Changes In DNA Supercoiling Research Articles

DNA Supercoiling and the Lrp Protein Determine the Directionality of fim Switch DNA Inversion in Escherichia coli K-12

Site-specific recombinases of the integrase family usually require cofactors to impart directionality in the recombination reactions that they catalyze. The FimB integrase inverts the Escherichia coli fim switch (fimS) in the on-to-off and off-to-on directions with approximately equal efficiency. Inhibiting DNA gyrase with novobiocin caused inversion to become biased in the off-to-on direction. This directionality was not due to differential DNA topological distortion of fimS in the on and off phases by the activity of its resident P(fimA) promoter. Instead, the leucine-responsive regulatory (Lrp) protein was found to determine switching outcomes. Knocking out the lrp gene or abolishing Lrp binding sites 1 and 2 within fimS completely reversed the response of the switch to DNA relaxation. Inactivation of either Lrp site alone resulted in mild on-to-off bias, showing that they act together to influence the response of the switch to changes in DNA supercoiling. Thus, Lrp is not merely an architectural element organizing the fim invertasome, it collaborates with DNA supercoiling to determine the directionality of the DNA inversion event.

Biosynthesis and Regulation of the Branched-Chain Amino Acids†.

This review focuses on more recent studies concerning the systems biology of branched-chain amino acid biosynthesis, that is, the pathway-specific and global metabolic and genetic regulatory networks that enable the cell to adjust branched-chain amino acid synthesis rates to changing nutritional and environmental conditions. It begins with an overview of the enzymatic steps and metabolic regulatory mechanisms of the pathways and descriptions of the genetic regulatory mechanisms of the individual operons of the isoleucine-leucine-valine (ilv) regulon. This is followed by more-detailed discussions of recent evidence that global control mechanisms that coordinate the expression of the operons of this regulon with one another and the growth conditions of the cell are mediated by changes in DNA supercoiling that occur in response to changes in cellular energy charge levels that, in turn, are modulated by nutrient and environmental signals. Since the parallel pathways for isoleucine and valine biosynthesis are catalyzed by a single set of enzymes, and because the AHAS-catalyzed reaction is the first step specific for valine biosynthesis but the second step of isoleucine biosynthesis, valine inhibition of a single enzyme for this enzymatic step might compromise the cell for isoleucine or result in the accumulation of toxic intermediates. The operon-specific regulatory mechanisms of the operons of the ilv regulon are discussed in the review followed by a consideration and brief review of global regulatory proteins such as integration host factor (IHF), Lrp, and CAP (CRP) that affect the expression of these operons.

A microarray-based antibiotic screen identifies a regulatory role for supercoiling in the osmotic stress response of Escherichia coli.

Changes in DNA supercoiling are induced by a wide range of environmental stresses in Escherichia coli, but the physiological significance of these responses remains unclear. We now demonstrate that an increase in negative supercoiling is necessary for transcriptional activation of a large subset of osmotic stress-response genes. Using a microarray-based approach, we have characterized supercoiling-dependent gene transcription by expression profiling under conditions of high salt, in conjunction with the microbial antibiotics novobiocin, pefloxacin, and chloramphenicol. Algorithmic clustering and statistical measures for gauging cellular function show that this subset is enriched for genes critical in osmoprotectant transport/synthesis and rpoS-driven stationary phase adaptation. Transcription factor binding site analysis also supports regulation by the global stress sigma factor rpoS. In addition, these studies implicate 60 uncharacterized genes in the osmotic stress regulon, and offer evidence for a broader role for supercoiling in the control of stress-induced transcription.

Effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.

Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat-induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature-dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.

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.

Separation of supercoiled DNA using capillary electrophoresis.

DNA supercoiling is a general phenomenon of almost all DNA in vivo. Plasmids, bacterial chromosomes, the mitochondrial genome and many viral genomes occur as closed circular DNA. Even some linear DNAs, such as eukaryotic chromosomes and yeast plasmids, behave like circular DNA because part of the DNA is anchored to the nuclear matrix creating closed-circular loops. Many DNA-associated processes are affected by changes in DNA supercoiling. These processes include the genome organization, transcription, gene expression, DNA replication, and recombination events (). Plasmids are double-stranded circular DNAs which are capable of replicating independent of the chromosome. This property allows plasmids to be carriers of desired DNA sequences and to be replicated in large quantity. Plasmids were actually the first successful DNA vectors used for molecular cloning in bacteria. Recently, the potential of plasmids in gene therapy has been explored extensively. The rising interest in gene therapy for treatment of human diseases, such as cancer and genetic disorders, is reflected in the growing number of clinical trials (,).

DNA topology and the thermal stress response, a tale from mesophiles and hyperthermophiles.

During heat shock and cold shock, plasmid DNA supercoiling changes transiently both in mesophilic bacteria and in hyperthermophilic archaea, despite a different overall topology (negative supercoiling versus relaxation to positive supercoiling). Transient changes in DNA supercoiling might be essential to generate the stress response, but they could also be a consequence of the physical effects of temperature on cellular components. Indeed, both appear intertwined. Comparison of the mechanisms acting in the two biological systems suggests that the dependence on temperature of the activity of different DNA topoisomerases, as well as of protein binding, are key factors for the control of DNA topology during stress, which may in turn be relevant for the expression of stress-induced genes.

DNA topology and the thermal stress response, a tale from mesophiles and hyperthermophiles

During heat shock and cold shock, plasmid DNA supercoiling changes transiently both in mesophilic bacteria and in hyperthermophilic archaea, despite a different overall topology (negative supercoiling versus relaxation to positive supercoiling). Transient changes in DNA supercoiling might be essential to generate the stress response, but they could also be a consequence of the physical effects of temperature on cellular components. Indeed, both appear intertwined. Comparison of the mechanisms acting in the two biological systems suggests that the dependence on temperature of the activity of different DNA topoisomerases, as well as of protein binding, are key factors for the control of DNA topology during stress, which may in turn be relevant for the expression of stress-induced genes.

DNA topology and adaptation of Salmonella typhimurium to an intracellular environment.

The expression of genes coding for determinants of DNA topology in the facultative intracellular pathogen Salmonella typhimurium was studied during adaptation by the bacteria to the intracellular environment of J774A.1 macrophage-like cells. A reporter plasmid was used to monitor changes in DNA supercoiling during intracellular growth. Induction of the dps and spv genes, previously shown to be induced in the macrophage, was detected, as was expression of genes coding for DNA gyrase, integration host factor and the nucleoid-associated protein H-NS. The topA gene, coding for the DNA relaxing enzyme topoisomerase I, was not induced. Reporter plasmid data showed that bacterial DNA became relaxed following uptake of S. typhimurium cells by the macrophage. These data indicate that DNA topology in S. typhimurium undergoes significant changes during adaptation to the intracellular environment. A model describing how this process may operate is discussed.

Intercalation-induced changes in DNA supercoiling observed in real-time by atomic force microscopy

Atomic force microscopy (AFM) has been employed to observe in real-time and in an aqueous environment the process of ethidium bromide induced supercoiling in individual DNA plasmid molecules. Image data reveal both the onset and the progressive presence of plectonemic DNA supercoiling. In addition, significant molecular motion of the surface adsorbed DNA is observed. These data illustrate the potential of AFM in the time-resolved study of biomolecular processes, and hence, provide new insights into biomolecular structure and function.

The Yersinia enterocolitica pYV virulence plasmid contains multiple intrinsic DNA bends which melt at 37 degrees C.

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (</=30 degrees C) and virulence plasmid-encoded Yop secretion (37 degrees C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the Y. enterocolitica virulence plasmid, pYV, undergoes a conformational transition between 30 and 37 degrees C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including virF, the positive activator of virulence genes. These bends are detectable at 30 degrees C but melt at 37 degrees C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from Y. enterocolitica were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the Y. enterocolitica pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in Y. enterocolitica and other enteric pathogens.

Changes in DNA supercoiling in fibroblasts cultured in the presence of hydralazine

We have analyzed changes in the supercoiling of nucleoid DNA of murine fibroblasts cultured in the presence of hydralazine. The entire DNA attached to the nuclear matrix was extracted from the cells and sedimented in neutral sucrose density gradients containing ethidium bromide. Nucleoids from cells treated with hydralazine responded to increasing ethidium bromide concentrations in a different way than those from control cultures. That is, supercoiled loops of DNA unwound with lower concentrations of ethidium bromide sedimented less rapidly than those of control cells, indicating that hydralazine reduced the degree of DNA supercoiling. Also, nucleoids from the drug-treated cells resisted the transition from relaxed to positive supercoiling at higher concentrations of ethidium bromide. Changes in nucleoid DNA supercoiling correlated directly with the dose of hydralazine in the fibroblast culture.

Synchronous block in DNA synthesis initiation with change in chromatin topology mediated by VP16.

The impact of chromatin topology on the DNA synthetic process was studied in the human squamous-cell carcinoma cell line SQ-20B. A 1-h exposure < or = 10 microM VP16 produced an increase in DNA supercoil tension, measured by recording laser light scatter from salt-extracted nuclei. This change was precisely paralleled by a decrease in DNA synthesis. The effects on both DNA supercoiling and DNA synthesis were suppressed at VP16 concentrations between 10 and 20 microM. The changes in DNA supercoiling and synthesis at VP16 concentrations -10 microM were eliminated by coincubation with mimosine, a DNA synthesis initiator poison. We conclude that brief exposure to low concentrations of VP16 disturbs the balance of torsional energy within discrete replicon domains by affecting normal topoisomerase II activity at sites of replication initiation. The resultant increase in negative supercoil tension mediates a topologic checkpoint, limiting the initiation of DNA synthesis. Such a checkpoint may be a common pathway for control, both during the normal replicative cycle and subsequent to DNA damage.

Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster: structural and functional characterization of an upstream untranslated mRNA sequence.

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Overview of the Proceedings: From Loose Loops to Sticky Ends

a chromatid break is obviously not a simple matter, since the latter can involve the loss of up to 20 Mbp, and evidence from some radiosensitive sublines suggests that secondary chromatin configuration influences conversion of DNA damage to chromosome breaks. These and other questions were addressed at a meeting held in Montreal in June 1997 which brought together experts in DNA damage and repair, chromatin structure and cytogenetics to delineate the route from a DNA lesion to a dead cell. The topics discussed moved through increasing levels of complexity from damage distribution at the molecular level to nucleoprotein structure to the organization of nuclear domains. Several presentations described investigation of the genomic distribution of DNA lesions. In one of these (M-E. Mirault, Universiti Laval), a single-strand break (SSB)-specific S1 nuclease was used as a probe. Similar cleavage patterns were found with a variety of chemical agents and with ionizing radiation with preferential localization of S1 cleavage in matrix-associated DNA. In another study (A. I. Kassis, Harvard Medical School), 1251 was used to assess the effects of OH radicals produced in proximity to DNA. Results were consistent with DSBs being produced by a single-event mechanism when a high radical density was generated close to naked DNA, while for higher-order chromatin both production of DSBs and toxicity appeared to be largely indirect in nature. The genomic distribution of naturally occurring DNA DSBs was examined using a technique in which illegitimate integration of a transgene in murine fibroblast DNA was localized by fluorescence in situ hybridization (FISH) analysis (G. Dellaire and P. Chartrand, Institut du Cancer de Montrial). Results indicated that illegitimate integration occurs randomly at megabase resolution in murine fibroblasts and, since the initiation of illegitimate integration would require a DNA DSB, the corollary is that DSBs occur randomly within the murine genome at megabase resolution. An important aspect of the organization of the eukaryotic chromosome which was addressed by several speakers at this meeting is the arrangement of DNA supercoils into looped domains anchored at intervals of 30-60 kbp to the nuclear matrix through matrix attachment region (MAR)-specific sequences (J. L. Roti Roti, Washington University, St. Louis; J. L. Schwartz, University of Washington, Seattle). These structures create independent domains of torsional stress in chromatin which are important for spatial control of DNA replication, gene transcription and DNA repair. Based on data obtained with assays that use high-salt extraction of nuclei (nucleoids), it is hypothesized that there are two categories of looped domains: those with loose (protease-sensitive) attachments to the nuclear matrix and those which are tightly attached to the nuclear matrix and are resistant to disruption. In the first case breaks can affect rewinding both in the loop where they occur and in adjacent loops, while in the second case breaks affect rewinding only in the loop where they occur and the adjacent loops are insulated from damage. In one subgroup of cell lines, differences in radiosensitivity could be explained in terms of the ability of anchoring regions to isolate DNA domains from changes in DNA supercoiling, whereas in other cell lines rewinding and radiation resistance are associated with a subset of specific proteins located at the anchoring regions. In another study (P. J. Johnston, British Columbia Cancer Research Centre), selective examination of DSBs occurring as multiples within looped DNA structures revealed identical subsets of DSBs repairing with slow kinetics through the DNA-dependent protein kinase (PK)/Ku pathway. Correction of the results to account for differences in the rate of elution of cells actively replicating DNA gave a looped domain size of 3.0 Mbp for all cells examined. It was suggested that this is a fundamental unit of chromatin structure which may play an important part in the processing of DNA DSBs. 1Selected papers from this meeting will appear in this and future issues of Radiation Research.

Dual function of the copR gene product of plasmid pIP501.

Replication of plasmid pIP501 is regulated at a step subsequent to transcription initiation by an antisense RNA (RNAIII) and transcriptionally by a repressor protein, CopR. Previously, it had been shown that CopR binds to a 44-bp DNA fragment upstream of and overlapping the repR promoter pII. Subsequently, we found that high-copy-number pIP501 derivatives lacking copR and low-copy-number derivatives containing copR produced the same intracellular amounts of RNAIII. This suggested a second, hitherto-unknown function of CopR. In this report, we show that CopR does not affect the half-life of RNAIII. Instead, we demonstrate in vivo that, in the presence of both pII and pIII, CopR provided in cis or in trans causes an increase in the intracellular concentration of RNAIII and that this effect is due to the function of the protein rather than its mRNA. We suggest that, in the absence of CopR, the increased (derepressed) RNAII transcription interferes, in cis, with initiation of transcription of RNAIII (convergent transcription), resulting in a lower RNAIII/plasmid ratio. When CopR is present, the pII promoter is repressed to >90%, so that convergent transcription is mostly abolished and RNAIII/plasmid ratios are high. The hypothesis that RNAII transcription influences promoter pIII through induced changes in DNA supercoiling is supported by the finding that the gyrase inhibitor novobiocin affects the accumulation of both sense and antisense RNA. The dual role of CopR in repression of RNAII transcription and in prevention of convergent transcription is discussed in the context of replication control of pIP501.

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 supercoiling changes and nuclear matrix-associated proteins: Possible role in oncogene-mediated radioresistance

Purpose : Transfection with either H- ras or H- ras and c- myc has been shown to confer radioresistance in rat embryonal cells (REC). REC primary, transfected with either c- myc or cotransfected with c- myc and H- ras (in ascending order of radioresistance and tumorigenicity), were used as an in vitro model system to determine if nuclear matrix-mediated higher order DNA organization contributes to oncogene-mediated radioresistance. Methods and Materials : DNA damage induction and repair were measured by the alkaline and neutral filter elution assays. Analysis of the ability of DNA loop domains to undergo supercoiling changes in the presence of radiation-induced damage was determined by the fluorescent halo assay 9FHA). Because DNA loops are organized by the nuclear matrix (NM), a study of NM-associated proteins by high resolution two0dimensional gel electrophoresis was performed. Results: Induction and repair rates of DNA single- and double-strand breaks were similar for the relatively radiosensitive c- myc transfected and the radioresistant c- myc + H- ras transfected cells. However, the degree of inhibition of DNA supercoil rewinding in the presence of radiation-induced damage was less in the radioresistant cells and was inversely correlated with survival. A. progressive loss of NM-associated proteins was observed, which correlated with increasing radioresistance and tumorigenicity in these cell lines. In addition, some protein changes were consistent with the possibility that these changes could be involved in DNA anchoring. Conclusions : Increased radioresistance associated with increasing tumorigenicity in these oncogene-transfected cell lines could be due to changes in NM-mediated DNA organization, possibly via differences in NM protein composition that occur following oncogenic transfection.

DNA Supercoiling Changes and Nucleoid Protein Composition in a Group of L5178Y Cells of Varying Radiosensitivity

Cell of the radioresistant L5178Y-R, -S35, -SR and M10(neo 5)-1 and radiosensitive L5178Y-S, M10 and LX830 cell lines were used to investigate the relationship between radiosensitivity and DNA supercoiling ability mediated by the nuclear matrix within chromatin loops containing DNA damage. The ability of DNA loops to undergo changes in supercoiling in the presence of radiation-induced damage revealed that in all cases the degree of inhibition of supercoil rewinding was greater in the radiosensitive cells. Since the amount of DNA damage induced per unit dose is known to be equal in all these cell lines, the same number of DNA lesions produced a greater loss of topological constraint in the radiosensitive cells. The differential loss of DNA supercoiling ability could be due to differences in DNA-nuclear matrix anchor points. High-resolution two-dimensional gel electrophoresis of nucleoid proteins showed numerous reproducible differences in nuclear matrix protein between the cell lines studied. A total of nine proteins were associated with nucleoids from L5178Y-R cells and absent from L5178Y-S nucleoids. None of them, however, correlated absolutely with radioresistance. Thus, unlike previous studies in CHO cells, no candidates for the conveyance of cellular radiosensitivity that were single proteins were detected. However, these results are consistent with the hypothesis that stability of DNA loop domains in the presence of DNA damage is a determinant of the outcome of radiation-induced DNA damage.

DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium.

Transcription from many bacterial promoters is sensitive to the level of DNA supercoiling. We have investigated the mechanism by which environmentally induced changes in DNA supercoiling might regulate transcription. For the proU promoter of Salmonella typhimurium, osmotically induced changes in DNA topology appear to play a primary regulatory role. Changes in DNA supercoiling (linking number; delta Lk) are partitioned into changes in the winding of the strands of the double helix about themselves (twist; delta Tw) and/or elastic deformations or flexibility of the DNA helix (writhe; delta Wr). Mutations of the proU promoter were isolated in vivo, or generated in vitro, which altered the spacing between the -10 and -35 motifs. Studies on these mutant promoters, both in vivo and in vitro, exclude models in which changes in DNA twist play a regulatory role. Instead, our data suggest that increased DNA flexibility, reflecting the osmotically induced increase in negative supercoiling of DNA, is required for promoter activation.