Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase. This enzyme has been purified from Escherichia coli cells. The reaction requires ATP and Mg++ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin El, phage X, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA. In the course of studies on integrative recombination of phage X DNA in a cell-free system from Escherichia coli (1, 2) we became aware that the process required a negatively supercoiled DNA substrate. This substrate could be replaced by relaxed closed-circular DNA only if the latter was incubated with an E. cali cell fraction and ATP (K. Mizuuchi, M. Gellert, and H. Nash, manuscript in preparation). The simplest interpretation of these results was that the E. coli extract contains an ATP-dependent activity capable of converting relaxed closed-circular DNA to the supercoiled form. We have obtained a considerable purification of this enzyme, which we call DNA gyrase, and report here the purification procedure and preliminary characterization of the enzyme. was prepared by sealing hydrogen-bonded circular DNA with DNA ligase (2). These DNA samples were deproteinized by shaking with chloroform-isoamyl alcohol (24:1 vol/vol) and repurified by centrifugation in cesium chloride-ethidium bromide density gradients. All DNA samples were dialyzed and stored in 0.01 M Tris.HCl at pH 8.0, 1 mM Na3EDTA, at 4°. MATERIALS AND METHODSAssay of DNA Supercoiling. The assay measures the conversion of relaxed closed-circular Col El DNA to the supercoiled form, as demonstrated by agarose gel electrophoresis. The standard reaction mixture (70,ul) contained 35 mM TrisHCl at pH 7.5, 1.6 mM MgCl2, 18 mM potassium phosphate at pH 7.5, 5 mM spermidine-HCl, 1.4 mM ATP, 90 jig/ml of E. coli tRNA (Calbiochem), 3.6 mg/ml of bovine serum albumin (Armour, crystalline), and 0.4 ,g of relaxed covalently circular Col El DNA. Enzyme (1-5 ,l) was diluted when necessary into 0.2 M potassium phosphate at pH 6.8, 1 mM Na3EDTA, 1 mM dithiothreitol, 10% glycerol (wt/vol), and 3.6 mg/ml of bovine serum albumin.The solution was incubated at 250 for 60 min, and then extracted with an equal volume of chloroform-isoamyl alcohol (24:1 vol/vol). After brief centrifugation, 50 ,l of the aqueous phase were added to 12.5 gl of a mixture of 5% sodium dodecyl sulfate, 25% glycerol, and 0.25 mg/ml of bromphenol blue, and the sample was loaded onto an agarose gel. Up to 30 samples at a time were electrophoresed in a 6 X 230 X 160 mm slab (E-C Apparatus Corp.) of 0.8% agarose (Type II, Sigma Chemical Co.) with Tris-borate-EDTA buffer (10.8 g of Tris base, 5.5 g of boric acid, and 0.93 g of Na2EDTA per liter). After 16 hr of electrophoresis at 40 V, the slab was stained with 1 gg/ml of ethidium bromide in the electrophoresis buffer for 1 hr, and destained in electrophoresis buffer for at least an hour. The slab was photographed using t...

ATP-dependent DNA supercoiling catalyzed by Escherichia coli DNA gyrase was inhibited by oxolinic acid, a compound similar to but more potent than nalidixic acid and a known inhibitor of DNA replication in E. coil. The supercoiling activity of DNA gyrase purified from nalidixic acidresistant mutant (naL4U) bacteria was resistant to oxolinic acid. Thus, the nalA locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (cou). Supercoiling of X DNA in E. coli cells was likewise inhibited by oxolinic acid, but was resistant in the nalAR mutant. The inhibition by oxolinic acid of colicin El plasmid DNA synthesis in a cell-free system was largely relieved by adding resistant DNA gyrase.In the absence of ATP, DNA gyrase preparations relaxed supercoiled DNA; this activity was also inhibited by oxolinic acid, but not by novobiocin. It appears that the oxolinic acid-sensitive component of DNA gyrase is involved in the nicking-closing activity required in the supercoiling reaction. In the presence of oxolinic acid, DNA gyrase forms a complex with DNA, which can be activated by later treatment with sodium dodecyl sulfate and a protease to produce double-strand breaks in the DNA. This process has some similarities to the known properties of relaxation complexes. Previous work (1-3) has described an enzyme activity, DNA gyrase, that is responsible for the supercoiling of DNA in Escherichia colh. As isolated from extracts of E. colh, the enzyme introduces negative superhelical turns into covalently closed circular DNA in an ATP-dependent reaction; the hydrolysis of ATP presumably provides the free energy needed to accumulate mechanical strain energy in the DNA.One genetic locus (cou), which determines resistance to coumermycin A1 and novobiocin, has been identified as controlling the activity of DNA gyrase (2). The enzyme isolated from wild-type cells is inhibited by both these antibiotics, while DNA gyrase from a coumermycin-resistant mutant strain is unaffected. Intracellular DNA supercoiling is similarly blocked by coumermycin.In this paper we report the involvement of a second genetic locus (nalA), which determines resistance to nalidixic acid and oxolinic acid (4, 5), in controlling DNA gyrase activity. These two drugs are inhibitors of DNA replication in E. coli (4, 5). They also inhibit replication in cell-free systems of colicin El plasmid (ColEl) DNA (6, 7) and of phage qX174 replicative form DNA (8), but they do not inhibit the synthesis of the complementary strand of /X174 single-stranded DNA (8). These properties are parallel to those described for coumermycin A1 and novobiocin.Nalidixic acid-resistant mutants of two classes have been identified and mapped (9). Mutations at one locus (naIB, 57 min on the standard E. coli map) are responsible for low-level resistance and have been characterized as interfering with the permeability of the cells to nalidixic acid. Mutations ...

Novobiocin and coumermycin are known to inhibit the replication of DNA in Escherichia coli. We show that these drugs inhibit the supercoiling of DNA catalyzed by E. coli DNA gyrase, a recently discovered enzyme that introduces negative superhelical turns into covalently circular DNA. The activity of DNA gyrase purified from a coumermycin-resistant mutant strain is resistant to both drugs. The inhibition by novobiocin of colicin El plasmid DNA replication in a cell-free system is partially relieved by adding resistant DNA gyrase. Both in the case of colicin El DNA in E. coli extracts and of phage X DNA in whole cells, DNA molecules which are converted to the covalently circular form in the presence of coumermycin remain relaxed, instead of achieving their normal supercoiled conformation. We conclude that DNA gyrase controls the supercoiling of DNA in E. coli.Novobiocin and the related drug coumermycin are preferential inhibitors of DNA replication in intact Escherichia coli cells (1, 2). In toluenized cells of E. coli, these drugs inhibit replicative DNA synthesis but not repair synthesis (3, 4). They are also effective inhibitors in cell-free systems for the replication of colicin El (Col El) DNA (5) and of phage OX174 replicative form DNA (6), but they do not inhibit the synthesis of the complementary strand of OX174 single-stranded DNA (6).Coumermycin-resistant mutants of E. coli have been isolated, and the mutation has been mapped near the dnaA locus (2).An enzyme, DNA gyrase, that introduces negative superhelical turns into double-stranded closed circular DNA, has recently been purified from E. coli (7). In this paper, we show that DNA gyrase activity in vitro as well as in vivo is specifically inhibited by coumermycin and novobiocin. Among several spontaneous coumermycin-resistant mutants of NI708 which were tested, all gave resistant extracts for the in vitro Col El DNA replication system. One of these mutants, N1741, was chosen for further work because of its good growth. This strain apparently has a partial reversion of the permeability mutation of N1708. The couR mutation of NI741 was transferred, by phage P1 cotransduction with dnaA+, to strain CRT46 dnaA (10). The resulting strain, N1748, was used as a source for purification of drug-resistant DNA gyrase. DNA gyrase sensitive to both novobiocin and coumermycin was isolated from N99 recB21 (7). Strain N1071(Xind-) (11) was used for experiments of superinfection by phage X, and strain YSl (8) for testing supercoiling of Col El DNA in extracts. MATERIALSOf these strains, the coumermycin-resistant isolates NI741 and N1748 are able to grow in liquid culture containing 60 gg/ml of coumermycin; growth of the other strains is blocked by 15 gg/ml of the drug.Chemicals. Novobiocin was obtained from Sigma Chemical Co. Samples of coumermycin A1 (referred to as coumermycin throughout this paper) were gifts from W. F. Minor (Bristol Laboratories) and J. Davies (University of Wisconsin). Sources of other materials have been described previously (7,8,12).Meth...

DNA gyrase from Escherichia coli, in the presence of ATP, can both separate catenated DNA: circles and unknot knotted DNA. Both these reactions require passage of a DNA segment through a transient double-strand break in DNA. Evidence that transient double-strand breaks are also involved in the supercoiling and relaxing activities of DNA gyrase is derived from experiments showing that the linking number of circular DNA is changed in steps of two. A mechanism is proposed for the action of the enzyme. We have investigated the action of DNA gyrase on catenated (interlocked) and knotted DNA molecules. It was previously reported that catenanes are produced in a cell-free system by integrative recombination between X phage attachment sites contained in the same supercoiled DNA molecule (1). In the present work, this reaction has been used to make either catenated or knotted circular DNA molecules. The relative orientation of the attachment sites in the substrate DNA determines whether the product is exclusively a knotted circular DNA molecule or a pair of catenated DNA circles. We show here that DNA gyrase from Escherichia coli (2) is able both to separate catenated circles and to unknot knotted DNA. Unknotting of knotted DNA made by other means has recently been reported by Liu et al. (3,4).Unknotting and unlinking of catenanes of double-stranded circular DNA, giving circular products, can occur only by passage of a DNA segment through a transient double-strand break, which is then resealed. It therefore seemed possible that such a mechanism is utilized in the supercoiling reaction catalyzed by DNA gyrase. A test of this class of mechanisms is that the linking number of the DNA should change in steps of two (5), whereas mechanisms involving single-strand breakage would predict unit changes of linking number. We find that DNA gyrase-catalyzed supercoiling and relaxation do indeed change linking numbers in steps of two, and we propose a mechanistic model for the enzyme. Similar experiments have been reported recently for phage T4 DNA topoisomerase (4) and, since the completion of this work, for DNA gyrase (6). MATERIALS AND METHODSEnzymes and Substrates. DNA gyrase A and B proteins were purified separately from E. coli strains in which the gyrA and gyrB genes had been cloned on plasmids (unpublished results). Both protein preparations were homogeneous (>99%), and each protein had a specific activity in the supercoiling assay (7) of about 1 X 10r' units/mg in the presence of an excess of the other. Pancreatic DNase was obtained from Worthington, and restriction endonucleases from New England BioLabs.The supercoiled DNA of plasmid pBR322 (8) and its relaxed form were prepared by standard methods (2, 9). Supercoiled and relaxed DNA samples with a single linking number were purified by agarose gel electrophoresis in the presence of chloroquine (10) (0.9 ,ug/ml for relaxed DNA, 8 ,g/ml for supercoiled DNA). Single bands were excised from the gel, and the DNA was electrophoretically eluted and purified by adsorption ...