Proc. Natl. Acad. Sci. USA Vol. 75, No.4, pp. 1773-1777, April 1978 Biochemistry Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity (DNA supercoiling/DNA relaxation/novobiocin/nalidixic acid/DNA topoisomerase II) N. PATRICK HIGGINS*, CRAIG L. PEEBLESt, AKIO SUGINO*f, AND NICHOLAS R. COZZARELLI*t * Departments of Biochemistry and t Biophysics and Theoretical Biology, The University of Chicago, Chicago, Illinois 60637; and t National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709. Communicated by Donald F. Steiner, February 9,1978 ABSTRACT Extensively purified DNA gyrase from Esch- and a free pool of cou coded subunits might exist to comple- erichia coli is inhibited by nalidixic acid and by novobiocin. ment the free pool of nalA subunits. The enzyme is composed of two subunits, A and B, which were We now that Pnal is a subunit purified as separate components. Subunit A is the product of the have proven of DNA gyrase gene controlling sensitivity to nalidixic acid (nal4) because: (I) and that the subunit controlled by the cou gene can also be the electrophoretic mobility of subunit A in the presence of so- purified independently. Each subunit contains one of two dium dodecyl sulfate is identical to that of the 105,000-dalton polypeptides found in extensively purified gyrase. Optimal nalA gene product; (ii) mutants that are resistant to nalidixic reconstitution of all four activities of DNA gyrase requires both acid (nalA r) produce a drug-resistant subunit A; and (iii) wild- subunits. type subunit A confers drug sensitivity to in vitro synthesis of 4X174 DNA directed by nalA r mutants. Subunit B contains a MATERIALS AND METHODS 95,000-dalton polypeptide and is controlled by the gene speci- fying sensitivity to novobiocin (cou) because cou' mutants Bacteria. The strains used were E. coil H560 polA endA, produce a novobiocin-resistant subunit B and novobiocin-resi- H560-1 polA endA nalAr constructed by R. Sternglanz (6), and tant gyrase is made drug sensitive by wild-type subunit B. Sub- N1748 cour (5). units A and B associate, so that gyrase was also purified as a Enzyme Assays. The Pnal assay was essentially that devised complex containing 105,000- and 95,000-dalton polypeptides. This enzyme and gyrase reconstructed from subunits have the by C. Sumida-Yasumoto and measures conferral of Oxo sensi- same drug sensitivity, K, for ATP, and catalytic properties. The tivity to OX174 DNA replication (6). Supertwisting activity of same ratio of subunits gives efficient reconstitution of the re- DNA gyrase was measured at 300 in a 17-Ml reaction mixture actions intrinsic to DNA gyrase, including catalysis of super- containing 35 mM Tris-HCl (pH 7.6), 18 mM potassium phos- coiling of closed duplex DNA, relaxation of supercoiled DNA phate, 5 mM dithiothreitol, 6.7 mM MgCl2, 5 mM spermi- in the absence of ATP, and site-specific cleavage of DNA in- dine-HCl, 50 Ag of bovine serum albumin per ml, 1.5 mM ATP, duced by sodium dodecyl sulfate. and 23 fmol of relaxed ColEl DNA. The reaction was stopped DNA gyrase [Eco DNA topoisomerase II (1)] is an ATP-re- (6) and the products were displayed by 1% agarose gel elec- quiring enzyme that introduces negative supertwists into closed trophoresis, stained with ethidium bromide, and photographed duplex DNA (2, 3). It is implicated in DNA replication and (2). Negatives were traced with a microdensitometer to quan- transcription and in phage A integrative recombination (2, 4). titate the supercoiled product. One gyrase unit catalyzes the In Escherichia coli the two genes involved in its activity (5-7) supertwisting of 23 fmol of relaxed ColEl DNA in 30 min at are nalA, which controls resistance to the related drugs nalidixic 30°. The A and B subunit assay was the same except that the (Nal) and oxolinic (Oxo) acids (8), and cou, which controls re- reaction contained an excess of the complementary subunit. sistance to coumermycin Al and novobiocin (9). Gyrase from DNA relaxation activity was measured with 70 fmol of native wild-type cells is highly sensitive to these drugs whereas the ColEl DNA in a reaction mixture lacking ATP but otherwise enzyme from mutant cells resistant to these agents is not. Three identical to that used for supercoiling. additional reactions carried out by DNA gyrase are the Oxo- Enzymes. Buffers used in enzyme purification contained 10 and Nal-sensitive relaxation of supertwisted DNA (6, 7), the mM 2-mercaptoethanol and, except in hydroxylapatite steps, Oxo-dependent, site-specific cleavage of DNA induced by so- 1 mM EDTA. DNA gyrase from 610 g of H560 cells was puri- dium dodecyl sulfate (NaDodSO4) (6, 7), and the novobiocin- fied through the first four steps as described (6). This prepa- sensitive, DNA-dependent hydrolysis of ATP (A. Sugino and ration (42 mg of protein) was filtered through a 220-ml Ultrogel N. R. Cozzarelli, unpublished data). AcA34 (LKB) column equilibrated with 0.2 M potassium Sugino et al. (6) and Gellert et al. (7) suggested that gyrase phosphate, pH 7.4/50% glycerol. Fractions containing gyrase might contain subunits coded by nalA and cou. We reported (Kav 0.22, 5.4 mg of protein) were purified by phosphocellulose that the nalA gene product can be isolated as a protein termed chromatography as described (6). Activity eluting at 0.25 M Pnal, which was purified using a complementation assay for potassium phosphate, pH 6.8 (0.2 mg of protein), was concen- OX174 DNA synthesis (6). It had only a trace of gyrase activity, trated by dialysis against polyethylene glycol to 0.2 ml and but addition of Pnal to some DNA gyrase preparations mark- sedimented through a 3.8-ml 15-30% glycerol gradient con- edly stimulated activity and made DNA gyrase from nalAr cells taining 50 mM Tris-HCI, pH 7.5/0.1 M KCI at 55,000 rpm at sensitive to Oxo. These observations suggested that if Pnal is a 20 for 15 hr.in a Spinco SW56 rotor. Peak fractions were di- gyrase subunit, then the subunits of gyrase exchange readily alyzed against 50 mM Tris-HCI, pH 7.5/0.1 M KCI/50% glycerol and stored at -20° (0.15 mg of protein). This fraction The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Abbreviations: Nal, nalidixic acid; Oxo, oxolinic acid; NaDodSO4, so- "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate dium dodecyl sulfate. An allele conferring resistance to a drug is in- this fact. dicated by r. 1773 Downloaded by guest on September 23, 2021 1774 Biochemistry: Higgins et al. Proc. Natl. Acad. Sci. USA 75 (1978) was used exclusively and has a specific activity of 8 X 104 i 2 3 4 units/mg of protein. Purification of B and A subunits of gyrase from 720 g of H560 followed the gyrase purification to step III, DEAE-cellulose chromatography, which resolved the subunits. A pool of subunit B with limited gyrase activity eluted between 110 and 140 mM RNAP /3 NaCl (850 mg of protein) and was precipitated with (NH4)2SO4, RNAP /3 resuspended, and dialyzed against 20mhM potassium phosphate, pH 6.8/10% glycerol (buffer B). The dialysate was applied to a 2 X 25-cm hydroxylapatite column in buffer B and eluted with a 20-500 mM potassium phosphate (pH 6.8) linear gra- dient containing 10% glycerol. Subunit B activity eluted at 250 /3-gd ---p mM phosphate (36 mg of protein). After concentration and w8 - dialysis against 200 mM potassium phosphate, pH 7.4/10% glycerol, the enzyme was applied to a 2.6 X 87-cm Sephacryl T4 DNAP S-200 (Pharmacia) column. The subunit B activity was nearly phos excluded (2.9 mg of protein). After dialysis against buffer B it RNAP a- was applied to a 0.8 X 4-cm phosphocellulose column. A 30-ml 0-0.5 M KC1 linear gradient in buffer B eluted one peak of activity (16% of total) at 130 mM KC1 and a second (52% of total) at 260 mM KC1. The material in the two peaks had the same catalytic properties, and the more pure material which eluted first was used exclusively. The specific activity is 1 X 105 units/mg of protein. Subunit B from NI748 cells was purified BSA -- through the hydroxylapatite step. For subunit A purification, material eluted between 180 and 220 mM NaCl from the DEAE-cellulose column (1.2 g of pro- tein) was precipitated with (NH4)2SO4, dialyzed against buffer B, and adsorbed to a;2.6 X 25-cm hydroxylapatite column. A 1-liter 20-500 mM potassium phosphate (pH 6.8) gradient eluted subunit A activity at 140 mM phosphate. The pool (206 mg of protein) was concentrated, dialyzed against buffer B, and GDH applied to a 2.6 X 25-cm phosphocellulose column. Of the re- covered activity, 60% was eluted late in the 350-ml 20 mM CPK - potassium phosphate (pH 6.8) wash. A 1.4-liter 20-500 mM potassium phosphate (pH 6.8) gradient was applied, and the 2RNA P a. remaining 40% eluted at 140 mM phosphate. With the excep- FIG. 1. NaDodSO4 gel electrophoresis of DNA gyrase and its tion of their behavior on phosphocellulose, the two pools were subunits. Applied to a 10% polyacrylamide slab gel were: 2 gg ofDNA gyrase (lane 1); 1 ,g of subunit B (lane 2); 1.5 ,ug of subunit A (lane 3); similar and the larger one (6 mg of protein) was purified by and 1.5 jg of subunit A plus 400 cpm of [14C]Pnal (lane 4).