Proc. Natl. Acad. Sci. USA Vol. 75, No.4, pp. 1773-1777, April 1978 Biochemistry Purification of subunits of DNA gyrase and reconstitution of enzymatic activity (DNA supercoiling/DNA relaxation///DNA 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 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 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 . 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- and in phage A integrative recombination (2, 4). titate the supercoiled product. One gyrase unit catalyzes the In Escherichia coli the two 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- . 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, ; 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). The Coo- Sephacryl S-200 chromatography (Fig. 3) as described for massie blue staining pattern is shown in lanes 1-3 and the fluorograph subunit B. This preparation has a specific activity of 1 X 106 of the labeled Pnal in lane 4. The reference proteins, indicated by units/mg of protein and was used throughout. The preparation arrows, are: E. coli RNA (RNAP) a, ,B, ,', and a subunits; of Pnal from H560 and DNA gyrase from H560-1 has been fl-galactosidase (,B-gal); E. coli w protein (w); T4 DNA polymerase K. N. (DNAP); phosphorylase b (phos b); bovine serum albumin (BSA); described (6). Radiochemically pure Pnal from Kreuzer glutamic dehydrogenase (GDH); and creatine phosphokinase was prepared by sedimenting through a glycerol gradient (CPK). 14C-labeled proteins from a XdnalA-infected, light-irradiated, X lysogen (6). Protein concentrations were determined by the method of Lowry et al. (10). rate as E. coli w protein, and the smaller polypeptide migrated Electrophoresis of Proteins. Polyacrylamide gel electro- slightly faster than the a subunit of E. colf RNA polymerase. phoresis containing NaDodSO4 has been described (gel III in Although this preparation of DNA gyrase was the purest ref. 6). The reference proteins were: RNA polymerase from available, a low enzyme yield frustrated verification that the Boehringer Mannheim, E. coli w protein (11) from R. E. Depew two polypeptides were gyrase protomers. Since a large loss of and J. C. Wang, T4 DNA polymerase from S. Rashbaum, and activity was sometimes observed after the first chromatographic beef liver catalase, rabbit muscle phosphorylase b, bovine serum column, DEAE-cellulose, we sought to reconstitute DNA gyrase albumin, bovine liver L-glutamic dehydrogenase, rabbit muscle at this step by addition of Pnal to the column fractions. A peak creatine phosphokinase, and E. coli f3-galactosidase from of supercoiling activity which was resolved from DNA gyrase Sigma. eluted at 0.15 M NaCl. This activity was over an order of magnitude greater than the unsupplemented activity and is RESULTS designated subunit B. Addition of subunit B to the column Purification of DNA Gyrase and Its Subunits. E. coil DNA fractions revealed an even larger activity peak at 0.2 M NaCl, gyrase was extensively purified and analyzed by electrophoresis which is designated subunit A. DNA gyrase was between the through a polyacrylamide gel containing NaDodSO4 (Fig. 1, A and B peaks. Using reconstruction of supercoiling activity as lane 1). The two polypeptides found had molecular weights of an assay (Fig. 2, lanes 1-7), we purified subunits A and B. 105,000 and 95,000, as determined from the position of the Subunit A Is Pnal, the nal4 Gene Product. Subunit A is standards shown. The larger polypeptide migrated at the same pure. Electrophoresis in the presence of NaDodSO4 revealed Downloaded by guest on September 23, 2021 Biochemistry: Higgins et al. Proc. Natl. Acad. Sci. USA 75 (1978) 1775

--2 3 4I 5 6 7 8 9 10 11 12 13

rn L 0~ ~ ~ ~ ~~~~6 X40-~~~~~~6

FIG.C30Spc30-10craoahosbiAfD40Z < ~~~~~~~~~02 C n ~~~~~~~~20 FIG. 2. Reconstruction of supercoiling and DNA cleavage with subunits A and B. Supercoiling reactions were preincubated at 300 for 40 min in the absence of ATP to allow gyrase complex formation. After 10 min at 30° in the presence ofATP, the reactions were stopped by addition of NaDodSO4 to 1%. Reaction mixtures contained 0.5 unit 0 Q05 0.10 0.15 0Q20 0.25 of A plus 0.5 unit of B (lane 1); 1 unit of A plus 0.5 unit of B (lane 2); 2 units of A plus 0.5 unit of B (lane 3); 4 units of A plus 0.5 unit of B (lane 4); 25 units of B alone (lane 5); 1000 units ofA alone (lane 6); and FIG. 3. SephacrylI5-200 chromatography of subunit Aof DNA no enzyme (lane 7). Cleavage reactions (lanes 8-13) contained 23 fmol gyrase. The subunit A phosphocellulose pool (13 ml) was passed of native ColEl DNA, no ATP, and 100 yg ofOxo per ml (6). After 2.5 through a 2.6 X 87-cm Sephacryl S-200 column. The 5-ml fractions hr at 300 NaDodSO4 and proteinase K were added to 1% and 10,ug/ml, were assayed for subunit A (@) and Pnal activities (peak indicated respectively, and incubation continued for 15 min at 370. Reaction by arrow). The 105,000-dalton protomer in the fractions indicated mixtures contained: 0.1 unit of A plus 1 unit of B (lane 8); 2 units of was analyzed by NaDodSO4 gel electrophoresis (bars). The column A plus 1 unit ofB (lane 9); 8 units ofA plus 1 unit ofB (lane 10); 2 units was calibrated, and the position of catalase (cat) and bovine serum of B alone (lane 11); 16 units of A alone (lane 12); and no enzyme (lane albumin (BSA) references are indicated with arrows. 13). The products of the supercoiling and cleavage reactions were analyzed by agarose gel electrophoresis. In lane 10 the order of DNA species from top to botton is relaxed plus nicked DNA, linear DNA, wild-type subunit A, subunit B from the Cour cells reconstituted and fully supercoiled DNA. gyrase that was novobiocin-resistant (Fig. 4B) but Oxo-sensitive. Wild-type subunit B made this activity novobiocin-sensitive (Fig. 4B). Even though wild-type subunit B increased gyrase a single polypeptide with the same mobility as the larger DNA activity, novobiocin reduced the absolute activity below that gyrase polypeptide (Fig. 1, lane 3). The ratio of this 105,000- obtained with the couT B subunit alone. Gyrase reconstituted dalton protomer to subunit A activity was constant across the with the A subunit from cout cells had novobiocin sensitivity peak eluting from Sephacryl S-200, the final purification step identical to that of the wild-type enzyme (data not shown). (Fig. 3). Properties of Reconstituted DNA Gyrase. DNA gyrase Subunit A and Pnal are identical in physical and functional reconsititued with purified subunits A and B has the same tests: (i) Subunit A and radioactively labeled nalA gene product properties as DNA gyrase purified in the associated form. The had identical electrophoretic mobility (Fig. 1, lanes 3 and 4). two enzymes were identically sensitive to novobiocin and Oxo The molecular weight of 110,000 reported previously for the (Fig. 5) and had the same Km, 0.3 mM, for ATP. Both subunits nalA gene product (6) was obtained with different reference were required for reconstitution of all four activities of DNA proteins and is not significantly different from the 105,000 value gyrase although the supercoiling rate was about 20 times greater estimated here. (tt) Subunit A and Pnal eluted identically from than cleavage and relaxation in terms of DNA molecules con- several columns, including Sephacryl S-200 (Fig. 3). The spe- verted. Gel electrophoresis patterns with varying amounts of cific activity of subunit A in the Pnal DNA synthesis assay is subunits A and B are shown for supertwisting (Fig. 2, lanes 1-7), 16,000 units/mg, which is, within error, the same reported for cleavage (Fig. 2, lanes 8-13), and relaxation (Fig. 6). With each homogeneous Pnal (6). (Mit) Like Pnal (6), subunit A is appar- subunit alone, no supertwisting activity was seen with 2000 ently a dimer of identical 105,000-dalton polypeptides because times the amount of subunit A or 50 times the amount of subunit the native molecular weight calculated assuming a partial B, which reconstitutes supertwisting activity in the presence specific volume of 0.725 (12) is 220,000. (tv) A partially purified of the complementary subunit (Fig. 2). Reaction rates ap- DNA gyrase preparation fron nalA" cells was a rich source of proached a limiting value as the concentration of one subunit subunit A. Subunit BWincreased activity by over an order of was increased (Fig. 7). These plateau values are a measure of magnitude and the reconsructed gyrase was fully Oxo-resistant the activity of each subunit when fully associated with its (Fig. 4A), but still novobiocin-sensitive. complement and, therefore, the subunit ratio for reconstitution. Subunit B Is Controlled by the cou Gene. Electrophoresis For supercoiling, this ratio was about 1-2 B protomers per A of subunit B through a NaDodSO4/polyacrylamide gel revealed protomer (Fig. 7 A and B and unpublished data). Significantly, a single prominent band with the same mobility as the an approximate equivalence of subunits was also optimal for 95,000-dalton polypeptide found in DNA gyrase (Fig. 1, lanes nicking-closing (Fig. 6), cleavage (Fig. 7C), and ATPase (data 1 and 2). Minor bands were observed on a more heavily loaded not shown). There appears to be about twice as much A as B gel, and the 95,000-dalton polypeptide represents, conserva- protomer in DNA gyrase purified in the associated form (Fig. tively, 40% of the total protein. In preparations of subunit B the 1, lane 1). However, subunit B but not subunit A stimulated the ratio of activity to the 95,000-dalton polypeptide was constant. activity of this gyrase preparation and thus the implied stoi- Therefore, the 95,000-dalton polypeptide is probably a pro- chiometry may just reflect selective recoveries during purifi- tomer of subunit B. cation. Subunit B is specified by the cou gene. Subunit B was par- Turnover Rate of DNA Gyrase. The reconstituted enzyme tially purified from a cou" mutant, N1748, that contains a no- turns over. In 5.5 hr at 300, 250 fmol of relaxed ColEl DNA was vobiocin-resistant DNA gyrase (5). In the presence of excess maximally supertwisted by 12 fmol each of subunit A and B Downloaded by guest on September 23, 2021 1776 Biochemistry: Higgins et al. Proc. Natl. Acad. Sci. USA 75 (1978)

-0 20 40 60 0

Cn08060

~.40

20

0 20 40 60 0 0.015 0.03 Q045 0.06 [Oxo], tMM [Novobiocinl, MM FIG. 4. Genetic specification for novobiocin and Oxo resistance resides in different subunits of DNA gyrase. (A) DNA gyrase reaction mixtures contained 46 fmol of DNA substrate, the indicated amounts of Oxo, and 0.5 unit of H560-1 (nalAr) gyrase (a), 0.1 unit of H560-1 gyrase plus 1.3 units of H560 (wild-type) subunit B (0), or 0.5 unit of H560 subunit A plus 1.3 units of H560 subunit B (0). One hundred percent values are 2.7, 6.1, and 20 fmol of DNA supercoiled, respectively. (B) The assays contained the indicated amounts of novobiocin, 1 unit of H560 subunit A, and 0.5 unit of NI784 (cour) subunit B (A), 0.5 unit of H560 subunit B (0), or 0.5 unit of NI748 subunit B and 0.5 unit of H560 subunit B (0). One hundred percent values are 4.1, 3.5, and 9.3 fmol of DNA supercoiled, respectively. protomers. The turnover number is greater than this experiment purified subunits. They are required in the same ratio for ef- implies. Preincubation of the gyrase reaction mixture in the ficient reconstitution of four separate reactions carried out by absence of ATP increased the initial rate about an order of DNA gyrase: supertwisting (Figs. 2 and 7), DNA cleavage (Figs. magnitude over that obtained in the standard assay (Fig. 7 A 2 and 7), nicking-closing (Fig. 6), and ATPase. Each subunit and B). Under these conditions, one molecule of subunit A contains one of two polypeptides found in DNA gyrase purified protomer introduced about 102 supertwists per min at 30° in in the associated form (Fig. 1). By purification of the individual the presence of excess subunit B. The estimated 500 molecules subunits we obtained over an order of magnitude more gyrase of A per cell have the capacity to introduce in one generation activity than by purification of the associated form. The Mi- two orders of magnitude more supertwists than are present in crococcus luteus DNA gyrase appears to contain two subunits E. coli DNA. similar in size to the E. colt subunits (13). The M. luteus enzyme is sensitive to Nal and novobiocin but the controlling genes have DISCUSSION not yet been determined. E. coli DNA gyrase has been resolved into two individually 1 2 3 4 5 6 7 8 [Novobiocin], WM 0 0.05 0.1 16-

/ + Novobiocin 12- *0 / + X0

> 8

4 U

0

FIG. 6. Reconstitution of nicking-closing activity from subunits o 4 8 12 16 20 A and B. The standard nicking-closing reaction mixtures contained [Oxo], AM 10 units of subunit A plus 1 unit of subunit B (lane 1), 10 units of A FIG. 5. Drug sensitivity ofpurified DNA gyrase and reconstituted plus 3.3 units of B (lane 2), 10 units of A plus 5 units of B (lane 3), 10 DNA gyrase. The standard supercoiling assays contained either 0.8 units of A plus 10 units of B (lane 4), 10 units of A plus 20 units of B unit of DNA gyrase (filled symbols) or 1.5 units of subunit A and 0.8 (lane 5), 10 units of A (lane 6), 25 units ofB (lane 7), 10 units ofA (lane unit of subunit B (open symbols) and the indicated drug concentra- 6), 25 units of B (lane 7), or no enzyme (lane 8). After 2 hr, the samples tions. vo is the reaction rate (v) in the absence of drug. Incubation was were treated with 60 gg of proteinase K per ml for 15 min and dis- for 40 min at 300. played by agarose gel electrophoresis. Downloaded by guest on September 23, 2021 Biochemistry: Higgins et al. Proc. Natl. Acad. Sci. USA 75 (1978) 1777 95,000-dalton polypeptide is probably the cou gene product, but alternatives-e.g., the cou gene product modifies the 95,000-dalton polypeptide to a form that is novobiocin-sensi-

E tive-are not ruled out. C.,. It is not known whether the A and B subunits are independent 8 entities in the cell that intermittently form gyrase or if they exist 20 in a complex that dissociates during isolation. Three lines of evidence support the conclusion that the subunits occasion intimate contact and that gyrase is not a coincidence of activities 'a contained in noninteracting subunits: (i) the associated form of gyrase was purified several thousand-fold; (Ui) the subunits by themselves did not catalyze efficiently any of the four gyrase 0. reactions; and (Ili) preincubation of subunits greatly enhanced reaction rates. However, the subunits readily dissociate because addition of wild-type subunits rendered a drug-resistant gyrase Subunit A, unitSubunit A us t inhibitable by drugs (ref. 6 and Fig. 4). E. coil DNA polymerase III and the three associated factors can also be purified as a complex termed holoenzyme or purified separately and re- constructed (14, 15). For both gyrase and polymerase III ho- C A-C 0 6- loenzyme the reversible association of subunits may have functional significance in the mechanism of supercoiling and 4 of DNA replication. Subunits A and B could function in reac- 20 tions other than supercoiling and, thus, DNA gyrase may not be the only target for Nal, Oxo, coumermycin, and novobiocin. amout A SubunitB This could explain the different consequences of the drugs, such ofsubunitC unitso ()0.untosbunitsB() -j as the apparently greater inhibition of RNA synthesis by no- 0 vobiocin and coumermycin (4).

0 2 4 6 8 16 Since a single gyrase molecule can supercoil more than one Subunit A, units DNA molecule, the enzyme must supercoil DNA by a mecha- to in FIG. 7. Dependence of supercoiling and cleavage reactions on nism different from the one postulated operate higher subunits A and B. Supercoiling (A and B) was assayed as described organisms-stoichiometric binding of histones coupled with in the legend of Fig. 2 except that 100 fmol of DNA was used and in- a nicking-closing enzyme (16). We envision that supercoiling cubation in the presence of ATP was for 2 mm. DNA cleavage (C) was is catalyzed by a complex of both DNA gyrase subunits acting assayed as in the legend of Fig. 2. In A and C were the indicated at specific nucleotide sequences. amount of subunit A and no subunit B (o), 0.5 unit of subunit BA or 1 unit of subunit B (0). In B was the indicated amount of subunit This work was supported by National Institutes of Health Grants B and no subunit(w),A 1 unit of subunit(o),A or 2 units of subunit GM-21397 and CA-19265. N.P.H. and C.L.P. were supported by A (e). National Institutes of Health Fellowships GM-7190 and GM-780, re- spectively. 1. Wang, J. C. & Liu, L. F. (1978) in Molecular Genetics, ed. Taylor, Genetic and converge in demon- biochemical evidence J. H. (Academic Press, New York), Part III, in press. strating that subunit A consists of 105,000-dalton protomers 2. Gellert, M., Mizuuchi, K., O'Dea, M. H. & Nash, H. A. (1976) coded by nalA. Subunit A had the same electrophoretic Proc. Natl. Acad. Sci. USA 73, 3872-3876. mobility though a NaDodSO4/polyacrylamide gel as authentic 3. Marians, K. J., Ikeda, J., Schlagman, S. & Hurwitz, J. (1977) Proc. nalA gene product prepared using a specialized nalA trans- Nati. Acad. Sci. USA 74, 1965-1968. ducing phage (Fig. 1). (ii) Subunit A from a nalAr mutant re- 4. Cozzarelli, N. R. (1977) Annu. Rev. Biochem. 46,641-668. constituted an Oxo-resistant gyrase (Fig. 4A). ( m)Subunit A 5. Gellert, M., O'Dea, M. H., Itoh, T. & Tomizawa, J. (1976) Proc. conferred Oxo sensitivity to in vltro DNA synthesis directed Natl. Acad. Sci. USA 73,4474-4478. by nalAy extracts (Fig. 3). We used this assay previously to 6. Sugino, A., Peebles, C. L., Kreuzer, K. N. & Cozzarelli, N. R. (1977) Proc. Natl. Acad. Sci. USA 74,47674771. purify Pnal (6), which is identical to subunit A. (iv) A temper- 7. Gellert, M., Mizuuchi, K., O'Dea, M. H., Itoh, T. & Tomizawa, ature-sensitive in nalA caused a loss of gyrase activity J. (1977) Proc. Natl. Acad. Sci. USA 74,4772-4776. in extracts which was restored specifically by addition of sub- 8. Bourguignon, G. J., Levitt, M. & Sternglanz, R. (1973) Antimi- unit A (K. N. Kreuzer and N. R Cozzarelli, unpublished data). crob. Agents Chemother. 4,479-486. The B subunit is controlled by the cots gene: (i) The cots gene 9. Ryan, M. J. (1976) Biochemistry 15,3769-3777. product has been implicated as the target for novobiocin (4, 9), 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. and minute levels of novobiocin identically inhibited gyrase, (1951) J. Biol. Chem. 193,265-275. purified in the associated form and gyrase, reconstituted from 11. Wang, J. C. (1971) J. Mol. Biol. 55,523-53. purified subunits (Fig. 5). (ii) The B subunit from a cous? mutant 12. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta 112,

reconstituted a novobiocin-resistant which is made 346-362. enzyme 13. Liu, L. F. & Wang, J. C. (1978) Proc. Natl. Acad. Sci. USA 75, drug-sensitive by addition of wild-type subunit B (Fig. 4B). It in press. is likely that subunit B contains a 95,000-dalton protomer. This 14. McHenry, C. & Kornberg, A. (1977) J. Biol. Chem. 252, is the only prominent polypeptide in subunit B preparations and 6478-6484. is one of two polypeptides discernable in DNA gyrase, (Fig. 1). 15. Wickner, S. (1976) Proc. Natl. Acad. Sci. USA 73,3511-3515. Subunit B activity was proportional to the concentration of the 16. Germond, J. E., Hirt, B., Oudet, P., Gross-Bellard, M. & Cham- 95,000-dalton polypeptide in the preparations analyzed. The bon, P. (1975) Proc. Natl.Acad. Sci. USA 72, 1843-1847. Downloaded by guest on September 23, 2021