Torsional tension in intracellular bacteriophage T4 DNA. Evidence that a linear DNA duplex can be supercoiled in vivo

Torsional tension in intracellular bacteriophage T4 DNA and host cell DNA was measured in infected Escherichia coli cells using the trimethylpsoralen photobinding assay. Early in infection superhelical tension in the host E. coli DNA was gradually reduced until at 8 min post-infection there was no detectable tension. Negative torsional tension in the T4 DNA appeared transiently, reaching a maximum 4 to 6 min post-infection (at 32 °C) and declined to undetectable levels by 10 min. The maximum level of tension averaged over all infecting T4 DNA molecules was equivalent to superhelical density of about σ = ?0.03.Sedimentation studies of the psoralen-associated T4 DNA isolated from infected cells at 5 min post-infection indicated that this DNA was primarily in an intact linear form. This is the first evidence indicating that a linear DNA molecule can acquire torsional tension in vivo: the finding suggests that intracellular T4 DNA can be topologically restrained probably by interaction with other structures in the cell. Effects of inhibitors of DNA gyrase and effects of mutations in T4 gene 39 indicated that the observed torsional tension was introduced by E. coli DNA gyrase, not by the T4 topoisomerase. Studies of the number of nicks required to relax the tension suggest that the entire T4 genome is organized into one topological domain of supercoiling.The possible role of the negative superhelical tension in the initiation of T4 DNA replication was examined. Initiation of DNA replication occurred shortly after the accumulation of tension in T4 DNA. However, replication occurred at near-normal levels under conditions where acquisition of tension was blocked in T4 DNA. Results suggest that although a topoisomerase activity is required for the initiation of DNA replication, the observed torsional tension is not a prerequisite.     

Relaxation of DNA torsional tension in defined domains of bacterial chromosomes in vivo

A procedure is described for selectively relaxing the DNA torsional tension in defined regions of the chromosome of living bacterial cells. Regions of the chromosomal DNA labelled with bromodeoxyuridine are selectively nicked by irradiation of the cells with long-wavelength ultraviolet light and then trimethylpsoralen residues are photobound to the chromosome in vivo. It is demonstrated that the rate of photobinding to the bromouridine-labelled parts of the chromosomes declines relative to the unlabelled parts of the same chromosomes as nicks are introduced into the former regions. The maximal difference in photobinding rates is that expected for the difference between relaxed and negatively supercoiled DNA. Analysis of the number of DNA breaks required for minimizing the photobinding rates permits a calculation of the number of domains of supercoiling per Bacillus subtilis chromosome.     

Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells

The rate of covalent photobinding of trimethylpsoralen to DNA is greater when the DNA is wound with negative superhelical tension than when it is relaxed. In vitro the rate of photobinding is directly proportional to the negative superhelical density of the DNA. Thus measurement of the rate of photobinding provides an assay for probing in vivo unrestrained tension in the winding of the DNA double helix. This approach has been applied to measure torsional tension in DNA as it is packaged in living E. coli. Drosophila and HeLa cells. A method is described for measuring the rate of photobinding to intracellular DNA and rRNA, and for using the latter measurement as an internal control of the rate of me3-psoralen photobinding in vivo. This permits more accurate and reproducible measurement of changes in the DNA-psoralen photobinding reaction. The me3-psoralen probe interacts with intracellular bacterial DNA as expected for a purified DNA duplex wound with superhelical density sigma = -0.05 +/- 0.01. This superhelical tension is relaxed in cells when multiple single-strand breaks are introduced into the chromosomal DNA by gamma-irradiation. Similar relaxation occurs when cells are treated with the DNA gyrase inhibitor coumermycin. The results suggest that the DNA double helix is wound with torsional tension in vivo and that DNA supercoils which are equilibrated with this tension are not completely restrained in nucleosome-like structures. Torsional tension in the DNA of eucaryotic cells is not detectable in analogous measurements of the packaged DNA of HeLa and Drosophila cells. The simplest interpretation of this finding is that, within the limits of detection, all superhelical turns in the DNA are restrained in nucleosomes or nucleosome-like structures in these eucaryotic cells.     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.     

Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.     

Role of HU proteins in forming and constraining supercoils of chromosomal DNA inEscherichia coli

Induction of supercoiling in plasmid DNA by HU heterotypic and homotypic dimers, a mutant HU-2 (HupAN12), HBs and HB1 proteins with different DNA-binding affinities was investigated in vitro. The abilities of these proteins to induce supercoiling in DNA correlated with their affinities for DNA. Stoichiometrical analysis of HU heterodimers bound to DNA in the complex restraining the negative torsional tension of DNA showed that 12–13 dimers account for a single superhelical turn. The number of supercoils in the plasmid in vivo decreased on inhibition of DNA gyrase with coumermycin, reaching a steady-state level that indicated the existence of a compartment of restrained supercoils. The size of the restrained compartment was reduced in the absence of HU, indicating the participation of HU in constituting this fraction, and was larger on overproduction of HU-2 in the cells. An increased level of DNA gyrase, expressed from a plasmid carrying bothgyr genes, in the cells did not compensate for the deficit of the restrained supercoils caused by HU deficiency, indicating seeming distinct and unrelated action of HU and DNA gyrase in introducing and constraining supercoiling of intracellular DNA.     

Cross-linking and relaxation of supercoiled DNA by psoralen and light.

Photoreaction of 4,5',8-trimethylpsoralen with superhelical ColE1 and ColE1amp DNA was studied. Changes in mobilities in agarose gels, formation of interstrand cross-links, and DNA strand breaks were determined. Psoralen and light treatment removed negative superhelical turns, and extensive treatments failed to produce positive superhelical turns in covalently closed plasmid DNA. The rate of relaxation of superhelical turns by psoralen Photobinding appeared to be directly proportional to the number of superhelical turns remaining. A unique reaction mechanism is presented to explain these results. By this interpretation the initial rate of psoralen photobinding to superhelical DNA was estimated to be 3 times that for linear DNA, and the ratio of cross-linking to monofuctional adducts appears to be dependent on the superhelical conformation of the DNA. The estimated ratio of psoralen molecules bound to DNA strand breaks was 1.7 . 10(4):1, and 70% of this breakage is caused by the light alone.     

Control of bacterial DNA supercoiling

Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, supercoiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady-state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.     

Effects of supercoiling on enhancer–promoter contacts

Using Brownian dynamics simulations, we investigate here one of possible roles of supercoiling within topological domains constituting interphase chromosomes of higher eukaryotes. We analysed how supercoiling affects the interaction between enhancers and promoters that are located in the same or in neighbouring topological domains. We show here that enhancer–promoter affinity and supercoiling act synergistically in increasing the fraction of time during which enhancer and promoter stay in contact. This stabilizing effect of supercoiling only acts on enhancers and promoters located in the same topological domain. We propose that the primary role of recently observed supercoiling of topological domains in interphase chromosomes of higher eukaryotes is to assure that enhancers contact almost exclusively their cognate promoters located in the same topological domain and avoid contacts with very similar promoters but located in neighbouring topological domains.     

DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor.

We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/- 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = -0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle-repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non-integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.     

Coarse-grained modeling reveals the impact of supercoiling and loop length in DNA looping kinetics

Measurements of protein-mediated DNA looping reveal that in vivo conditions favor the formation of loops shorter than those that occur in vitro, yet the precise physical mechanisms underlying this shift remain unclear. To understand the extent to which in vivo supercoiling may explain these shifts, we develop a theoretical model based on coarse-grained molecular simulation and analytical transition state theory, enabling us to map out looping energetics and kinetics as a function of two key biophysical parameters: superhelical density and loop length. We show that loops on the scale of a persistence length respond to supercoiling over a much wider range of superhelical densities and to a larger extent than longer loops. This effect arises from a tendency for loops to be centered on the plectonemic end region, which bends progressively more tightly with superhelical density. This trend reveals a mechanism by which supercoiling favors shorter loop lengths. In addition, our model predicts a complex kinetic response to supercoiling for a given loop length, governed by a competition between an enhanced rate of looping due to torsional buckling and a reduction in looping rate due to chain straightening as the plectoneme tightens at higher superhelical densities. Together, these effects lead to a flattening of the kinetic response to supercoiling within the physiological range for all but the shortest loops. Using experimental estimates for in vivo superhelical densities, we discuss our model’s ability to explain available looping data, highlighting both the importance of supercoiling as a regulatory force in genetics and the additional complexities of looping phenomena in vivo.     

Effects of salt and temperature on plasmid topology in the halophilic archaeon Haloferax volcanii.

We report here the effect of environmental parameters, salinity, temperature, and an intercalating drug on plasmid topology in the halophilic archaeon Haloferax volcanii. We first studied the topological state of the plasmid pHV11 in media of different salt compositions and concentrations. The superhelical density of plasmid PHV11 varies in a way that depends on the kind of salt and on the concentrations of individual salts. With respect to growth temperature, the plasmid linking number increased at higher temperature in a linear way, contrary to what has been reported for Escherichia coli, in which the plasmid linking number decreased at higher temperature. These results suggest that some of the mechanisms that control DNA supercoiling in halophilic Archaea may be different from those described for E. coli. However, homeostatic control of DNA supercoiling seems to occur in haloarchaea, as in Bacteria, since we found that relaxation of DNA by chloroquine triggers an increase in negative supercoiling.     

Hyperreactivity of B-Z junctions to 4,5',8-trimethylpsoralen photobinding assayed by an exonuclease III/photoreversal mapping procedure

We have developed an exonuclease III/photoreversal procedure to map, with base-pair resolution, the bases that have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand crosslinks in DNA. This assay allows quantification of relative rates of Me3-psoralen photobinding to bases in DNA at levels less than one crosslink per 8000 base-pairs. We demonstrate the applicability of the Me3-psoralen mapping procedure on the Z-forming sequence GAATT(CG)6-TA(CG)6AATTC. The results confirm our previous findings that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation. In addition, with increasing superhelical density we observe at least a hundred-fold increased Me3-psoralen presumably represent B-Z junctions. The two presumed junctions respond differently with increasing negative superhelical tension, however, suggesting that the structures of these negative superhelical tension, however, suggesting that the structures of these junctions differ. This increased Me3-psoralen photoreactivity provides a positive signal for the presence of Z-DNA. The sequence and assay described here provide a "torsionally tuned probe" for determining the effective superhelical density of DNA in vivo.     

The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA

The Escherichia coli DNA architectural protein FIS is a pleiotropic regulator, which couples the cellular physiology with transitions in the superhelical density of bacterial DNA. Recently, we have shown that this effect is in part mediated via DNA gyrase, the major cellular topoisomerase responsible for the elevation of negative supercoiling. Here, we demonstrate that, in turn, the expression of the fis gene strongly responds to alterations in the topology of DNA in vivo, being maximal at high levels of negative supercoiling. Any deviations from these optimal levels decrease fis promoter activity. This strict dependence of fis expression on the superhelical density suggests that fis may be involved in 'fine-tuning' the homeostatic control mechanism of DNA supercoiling in E. coli.