Role Of DNA Supercoiling Research Articles

Protein-Mediated Loops in Supercoiled DNA Generate Large, Dynamic Topological Domains

Proteins that act at a distance along DNA by binding at one site and contacting another create loops that form topological domains and influence regulation. Such loops are affected by the torsional state of DNA, which dramatically modulates topology, driving the DNA from extended and accessible, to more compact and genetically secured forms. Furthermore, the response of the DNA filament to supercoiling is biased by accessory factors. For example, small molecules like polyamines, which neutralize the negative charge repulsions along the phosphate backbone, enhance flexibility and promote writhe over twist in response to torsion. On the other hand, stiffer DNA antagonizes looping and bending. Recent experiments quantitatively reveal the extent to which negatively supercoiling DNA lowers the free energy of looping, and could therefore bias the operation of genetic switches. A new role for DNA supercoiling has emerged; it synergizes with protein-mediated looping to create large, dynamic topological domains that extend beyond the length of the loop. Such domains may coordinate gene regulation and other DNA transactions across spans in the genome that exceed the separation between the protein binding sites.

Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus

BackgroundIt has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling.ResultsHere, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location.ConclusionsThe results indicate that the molecular properties of a given promoter, rather than the chromosomal topology, dictate the responsiveness to changes in supercoiling in the pathogen Staphylococcus aureus.

Determining the topology of stable protein–DNA complexes

Difference topology is an experimental technique that can be used to unveil the topological structure adopted by two or more DNA segments in a stable protein-DNA complex. Difference topology has also been used to detect intermediates in a reaction pathway and to investigate the role of DNA supercoiling. In the present article, we review difference topology as applied to the Mu transpososome. The tools discussed can be applied to any stable nucleoprotein complex.

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.

A role for DNA supercoiling in the regulation of the cytochrome bd oxidase of Escherichia coli.

The cydAB operon of Escherichia coli encodes cytochrome bd, a terminal oxidase in the aerobic respiratory chain. The high oxygen affinity of this oxidase explains its increased synthesis under low-oxygen conditions. Expression of the cydAB operon is controlled by the ArcA/ArcB two-component system and the oxygen-sensing transcriptional regulator Fnr. However, cydAB expression is still induced upon entry into stationary phase or following a shift to anaerobic conditions in a mutant deleted for arcA and fnr [Cotter, P. A. & Gunsalus, R. P. (1992), FEMS Microbiol Lett 91, 31-36]. Indeed, such a mutant contains 60% of the wild-type levels of spectrally detectable cytochrome bd. A possible mechanism to account for this regulation is that changes in negative supercoiling, which occur during a shift to low-oxygen or anaerobic conditions, may contribute to the regulation of the cydAB operon. This paper reports several lines of evidence in support of this idea. Firstly, the expression of cydAB, and the final level of spectrally detectable cytochrome bd, is sensitive to inhibitors of DNA gyrase, the enzyme responsible for introducing negative supercoils into DNA. Both nalidixic acid and novobiocin reduce cydA-lacZ expression in a concentration-dependent manner. Secondly, in a gyrA mutant, defective in DNA gyrase activity, expression of cydAB is reduced to a basal level that is no longer sensitive to the oxygen status. Both gyrase inhibitors and the gyrA mutation reduce cydAB expression in a strain deleted for arcA and fnr, indicating that their effects are not mediated indirectly through ArcA or Fnr, but rather that they are likely to be direct effects on cydAB expression. In conclusion, the authors have shown that changes in DNA supercoiling play a role in the induction of cydAB expression and may provide a general way of increasing cytochrome bd levels in the cell in response to environmental stress.

Escherichia coli response to hydrogen peroxide: a role for DNA supercoiling, Topoisomerase I and Fis

Bacterial cells respond to the deleterious effects of reactive oxygen species by inducing the expression of antioxidant defence genes. Here we show that treatment with hydrogen peroxide leads to a transient decrease in DNA negative supercoiling. We also report that hydrogen peroxide activates topA P1 promoter expression. The peroxide-dependent topA P1 activation is independent of oxyR, but is mediated by Fis. This nucleoid-associated protein binds to the promoter region of topA. We also show that a fis deficient mutant strain is extremely sensitive to hydrogen peroxide. Our results suggest that topA activation by Fis is an important component of the Escherichia coli response to oxidative stress.

Role of DNA supercoiling and RpoS sigma factor in the osmotic and growth phase-dependent induction of the Gene osmE of Escherichia coli K12

Transcription of the gene osmE of Escherichia coli is osmotically inducible and regulated by the growth phase. In a medium of low osmotic pressure, expression of osmE is induced at the onset of stationary phase. At elevated osmotic pressure, a biphasic induction pattern is observed. The first step occurs during exponential phase, and this is followed by a strong induction at the onset of stationary phase. Both steps appear to result from stimulation of transcription at the same promoter, osmE p . In the absence of σ s, the stationary phase sigma factor encoded by rpoS, osmE p stationary phase induction is abolished, while the osmotic effect is still observed. Mutations that compensate for the absence of σ s mapped to the gene topA. The effect of such mutation and of novobiocin, an inhibitor of DNA gyrase, suggest that changes in DNA supercoiling are involved in the osmotic induction of osmE p . In addition, modulation of the supercoiling level of a reporter plasmid was observed during growth in rich media. The kinetics of osmE p transcription are discussed in light of the variations of DNA supercoiling.

DNA topology and gene expression in Clostridium acetobutylicum: Implications for the regulation of solventogenesis

The relationship between solventogenesis, heat shock, and DNA topology in Clostridium acetobutylicum was investigated by inhibiting DNA gyrase in vivo by high concentrations of novobiocin. Primer extension and Northern blot analyses revealed that expression of genes required for solventogenesis and heat shock response was induced in the presence of this drug, whereas the transcription rate of acidogenic genes decreased, suggesting a role for DNA supercoiling as a signal for onset of solventogenesis.

Mobilization protein-DNA binding and divergent transcription at the transfer origin of the Thiobacillus ferrooxidans pTF1 plasmid.

The possible interaction of the trans-acting mobilization proteins, MobL and MobS, at the cognate origin of transfer (oriT) region of the Thiobacillus ferrooxidans plasmid pTF1 has been investigated. In gel retardation assays with crude protein extracts from overproducing strains, a truncated MobL (c. 28 kDa) as well as its native protein (42 kDa), but not the 11 kDa MobS protein, were found to bind specifically to a 42-mer oligonucleotide which represents the transferred DNA strand of the minimal oriT fragment of pTF1. In vivo, the binding of MobL was studied by monitoring catechol 2,3-dioxygenase (xylE) activities driven by promoters of the divergently transcribed mobL and mobS genes. The mob promoter sequences were found to resemble the Escherichia coli sigma 70-dependent consensus promoter elements. The '-10' recognition sequences of mobL and one of the two mobS promoters overlap except for one base and they are positioned within the putative 'hairpin' structure in the minimal oriT sequence. In accordance with the twin supercoil-domain model of Liu and Wang (1987) which suggests that transcription can generate local variations in DNA superhelicity, we propose a possible physiological role of DNA supercoiling in the transfer origin with reference to divergent transcription of mobL and mobS genes.

Osmotic regulation of porin expression: a role for DNA supercoiling

The OmpC and OmpF porins are major outer membrane proteins of Escherichia coli and Salmonella typhimurium. Their expression is affected by many environmental factors and by mutations in a variety of independent genes. The pair of regulatory proteins, OmpR and EnvZ, are required for normal porin expression. Despite intensive investigation, the mechanisms by which porin expression is regulated remain unclear. Mutations which alter supercoiling, as well as inhibitors of DNA gyrase, show that porin expression is extremely and specifically sensitive to the level of DNA supercoiling. Our data lead us to suggest that environmentally induced changes in DNA supercoiling may play a role in determining the level of porin expression. These findings have implications for current models of porin regulation.

Changes in DNA topology can modulatein vitro transcription of certain RNA polymerase III genes

The role of DNA supercoiling in eukaryotic gene expression is not fully understood. The objective of this study was to examine the regulation of in vitro chromatin assembly by topological alterations in the DNA template using a cell-free extract from Xenopus laevis oocytes (S-150). The results suggest that input DNA topology may be a determining factor in controlling the transcriptional activity of the Xenopus tRNA and one particular 5S gene. When the input topology is supercoiled, high levels of transcription are observed, whereas input relaxed DNA is transcribed to a much lower extent. Transcription from an input relaxed template is stimulated by the addition of supercoiled nonspecific, vector DNA. Furthermore, in direct competition experiments, supercoiled DNA molecules were shown to be transcriptionally dominant over relaxed DNA molecules. Taken together, these data suggest that the efficiency with which a repressor or activator binding protein interacts with DNA may be significantly influenced by the topological status of its target. We demonstrate that modulation of reaction parameters which alter the normal topological processing events catalyzed by the S-150 can dramatically influence the level of gene expression.

The role of DNA supercoiling in site-specific recombination by Tn21 resolvase

The role of DNA supercoiling in site-specific recombination by Tn21 resolvase