Level Of Supercoiling Research Articles

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine ( CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.

Real-Time Detection of Cruciform Extrusion by Single-Molecule DNA Nanomanipulation

Cruciform extrusion in dsDNA can occur when a DNA palindrome is subjected to physiological levels of negative supercoiling. Here we use single-DNA nanomanipulation to explore the kinetic and structural properties of cruciform extrusion induced by negative supercoiling. Cruciform extrusion appears as an abrupt increase in the extension of negatively supercoiled DNA, and the amplitude of the change in extension is proportional to the number of bases in the cruciform. The kinetics of this two-state system, B-DNA and cruciform DNA, can be tuned by negative supercoiling which destabilizes the former and stabilizes the latter. The rate of extrusion is controlled by the size of the apical loop, decreasing as the loop size is increased from 5 to 8 bases. Cruciform rewinding is controlled by features in the stem. Perfect cruciforms will tend to extrude irreversibly, whereas shortening and addition of imperfections to a stem can render extrusion reversible. From measurements of the effect of torque on extrusion/rewinding kinetics we propose that in the transition state to cruciform extrusion the palindrome is unwound in the unpaired loop region of the cruciform. These results provide insight into the mechanism of cruciform extrusion and help to understand the potential role of these structures in processes of genomic instability as well as those underpinning the synthesis of non-coding RNAs.

Nucleosome Assembly Depends on the Torsion in the DNA Molecule: A Magnetic Tweezers Study

We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers. To verify our hypothesis that the lack of full nucleosome assembly on topologically constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we carried out experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Indeed, such rotation did lead to the same nucleosome saturation level as in the case of nicked tethers. We conclude that levels of positive supercoiling in the range of 0.025–0.051 (most probably in the form of twist) stall the nucleosome assembly process.

Mwr Xer site-specific recombination is hypersensitive to DNA supercoiling

The multiresistance plasmid pJHCMW1, first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis, includes a Xer recombination site, mwr, with unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium. An increase in supercoiling density of plasmid DNA was observed as the osmotic pressure of the growth culture decreased. Reporter plasmids containing directly repeated mwr, or the related cer sites were used to test if DNA topological changes were correlated with significant changes in efficiency of Xer recombination. Quantification of Holliday junctions showed that while recombination at cer was efficient at all levels of negative supercoiling, recombination at mwr became markedly less efficient as the level of supercoiling was reduced. These results support a model in which modifications at the level of supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites.

Human genomic Z-DNA segments probed by the Zα domain of ADAR1

Double-stranded DNA is a dynamic molecule that adopts different secondary structures. Experimental evidence indicates Z-DNA plays roles in DNA transactions such as transcription, chromatin remodeling and recombination. Furthermore, our computational analysis revealed that sequences with high Z-DNA forming potential at moderate levels of DNA supercoiling are enriched in human promoter regions. However, the actual distribution of Z-DNA segments in genomes of mammalian cells has been elusive due to the unstable nature of Z-DNA and lack of specific probes. Here we present a first human genome map of most stable Z-DNA segments obtained with A549 tumor cells. We used the Z-DNA binding domain, Zα, of the RNA editing enzyme ADAR1 as probe in conjunction with a novel chromatin affinity precipitation strategy. By applying stringent selection criteria, we identified 186 genomic Z-DNA hotspots. Interestingly, 46 hotspots were located in centromeres of 13 human chromosomes. There was a very strong correlation between these hotspots and high densities of single nucleotide polymorphism. Our study indicates that genetic instability and rapid evolution of human centromeres might, at least in part, be driven by Z-DNA segments. Contrary to in silico predictions, however, we found that only two of the 186 hotspots were located in promoter regions.

DNA Conformation and Biomolecular Motors: New Nanomedicine Research Targets

DNA supercoiling is a feature of almost all DNA molecules. It is a powerful thermodynamic force that drives and directs many DNA associated processes in vivo. The level of supercoiling or DNA spatial conformation is constantly changing due to the activities of proteins and the environmental conditions of the cell. Local and temporal changes in DNA supercoiling affect many cellular processes such as replication, transcription recombination and chromosome organization.DNA biomolecular motors such as DNA topoisomerases and DNA translocases are responsible for maintaining the steady state of supercoiling essential for cell viability. In prokaryotes, DNA supercoiling is expected to play an important role in site-specific recombination, a fundamental process to achieve resolution of dimeric chromosomes, allowing plasmids and chromosome segregation and consequently cell division. During this process, DNA undergoes multiple conformational changes due to the activity of Tyrosine recombinases and a DNA translocase known as FtsK.I use cell biology, biochemical and biophysical techniques to study the role of DNA biomolecular motors and DNA topology in different cellular processes. In vitro, we demonstrate the topology dependence of the different steps in site-specific recombination events using DNA substrates with different superhelical density. By TIRFM, I characterize at the level of single molecule the activity of DNA molecular motors. Using high-resolution amplitude modulation atomic force microscopy (AM-AFM) in physiological buffer we characterize the nature of the forces that drive relevant DNA conformational changes by itself or after protein interaction during site-specific recombination events. Additionally, we observe for the first time the dynamics of DNA and the conformational changes of DNA during site-specific recombination events imaged by high-speed AFM at time resolutions up to 20 ms and sub-nm spatial resolution. Our current research is focus on DNA biomolecular motors as new nanomedice targets.

Topoisomerase 3α and RMI1 Suppress Somatic Crossovers and Are Essential for Resolution of Meiotic Recombination Intermediates in Arabidopsis thaliana

Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3α (TOP3α) functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3α in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3α-2), which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3α-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3α, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3α is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3α mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3α and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.

How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

DNA topoisomerases control the topology of DNA (e.g., the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e., more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower-than-equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analyzed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the supercoil-sensing C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topoisomerase VI (which is only distantly related to type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range ∼2–9 kbp and is not altered by reducing the free energy available from ATP hydrolysis by varying the ADP:ATP ratio. A direct test of one model (DNA tracking; i.e., sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect, but that it is possible that other kinetic factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance.

Impact of DNA gyrase inhibition by antisense ribozymes on rec A in E. coli

The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluoroquinolones.

DNA Supercoiling-Dependent Gene Regulation in Chlamydia

The intracellular pathogen Chlamydia has an unusual developmental cycle marked by temporal expression patterns whose mechanisms of regulation are largely unknown. To examine if DNA topology can regulate chlamydial gene expression, we tested the in vitro activity of five chlamydial promoters at different superhelical densities. We demonstrated for the first time that individual chlamydial promoters show a differential response to changes in DNA supercoiling that correlates with the temporal expression pattern. The promoters for two midcycle genes, ompA and pgk, were responsive to alterations in supercoiling, and promoter activity could be regulated more than eightfold. In contrast, the promoters for three late transcripts, omcAB, hctA, and ltuB, were relatively insensitive to supercoiling, with promoter activity varying by no more than 2.2-fold over a range of superhelicities. To obtain a measure of how DNA supercoiling levels vary during the chlamydial developmental cycle, we recovered the cryptic chlamydial plasmid at different times after infection and assayed its superhelical density. The chlamydial plasmid was most negatively supercoiled at midcycle, with an approximate superhelical density of -0.07. At early and late times, the plasmid was more relaxed, with an approximate superhelicity of -0.03. Thus, we found a correlation between the responsiveness to supercoiling shown by the two midcycle promoters and the increased level of negative supercoiling during mid time points in the developmental cycle. Our results support a model in which the response of individual promoters to alterations in DNA supercoiling can provide a mechanism for global patterns of temporal gene expression in Chlamydia.

II. Correlations between secondary structure stability and mutation frequency during somatic hypermutation

The role of secondary structures and base mutability at different levels of transcription and supercoiling is analyzed in variable region antibody genes VH5, VH94 and VH186.2. The data are consistent with a model of somatic hypermutation in which increasing levels of transcription and secondary structure stability correlate with the initial formation of successive mutable sites. Encoded differences exist in stem length and the number of GC pairs at low versus high levels of transcription in CDRs. These circumstances simplify the complexities of coordinating mutagenesis by confining this process to each mutable site successively, as they form in response to increasing levels of transcription during affinity maturation.

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.

DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites

The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.

Aerobic TMAO respiration in Escherichia coli

In the absence of oxygen, Escherichia coli can use alternative exogenous electron acceptors, including trimethylamine oxide (TMAO), to generate energy. In this study, we showed that in contrast to the other anaerobic respiratory systems, the TMAO reductase (Tor) system was expressed during both aerobiosis and anaerobiosis. By using a torA-lacZ fusion and quantitative reverse transcription polymerase chain reaction, we established that the torCAD operon encoding the Tor system was induced in the presence of TMAO mainly during exponential phase, and that optimal induction required a certain level of DNA supercoiling. We also showed that the presence of oxygen prevented neither the biogenesis of the Tor system nor the reduction of TMAO. The physiological role of TMAO reduction during aerobiosis has not been yet established, but our experiments suggest that alkaline TMA production could enhance the growth conditions by increasing the pH of the culture.

SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG)n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG)n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS– strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG)n, (CGG/CCG)n and (GAA/TTC)n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.

Secondary structures as predictors of mutation potential in the lacZ gene of Escherichia coli

Four independent nonsense mutations were engineered into the Escherichia coli chromosomal lacZ gene, and reversion rates back to LacZ(+) phenotypes were determined. The mutation potential of bases within putative DNA secondary structures formed during transcription was predicted by a sliding-window analysis that simulates successive folding of the ssDNA creating these structures. The relative base mutabilities predicted by the mfg computer program correlated with experimentally determined reversion rates in three of the four mutants analysed. The nucleotide changes in revertants at one nonsense codon site consisted of a triple mutation, presumed to occur by a templated repair mechanism. Additionally, the effect of supercoiling on mutation was investigated and, in general, reversion rates increased with higher levels of negative supercoiling. Evidence indicates that predicted secondary structures are in fact formed in vivo and that directed mutation in response to starvation stress is dependent upon the exposure of particular bases, the stability of the structures in which these bases are unpaired and the level of DNA supercoiling within the cell.

Thirty years of Escherichia coli DNA gyrase: From in vivo function to single-molecule mechanism

The level of negative DNA supercoiling of the Escherichia coli chromosome is tightly regulated in the cell and influences many DNA metabolic processes including DNA replication, transcription, repair and recombination. Gyrase is the only type II topoisomerase able to introduce negative supercoils into DNA, a unique ability that arises from the specialized C-terminal DNA wrapping domain of the GyrA subunit. Here, we review the biological roles of gyrase in vivo and its mechanism in vitro.

Chromatin structure can strongly facilitate enhancer action over a distance

Numerous DNA transactions in eukaryotic nuclei are regulated by elements (enhancers) that can directly interact with their targets over large regions of DNA organized into chromatin. The mechanisms allowing communication over a distance in chromatin are unknown. We have established an experimental system allowing quantitative analysis of the impact of chromatin structure on distant transcriptional regulation. Assembly of relaxed or linear DNA templates into subsaturated chromatin results in a strong increase of the efficiency of distant enhancer-promoter E-P communication and activation of transcription. The effect is directly proportional to the efficiency of chromatin assembly and cannot be explained only by DNA compaction. Transcription activation on chromatin templates is enhancer- and activator-dependent, and must be accompanied by direct E-P interaction and formation of a chromatin loop. Previously we have shown that DNA supercoiling can strongly facilitate E-P communication on histone-free DNA. The effects of chromatin assembly and DNA supercoiling on the communication are quantitatively similar, but the efficiency of enhancer action in subsaturated chromatin does not depend on the level of unconstrained DNA supercoiling. Thus chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state characteristic for prokaryotic DNA.

Roles for DNA supercoiling and the Fis protein in modulating expression of virulence genes during intracellular growth of Salmonella enterica serovar Typhimurium

Adaptation of bacterial pathogens to an intracellular environment requires resetting of the expression levels of a wide range of both virulence and housekeeping genes. We investigated the possibility that changes in DNA supercoiling could modulate the expression of genes known to be important in the intracellular growth of the pathogen Salmonella enterica serovar Typhimurium. Our data show that DNA becomes relaxed when Salmonella grows in murine macrophage but not in epithelial cells, indicating that DNA supercoiling plays a role in discrimination between two types of intracellular environment. The ssrA regulatory gene within the SPI-2 pathogenicity island that is required for survival in macrophage was found to be upregulated by DNA relaxation. This enhancement of expression also required the Fis nucleoid-associated protein. Manipulating the level of the Fis protein modulated both the level of DNA supercoiling and ssrA transcription. We discuss a model of bacterial intracellular adaptation in which Fis and DNA supercoiling collaborate to fine-tune virulence gene expression.

SOS Repair and DNA Supercoiling Influence the Genetic Stability of DNA Triplet Repeats in Escherichia coli

Molecular mechanisms responsible for the genetic instability of DNA trinucleotide sequences (TRS) account for at least 20 human hereditary disorders. Many aspects of DNA metabolism influence the frequency of length changes in such repeats. Herein, we demonstrate that expression of Escherichia coli SOS repair proteins dramatically decreases the genetic stability of long (CTG/CAG) n tracts contained in plasmids. Furthermore, the growth characteristics of the bacteria are affected by the (CTG/CAG) n tract, with the effect dependent on the length of the TRS. In an E. coli host strain with constitutive expression of the SOS regulon, the frequency of deletions to the repeat is substantially higher than that in a strain with no SOS response. Analyses of the topology of reporter plasmids isolated from the SOS+ and SOS– strains revealed higher levels of negative supercoiling in strains with the constitutively expressed SOS network. Hence, we used strains with mutations in topoisomerases to examine the effect of DNA topology upon the TRS instability. Higher levels of negative DNA supercoiling correlated with increased deletions in long (CTG/CAG) n, (CGG/CCG) n and (GAA/TTC) n. These observations suggest a link between the induction of bacterial SOS repair, changes in DNA topology and the mechanisms leading to genetic instability of repetitive DNA sequences.