DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.     

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.     

DNA gyrase is a host factor required for transposition of Tn5

We show that DNA gyrase is required for transposition of Tn5. Coumermycin, a potent inhibitor of DNA gyrase subunit B, inhibits transposition in a wild-type strain, but has no effect on strains carry ing a coumermycin-resistant allele in gyrB. In addition, strains containing a thermolabile subunit A of gyrase (gyrA43) are defective for transposition at a nonpermissive temperature. The requirement for gyrase is due to a requirement for supercoiled DNA. We showed this by introducing into the gyrA43 strain a deletion of the gene encoding topoisomerase I. The introduction of the second mutation caused an increase in the superhelical density of DNA as well as an increase in the transposition frequency. This also implies that if the DNA is supercoiled there is no further requirement for gyrase. Experiments with coumermycin support this, because the drug does not inhibit transposition if the recipient DNA remains supercoiled. This indicates that if the DNA acting as recipient of the transposon is deficient in supercoils, it will be a poor substrate for transposition. We also describe a system in which a gene on a multicopy plasmid can be efficiently introduced into the Escherichia coli chromosome.     

An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity

Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli, typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP-dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.     

The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea

Abstract: Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5'-type I DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri , the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type I DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3'-type I DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.     

Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.     

Drug-induced relaxation of supercoiled plasmid DNA in Bacillus subtilis and induction of the SOS response.

Whereas treatment with many different drugs led to induction of the SOS response in Bacillus subtilis, only inhibitors of DNA gyrase subunit B and, unexpectedly, polyether antibiotics (membrane ionophores) led to relaxation of supercoiled plasmid DNA. However, treatment with DNA gyrase subunit B inhibitors but not with polyethers led to SOS induction. Thus, the presence of underwound supercoiled DNA was not sufficient to induce the SOS response. Possible mechanisms by which polyethers induce relaxation of supercoiled DNA in vivo are discussed.     

Dissection of the nucleotide cycle of B. subtilis DNA gyrase and its modulation by DNA

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. While all type II DNA topoisomerases relax supercoiled DNA, DNA gyrase is the only enyzme that introduces negative supercoils into DNA at the expense of ATP hydrolysis. We present here a biophysical characterization of the nucleotide cycle of DNA gyrase from Bacillus subtilis, both in the absence and presence of DNA. B. subtilis DNA gyrase is highly homologous to its well-studied Escherichia coli counterpart, but exhibits unique mechanistic features. The active heterotetramer of B. subtilis DNA gyrase is formed by mixing the GyrA and GyrB subunits. GyrB undergoes nucleotide-induced dimerization and is an ATP-operated clamp. The intrinsic ATPase activity of gyrase is stimulated tenfold in the presence of plasmid DNA. However, in contrast to the E. coli homolog, the rate-limiting step in the nucleotide cycle of B. subtilis GyrB is ATP hydrolysis, not product dissociation or an associated conformational change. Furthermore, there is no cooperativity between the two DNA and ATP binding sites in B. subtilis DNA gyrase. Nevertheless, the enzyme is as efficient in negative supercoiling as the E. coli DNA gyrase. Our results provide evidence that the evolutionary goal of efficient DNA supercoiling can be realized by similar architecture, but differences in the underlying mechanism. The basic mechanistic features are conserved among DNA gyrases, but the kinetics of individual steps can vary significantly even between closely related enzymes. This suggests that each topoisomerase represents a different solution to the complex reaction sequence in DNA supercoiling.     

Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque

E. coli DNA gyrase uses the energy of ATP hydrolysis to introduce essential negative supercoils into the genome, thereby working against the mechanical stresses that accumulate in supercoiled DNA. Using a magnetic-tweezers assay, we demonstrate that small changes in force and torque can switch gyrase among three distinct modes of activity. Under low mechanical stress, gyrase introduces negative supercoils by a mechanism that depends on DNA wrapping. Elevated tension or positive torque suppresses DNA wrapping, revealing a second mode of activity that resembles the activity of topoisomerase IV. This 'distal T-segment capture' mode results in active relaxation of left-handed braids and positive supercoils. A third mode is responsible for the ATP-independent relaxation of negative supercoils. We present a branched kinetic model that quantitatively accounts for all of our single-molecule results and agrees with existing biochemical data.     

DNA stability at temperatures typical for hyperthermophiles.

We have studied the fate of covalently-closed circular DNA in the temperature range from 95 to 107 degrees C. Supercoiled plasmid was not denatured up to the highest temperature tested. However, it was progressively transformed into open DNA by cleavage and then denatured. Thermodegradation was not dependent on the DNA supercoiling density. In particular, DNA made positively supercoiled by an archaeal reverse gyrase was not more resistant to depurination and thermodegradation than negatively supercoiled DNA. Thermodegradation was similar in aerobic or anaerobic conditions but strongly reduced in the presence of physiological concentrations of K+ or Mg2+. These results indicate that the major problem faced by covalently closed DNA in hyperthermophilic conditions is not thermodenaturation, but thermodegradation, and that intracellular salt concentration is important for stability of DNA primary structure. Our data suggest that reverse gyrase is not directly required to protect DNA against thermodegradation or thermodenaturation.     

Internal Dynamics of Supercoiled DNA Molecules

The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length.     

Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

Novobiocin induces positive supercoiling of small plasmids from halophilic archaebacteria in vivo.

The halophilic archaebacterium Halobacterium strain GRB harbours a multicopy plasmid of 1.7 kb which is negatively supercoiled. After addition of novobiocin to culture medium all 1.7 kb plasmid molecules become positively supercoiled. Positive supercoiling occurs at the same dose of novobiocin inhibiting the eubacterial DNA gyrase in vitro. Novobiocin also induces positive supercoiling of pHV2, a 6.3 kb plasmid from Halobacterium volcanii. These results indicate the existence of a mechanism producing positive superturns in halobacteria. The 1.7 kb plasmid from Halobacterium GRB could be used to produce high amounts of pure positively supercoiled DNA for biophysical and biochemical studies.     

Characterization of the Nucleoid-associated Protein YejK

Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait. The purified protein likely exists in a monomer-dimer equilibrium in solution and can form tetramers. Cross-linking of the protein bound to DNA suggests that the active form could be either a dimer or tetramer. YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12–14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds. YejK binds RNA, but not single-stranded DNA, with 65% of the avidity with which it binds DNA. However, cells deleted for yejK do not show defects in either RNA or protein synthesis. YejK interacts with all the subunits of both DNA gyrase and topoisomerase IV and has measurable effects on their activities. In the presence of YejK, relaxation of negatively supercoiled DNA by topoisomerase IV becomes distributive, whereas relaxation of positively supercoiled DNA is stimulated. Relaxation of negatively supercoiled DNA by DNA gyrase is inhibited, whereas the extent of supercoiling of relaxed DNA is limited. A YejK-GFP chimera is an effective marker for the nucleoid in live cell imaging.     

Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro.

Trichosanthin, an abortifacient, immunosuppressive and anti-tumor protein purified from the traditional Chinese herb medicine Tian Hua Fen, is a potent inhibitor against HIV-1 replication. Under normal enzymatic digestion conditions, trichosanthin cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA. Trichosanthin has no effect on linear double-stranded DNA. Neither does it convert relaxed circular duplex DNA into a supercoiled form in the presence of ATP. Thus trichosanthin is not a DNA gyrase. However, trichosanthin can cleave the relaxed circular DNA into a linear form, indicating that both the circular as well as the supercoiled forms are essential for trichosanthin recognition. In addition, trichosanthin contains one calcium metal ion per protein molecule, which presumably is related to its endonucleolytic activity.     

Nucleotide- and stoichiometry-dependent DNA supercoiling by reverse gyrase

Reverse gyrase is a unique type IA topoisomerase that can introduce positive supercoils into DNA. We have investigated some of the biochemical properties of Archaeoglobus fulgidus reverse gyrase. It can mediate three distinct supercoiling reactions depending on the adenine nucleotide cofactor that is present in the reaction. Besides the ATP-driven positive supercoiling reaction, the enzyme can introduce negative supercoils with a nonhydrolyzable analog, adenylyl imidodiphosphate. In the presence of ADP the plasmid DNA is relaxed almost completely, leaving a very low level of positive supercoiling. Surprisingly, the final supercoiling extent for all three distinct reactions depends on the stoichiometry of enzyme to DNA. This dependence is not due to the difference of reaction rate, suggesting that the amount of enzyme bound to DNA is an important determinant for the final supercoiling state of the reaction product. Reverse gyrase also displays exquisite sensitivity toward temperature. Raising the reaction temperatures from 80 to 85 degrees C, both of which are within the optimal growth temperature of A. fulgidus, greatly increases enzyme activity for all the supercoiling reactions. For the reaction with AMPPNP, the product is a hypernegatively supercoiled DNA. This dramatic enhancement of the reverse gyrase activity is also correlated with the appearance of DNA in a pre-melting state at 85 degrees C, likely due to the presence of extensively unwound regions in the plasmid. The possible mechanistic insights from these findings will be presented here.