DNA supercoiling-dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.

The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons

The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons

Saturation mutagenesis of the haloarchaeal bop gene promoter: identification of DNA supercoiling sensitivity sites and absence of TFB recognition element and UAS enhancer activity.

Transcription from the bop promoter in the haloarchaeon Halobacterium NRC-1, is highly induced under oxygen-limiting conditions. A DNA gyrase inhibitor, novobiocin, was previously shown to block bop gene induction and suggested that DNA supercoiling mediates transcriptional induction. A region of non-B structure was found 3' to the TATA box within an 11 bp alternating purine-pyrimidine sequence (RY box), which correlated to both increased DNA supercoiling and transcriptional induction. Here, saturation mutagenesis of the RY box region has been used to show that single-base substitutions of A(r)G either 23 or 19 bp 5' to the transcription start site temper the effect of DNA supercoiling based on novobiocin insensitivity of transcription. Mutagenesis of the region 5' to the TATA box showed its involvement in DNA supercoiling modulation of transcription, defined the 3' end of the upstream activator sequence (UAS) regulatory element, and ruled out the requirement for a TFB (TFIIB) Recognition Element. Spacing between the TATA box and UAS was found to be critical for promoter activity because insertion of partial or whole helical turns between the two elements completely inhibited transcription indicating that the UAS element does not function as a transcriptional enhancer. The results are discussed in the context of DNA melting and flexibility around the TATA box region and the involvement of multiple regulatory and transcription factors in bop promoter activity.

Promoter Selectivity of Escherichia coli RNA Polymerase Eσ70 and Eσ38 Holoenzymes: EFFECT OF DNA SUPERCOILING

The functional specificity was compared between two sigma factors, sigma 70 (the major sigma at exponentially growing phase) and sigma 38 (the essential sigma at stationary growth phase), of Escherichia coli RNA polymerase. The core enzyme binding affinity of sigma 38 was less than half the level of sigma 70 as measured by gel filtration column chromatography or by titrating the concentration of sigma required for the maximum transcription in the presence of a fixed amount of core enzyme. In addition, the holoenzyme concentration required for the maximum transcription of a fixed amount of templates was higher for E sigma 38 than E sigma 70. The transcription by E sigma 38 was, however, enhanced with the use of templates with low superhelical density, in good agreement with the decrease in DNA superhelicity in the stationary growth phase. We thus propose that the selective transcription of stationary-specific genes by E sigma 38 holoenzyme requires either a specific reaction condition(s) or a specific factor(s) such as template DNA with low superhelical density.

Cryo-electron microscopy: The method of choice for direct study of 3D-shape of individual DNA molecules in a solution

3-D reconstruction of the trajectory of individual DNA molecules freely suspended in vitrified buffer opens new ways to study DNA. We showed that tightly supercoiled regions appearing as one thick filament can be reconstructed from their two projections. We present here use of this technique to trace normal double-stranded DNA regions to study the effect of DNA supercoiling on the overall shape of circular DNA molecules.

Effect of DNA supercoiling on in vitro transcription from the adenovirus early region 4

The effect of DNA supercoiling on in vitro transcription from the early region 4 (E4) promoter of adenovirus type 5 (Ad5) has been investigated by using a cell-free transcription system that maintains supercoiled DNA templates. The supercoiled DNAs yield several-fold higher levels of E4 transcripts and more faithfully reflect the regulation of in vivo transcription than the linear DNAs.

DNA supercoiling and temperature shift affect the promoter activity of the Escherichia coli rpoH gene encoding the heat-shock sigma subunit of RNA polymerase

The effects of DNA supercoiling and temperature shift on the activity of two major promoters of the Escherichia coli rpoH gene, which codes for the heat-shock sigma subunit of RNA polymerase, were examined using an in vitro transcription system. Upstream promoter P1 is not affected by DNA supercoiling nor temperature upshift but downstream promoter P2 is enhanced by both of these factors. Both factors facilitate the formation of an open complex on rpoH P2, and these two effects are not additive. Hence these two factors act at the same step of transcription, probably the local unwinding of the DNA double helix at the promoter region. The different sensitivities of the rpoH major promoters to DNA supercoiling and temperature shift indicate that these promoters are under differential control. Taken together with the in vivo enhancement by heat shock of rpoH P2 transcription, we propose that at least one of the mechanism(s) of induction of sigma 32 synthesis by heat shock is the activation of the rpoH P2 promoter by temperature up-shift.

Effects of DNA supercoiling on the topological properties of nucleosomes.

In the nucleosome core particle, at least 145 base pairs of DNA are bound to the histone octamer in a superhelical conformation. We have asked what effect the presence of these particles has on the ability of DNA gyrase to supercoil DNA. Synthetic minichromosomes, constructed by reconstituting complexes of core histones with the closed circular plasmid pBR322, were treated with various amounts of DNA gyrase. We have found that the maximum level of supercoiling that is attainable is nearly identical for protein-free plasmids and for plasmids half-saturated with core histones, even though supercoiling does not result in a loss of histones from the complex. It appears that, at sufficiently high levels of supercoiling, the core particle is disrupted in such a way that the DNA bound to histones is no longer constrained.

Phage P1 Cre- loxP site-specific recombination : Effects of DNA supercoiling on catenation and knotting of recombinant products

Bacteriophage P1 contains a site-specific recombination system consisting of a site, loxP, and a recombinase protein Cre. We have shown that with purified Cre protein we can carry out recombination between two loxP sites in vitro. When that recombination occurs between two sites in direct orientation on the same DNA molecule, we observed the production of free and catenated circular molecules. In this paper we show that recombination between sites in opposite orientation leads to both knotted and unknotted circular products. We also demonstrate that the production of catenanes and knots is influenced by two factors: (1) supercoiling in the DNA substrate, supercoiled DNA substrates yield significantly more catenated and knotted products than nicked circular substrates; and (2) mutations in the loxP site, a class of mutations have been isolated that carry out recombination but result in a distribution of products in which the ratio of catenanes to free circles is increased over that observed with a wild-type site. A more detailed analysis of the products from recombination between wild-type sites indicates: (1) that the catenanes or knots produced by recombination are both simple and complex; (2) that the ratio of free products to catenanes is independent of the distance between the two directly repeated loxP sites; and (3) that for DNA substrates with four loxP sites significant recombination between non-adjacent sites occurs to give free circular products. These observations provide insights into how two loxP sites are brought together during recombination.

Supercoiling response of a bacterial tRNA gene.

The effect of DNA supercoiling on transcription of a bacterial tRNA gene has been analysed in vitro and in vivo. The Escherichia coli tyrT gene is found to be completely dependent on negative supercoiling when transcription is assayed in vitro at physiological salt concentrations and this supercoiling dependence is shown to be a property of the primary promoter sequences. The supercoiling sensitivity can however be removed by reducing the salt concentration below 50 mM KCl. The effect of supercoiling in vivo was analysed by measuring the activity of the tyrT promoter on a galK fusion vector in E. coli strains that have mutations in either the DNA gyrase or topoisomerase I genes. Surprisingly, in view of the dramatic in vitro effects, supercoiling could be either increased or decreased relative to the wild-type in vivo level without causing any change in tyrT promoter activity.