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 Escherichia coli is inhibited by nalidixic acid and by novobiocin. The enzyme is composed of two subunits, A and B, which were purified as separate components. Subunit A is the product of the gene controlling sensitivity to nalidixic acid (nal4) because: (I) the electrophoretic mobility of subunit A in the presence of sodium dodecyl sulfate is identical to that of the 105,000-dalton nalA gene product; (ii) mutants that are resistant to nalidixic acid (nalA r) produce a drug-resistant subunit A; and (iii) wildtype subunit A confers drug sensitivity to in vitro synthesis of 4X174 DNA directed by nalA r mutants. Subunit B contains a 95,000-dalton polypeptide and is controlled by the gene specifying sensitivity to novobiocin (cou) because cou' mutants produce a novobiocin-resistant subunit B and novobiocin-resitant gyrase is made drug sensitive by wild-type subunit B. Subunits A and B associate, so that gyrase was also purified as a complex containing 105,000- and 95,000-dalton polypeptides. This enzyme and gyrase reconstructed from subunits have the same drug sensitivity, K, for ATP, and catalytic properties. The same ratio of subunits gives efficient reconstitution of the reactions intrinsic to DNA gyrase, including catalysis of supercoiling of closed duplex DNA, relaxation of supercoiled DNA in the absence of ATP, and site-specific cleavage of DNA induced by sodium dodecyl sulfate.

DNA gyrase [Eco DNA topoisomerase II (1)] is an ATP-requiring enzyme that introduces negative supertwists into closed duplex DNA (2, 3). It is implicated in DNA replication and transcription and in phage A integrative recombination (2, 4). In Escherichia coli the two genes involved in its activity (5-7) are nalA, which controls resistance to the related drugs nalidixic (Nal) and oxolinic (Oxo) acids (8), and cou, which controls resistance to coumermycin Al and novobiocin (9). Gyrase from wild-type cells is highly sensitive to these drugs whereas the enzyme from mutant cells resistant to these agents is not. Three additional reactions carried out by DNA gyrase are the Oxoand Nal-sensitive relaxation of supertwisted DNA (6, 7), the Oxo-dependent, site-specific cleavage of DNA induced by sodium dodecyl sulfate (NaDodSO4) (6, 7), and the novobiocinsensitive, DNA-dependent hydrolysis of ATP (A. Sugino and N. R. Cozzarelli, unpublished data). Sugino et al. (6) and Gellert et al. (7) suggested that gyrase might contain subunits coded by nalA and cou. We reported that the nalA gene product can be isolated as a protein termed Pnal, which was purified using a complementation assay for OX174 DNA synthesis (6). It had only a trace of gyrase activity, but addition of Pnal to some DNA gyrase preparations markedly stimulated activity and made DNA gyrase from nalAr cells sensitive to Oxo. These observations suggested that if Pnal is a gyrase subunit, then the subunits of gyrase exchange readily The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

and a free pool of cou coded subunits might exist to complement the free pool of nalA subunits. We have now proven that Pnal is a subunit of DNA gyrase and that the subunit controlled by the cou gene can also be purified independently. Each subunit contains one of two polypeptides found in extensively purified gyrase. Optimal reconstitution of all four activities of DNA gyrase requires both subunits. MATERIALS AND METHODS Bacteria. The strains used were E. coil H560 polA endA, H560-1 polA endA nalAr constructed by R. Sternglanz (6), and N1748 cour (5). Enzyme Assays. The Pnal assay was essentially that devised by C. Sumida-Yasumoto and measures conferral of Oxo sensitivity to OX174 DNA replication (6). Supertwisting activity of DNA gyrase was measured at 300 in a 17-Ml reaction mixture containing 35 mM Tris-HCl (pH 7.6), 18 mM potassium phosphate, 5 mM dithiothreitol, 6.7 mM MgCl2, 5 mM spermidine-HCl, 50 Ag of bovine serum albumin per ml, 1.5 mM ATP, and 23 fmol of relaxed ColEl DNA. The reaction was stopped (6) and the products were displayed by 1% agarose gel electrophoresis, stained with ethidium bromide, and photographed (2). Negatives were traced with a microdensitometer to quantitate the supercoiled product. One gyrase unit catalyzes the supertwisting of 23 fmol of relaxed ColEl DNA in 30 min at 30°. The A and B subunit assay was the same except that the reaction contained an excess of the complementary subunit. DNA relaxation activity was measured with 70 fmol of native ColEl DNA in a reaction mixture lacking ATP but otherwise identical to that used for supercoiling. Enzymes. Buffers used in enzyme purification contained 10 mM 2-mercaptoethanol and, except in hydroxylapatite steps, 1 mM EDTA. DNA gyrase from 610 g of H560 cells was purified through the first four steps as described (6). This preparation (42 mg of protein) was filtered through a 220-ml Ultrogel AcA34 (LKB) column equilibrated with 0.2 M potassium phosphate, pH 7.4/50% glycerol. Fractions containing gyrase (Kav 0.22, 5.4 mg of protein) were purified by phosphocellulose chromatography as described (6). Activity eluting at 0.25 M potassium phosphate, pH 6.8 (0.2 mg of protein), was concentrated by dialysis against polyethylene glycol to 0.2 ml and sedimented through a 3.8-ml 15-30% glycerol gradient containing 50 mM Tris-HCI, pH 7.5/0.1 M KCI at 55,000 rpm at 20 for 15 hr. in a Spinco SW56 rotor. Peak fractions were dialyzed 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 Abbreviations: Nal, nalidixic acid; Oxo, oxolinic acid; NaDodSO4, sodium dodecyl sulfate. An allele conferring resistance to a drug is indicated by r.

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Proc. Natl. Acad. Sci. USA 75 (1978)

Biochemistry: Higgins et al.

was used exclusively and has a specific activity of 8 X 104 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 NaCl (850 mg of protein) and was precipitated with (NH4)2SO4, resuspended, and dialyzed against 20 mhM 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 gradient containing 10% glycerol. Subunit B activity eluted at 250 mM phosphate (36 mg of protein). After concentration and dialysis against 200 mM potassium phosphate, pH 7.4/10% glycerol, the enzyme was applied to a 2.6 X 87-cm Sephacryl S-200 (Pharmacia) column. The subunit B activity was nearly excluded (2.9 mg of protein). After dialysis against buffer B it 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 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 protein) 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 applied to a 2.6 X 25-cm phosphocellulose column. Of the recovered activity, 60% was eluted late in the 350-ml 20 mM potassium phosphate (pH 6.8) wash. A 1.4-liter 20-500 mM potassium phosphate (pH 6.8) gradient was applied, and the remaining 40% eluted at 140 mM phosphate. With the exception of their behavior on phosphocellulose, the two pools were similar and the larger one (6 mg of protein) was purified by Sephacryl S-200 chromatography (Fig. 3) as described for subunit B. This preparation has a specific activity of 1 X 106 units/mg of protein and was used throughout. The preparation of Pnal from H560 and DNA gyrase from H560-1 has been described (6). Radiochemically pure Pnal from K. N. Kreuzer was prepared by sedimenting through a glycerol gradient 14C-labeled proteins from a XdnalA-infected, ultraviolet light-irradiated, X lysogen (6). Protein concentrations were determined by the method of Lowry et al. (10). Electrophoresis of Proteins. Polyacrylamide gel electrophoresis containing NaDodSO4 has been described (gel III in ref. 6). The reference proteins were: RNA polymerase from Boehringer Mannheim, E. coli w protein (11) from R. E. Depew and J. C. Wang, T4 DNA polymerase from S. Rashbaum, and beef liver catalase, rabbit muscle phosphorylase b, bovine serum albumin, bovine liver L-glutamic dehydrogenase, rabbit muscle creatine phosphokinase, and E. coli f3-galactosidase from Sigma. RESULTS Purification of DNA Gyrase and Its Subunits. E. coil DNA gyrase was extensively purified and analyzed by electrophoresis through a polyacrylamide gel containing NaDodSO4 (Fig. 1, lane 1). The two polypeptides found had molecular weights of 105,000 and 95,000, as determined from the position of the standards shown. The larger polypeptide migrated at the same

i

2

3

4

RNAP /3

RNAP /3

/3-gd

---p

-

w8 T4 DNAP phos RNAP a-

BSA

--

GDH

CPK 2RNA P a. FIG. 1. NaDodSO4 gel electrophoresis of DNA gyrase and its subunits. Applied to a 10% polyacrylamide slab gel were: 2 gg of DNA gyrase (lane 1); 1 ,g of subunit B (lane 2); 1.5 ,ug of subunit A (lane 3); and 1.5 jg of subunit A plus 400 cpm of [14C]Pnal (lane 4). The Coomassie blue staining pattern is shown in lanes 1-3 and the fluorograph of the labeled Pnal in lane 4. The reference proteins, indicated by arrows, are: E. coli RNA polymerase (RNAP) a, ,B, ,', and a subunits; fl-galactosidase (,B-gal); E. coli w protein (w); T4 DNA polymerase (DNAP); phosphorylase b (phos b); bovine serum albumin (BSA); glutamic dehydrogenase (GDH); and creatine phosphokinase

(CPK).

rate as E. coli w protein, and the smaller polypeptide migrated slightly faster than the a subunit of E. colf RNA polymerase. Although this preparation of DNA gyrase was the purest available, a low enzyme yield frustrated verification that the two polypeptides were gyrase protomers. Since a large loss of activity was sometimes observed after the first chromatographic column, DEAE-cellulose, we sought to reconstitute DNA gyrase at this step by addition of Pnal to the column fractions. A peak of supercoiling activity which was resolved from DNA gyrase eluted at 0.15 M NaCl. This activity was over an order of magnitude greater than the unsupplemented activity and is designated subunit B. Addition of subunit B to the column fractions revealed an even larger activity peak at 0.2 M NaCl, which is designated subunit A. DNA gyrase was between the A and B peaks. Using reconstruction of supercoiling activity as an assay (Fig. 2, lanes 1-7), we purified subunits A and B. Subunit A Is Pnal, the nal4 Gene Product. Subunit A is pure. Electrophoresis in the presence of NaDodSO4 revealed

Biochemistry: Higgins et al. 2 -3

-

4I

5

6

7

8

9

Proc. Natl. Acad. Sci. USA 75 (1978) 10 11

12

1775

13 rn

L

0 ~~

~

~

~~~~6

-~~~~~~6 X40 C30 -1 FIG. Spc 30 0craoahosbiAfD40Z < ~~~~~~~~~02 C

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 of ATP, the reactions were stopped by addition of NaDodSO4 to 1%. Reaction mixtures contained 0.5 unit 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 of A alone (lane 6); and no enzyme (lane 7). Cleavage reactions (lanes 8-13) contained 23 fmol of native ColEl DNA, no ATP, and 100 yg of Oxo per ml (6). After 2.5 hr at 300 NaDodSO4 and proteinase K were added to 1% and 10,ug/ml, respectively, and incubation continued for 15 min at 370. Reaction mixtures contained: 0.1 unit of A plus 1 unit of B (lane 8); 2 units of A plus 1 unit of B (lane 9); 8 units of A plus 1 unit of B (lane 10); 2 units of B alone (lane 11); 16 units of A alone (lane 12); and no enzyme (lane 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, and fully supercoiled DNA.

single polypeptide with the same mobility as the larger DNA polypeptide (Fig. 1, lane 3). The ratio of this 105,000dalton protomer to subunit A activity was constant across the peak eluting from Sephacryl S-200, the final purification step (Fig. 3). Subunit A and Pnal are identical in physical and functional tests: (i) Subunit A and radioactively labeled nalA gene product had identical electrophoretic mobility (Fig. 1, lanes 3 and 4). The molecular weight of 110,000 reported previously for the nalA gene product (6) was obtained with different reference proteins and is not significantly different from the 105,000 value estimated here. (tt) Subunit A and Pnal eluted identically from several columns, including Sephacryl S-200 (Fig. 3). The specific activity of subunit A in the Pnal DNA synthesis assay is 16,000 units/mg, which is, within error, the same reported for homogeneous Pnal (6). (Mit) Like Pnal (6), subunit A is apparently a dimer of identical 105,000-dalton polypeptides because the native molecular weight calculated assuming a partial specific volume of 0.725 (12) is 220,000. (tv) A partially purified DNA gyrase preparation fron nalA" cells was a rich source of subunit A. Subunit BWincreased activity by over an order of magnitude and the reconsructed gyrase was fully Oxo-resistant (Fig. 4A), but still novobiocin-sensitive. Subunit B Is Controlled by the cou Gene. Electrophoresis of subunit B through a NaDodSO4/polyacrylamide gel revealed a single prominent band with the same mobility as the 95,000-dalton polypeptide found in DNA gyrase (Fig. 1, lanes 1 and 2). Minor bands were observed on a more heavily loaded gel, and the 95,000-dalton polypeptide represents, conservatively, 40% of the total protein. In preparations of subunit B the ratio of activity to the 95,000-dalton polypeptide was constant. Therefore, the 95,000-dalton polypeptide is probably a protomer of subunit B. Subunit B is specified by the cou gene. Subunit B was partially purified from a cou" mutant, N1748, that contains a novobiocin-resistant DNA gyrase (5). In the presence of excess a

gyrase

~~~~~~~~20

n

0

FIG. 3.

Q05

0.10

0.15

0Q20 0.25

SephacrylI5-200 chromatography of subunit Aof DNA

gyrase. The subunit A phosphocellulose pool (13 ml) was passed through a 2.6 X 87-cm Sephacryl S-200 column. The 5-ml fractions were assayed for subunit A (@) and Pnal activities (peak indicated by arrow). The 105,000-dalton protomer in the fractions indicated was analyzed by NaDodSO4 gel electrophoresis (bars). The column was calibrated, and the position of catalase (cat) and bovine serum albumin (BSA) references are indicated with arrows.

wild-type subunit A, subunit B from the Cour cells reconstituted 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 activity, novobiocin reduced the absolute activity below that obtained with the couT B subunit alone. Gyrase reconstituted with the A subunit from cout cells had novobiocin sensitivity identical to that of the wild-type enzyme (data not shown). Properties of Reconstituted DNA Gyrase. DNA gyrase reconsititued with purified subunits A and B has the same properties as DNA gyrase purified in the associated form. The two enzymes were identically sensitive to novobiocin and Oxo (Fig. 5) and had the same Km, 0.3 mM, for ATP. Both subunits were required for reconstitution of all four activities of DNA gyrase although the supercoiling rate was about 20 times greater than cleavage and relaxation in terms of DNA molecules converted. Gel electrophoresis patterns with varying amounts of subunits A and B are shown for supertwisting (Fig. 2, lanes 1-7), cleavage (Fig. 2, lanes 8-13), and relaxation (Fig. 6). With each subunit alone, no supertwisting activity was seen with 2000 times the amount of subunit A or 50 times the amount of subunit B, which reconstitutes supertwisting activity in the presence of the complementary subunit (Fig. 2). Reaction rates approached a limiting value as the concentration of one subunit was increased (Fig. 7). These plateau values are a measure of the activity of each subunit when fully associated with its complement and, therefore, the subunit ratio for reconstitution. For supercoiling, this ratio was about 1-2 B protomers per A protomer (Fig. 7 A and B and unpublished data). Significantly, an approximate equivalence of subunits was also optimal for nicking-closing (Fig. 6), cleavage (Fig. 7C), and ATPase (data not shown). There appears to be about twice as much A as B protomer in DNA gyrase purified in the associated form (Fig. 1, lane 1). However, subunit B but not subunit A stimulated the activity of this gyrase preparation and thus the implied stoichiometry may just reflect selective recoveries during purification. Turnover Rate of DNA Gyrase. The reconstituted enzyme turns over. In 5.5 hr at 300, 250 fmol of relaxed ColEl DNA was maximally supertwisted by 12 fmol each of subunit A and B

Biochemistry: Higgins et al.

1776

Proc. Natl. Acad. Sci. USA 75 (1978)

40

20

-0

60

0

80 60 Cn0 ~.40

20

0.06 Q045 0.03 [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.

0

40

20

60

0

0.015

[Oxo], tMM

protomers. The turnover number is greater than this experiment implies. Preincubation of the gyrase reaction mixture in the absence of ATP increased the initial rate about an order of magnitude over that obtained in the standard assay (Fig. 7 A and B). Under these conditions, one molecule of subunit A protomer introduced about 102 supertwists per min at 30° in the presence of excess subunit B. The estimated 500 molecules of A per cell have the capacity to introduce in one generation two orders of magnitude more supertwists than are present in E. coli DNA. DISCUSSION E. coli DNA gyrase has been resolved into two individually

purified subunits. They are required in the same ratio for efficient reconstitution of four separate reactions carried out by DNA gyrase: supertwisting (Figs. 2 and 7), DNA cleavage (Figs. 2 and 7), nicking-closing (Fig. 6), and ATPase. Each subunit contains one of two polypeptides found in DNA gyrase purified in the associated form (Fig. 1). By purification of the individual subunits we obtained over an order of magnitude more gyrase activity than by purification of the associated form. The Micrococcus luteus DNA gyrase appears to contain two subunits similar in size to the E. colt subunits (13). The M. luteus enzyme is sensitive to Nal and novobiocin but the controlling genes have not yet been determined. 1

2

3

4

5

6

7

8

[Novobiocin], WM 0

0.05

0.1

16/ + Novobiocin

12*0 >

/

+

X0

8

4

U 0

o

4

8

12

16

20

[Oxo], AM FIG. 5. Drug sensitivity of purified DNA gyrase and reconstituted DNA gyrase. The standard supercoiling assays contained either 0.8 unit of DNA gyrase (filled symbols) or 1.5 units of subunit A and 0.8 unit of subunit B (open symbols) and the indicated drug concentrations. vo is the reaction rate (v) in the absence of drug. Incubation was for 40 min at 300.

FIG. 6. Reconstitution of nicking-closing activity from subunits A and B. The standard nicking-closing reaction mixtures contained 10 units of subunit A plus 1 unit of subunit B (lane 1), 10 units of A plus 3.3 units of B (lane 2), 10 units of A plus 5 units of B (lane 3), 10 units of A plus 10 units of B (lane 4), 10 units of A plus 20 units of B (lane 5), 10 units of A (lane 6), 25 units of B (lane 7), 10 units of A (lane 6), 25 units of B (lane 7), or no enzyme (lane 8). After 2 hr, the samples were treated with 60 gg of proteinase K per ml for 15 min and displayed by agarose gel electrophoresis.

Biochemistry: Higgins et al.

Proc. Natl. Acad. Sci. USA 75 (1978)

E

C.,

8

20

'a

0.

Subunit A, unitSubunit A us

t

C

A-C

0 6-

4

20

amout ofsubunit C

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Subunit B ()0.untosbunitsB()

-j

0

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0

6

4

Subunit A, FIG.

Dependence

7.

the

assayed

in the as

in

presence

the

of ATP

1

was

legend of Fig.

amount of subunit A and or

was

assayed

legend of Fig. 2 except that 100 fmol of DNA

cubation

no

for 2

mm.

A

and

2. In

subunit B

DNA

C

was

reactions as

used

cleavage

were

on

described and

(C)

in-

was

the indicated

(o), 0.5 unit of subunit BA

the indicated amount of subunit unit of subunit B (0). In B subunit(w), A 1 unit of subunit A 2 units of subunit (o),

and

B

16

of supercoiling and cleavage

subunits A and B. Supercoiling (A and B) in

8

units

was

no

or

A (e).

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-sensitive-are not ruled out. It is not known whether the A and B subunits are independent entities in the cell that intermittently form gyrase or if they exist 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 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 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 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 reconstructed (14, 15). For both gyrase and polymerase III holoenzyme the reversible association of subunits may have functional significance in the mechanism of supercoiling and of DNA replication. Subunits A and B could function in reactions other than supercoiling and, thus, DNA gyrase may not be the only target for Nal, Oxo, coumermycin, and novobiocin. This could explain the different consequences of the drugs, such as the apparently greater inhibition of RNA synthesis by novobiocin and coumermycin (4). Since a single gyrase molecule can supercoil more than one DNA molecule, the enzyme must supercoil DNA by a mechanism different from the one postulated to operate in higher organisms-stoichiometric binding of histones coupled with a nicking-closing enzyme (16). We envision that supercoiling is catalyzed by a complex of both DNA gyrase subunits acting at specific nucleotide sequences. This work was supported by National Institutes of Health Grants GM-21397 and CA-19265. N.P.H. and C.L.P. were supported by National Institutes of Health Fellowships GM-7190 and GM-780, re-

spectively. Genetic

strating

biochemical evidence

and

that subunit A

Subunit

coded by nalA.

mobility though

converge in demon-

consists of 105,000-dalton protomers A had the same electrophoretic

NaDodSO4/polyacrylamide gel as authentic nalA gene product using a specialized nalA transphage (Fig. 1). (ii) Subunit A from a nalAr mutant reconstituted an Oxo-resistant gyrase (Fig. 4A). ( m) Subunit A a

prepared

ducing

sensitivity to in vltro DNA synthesis directed (Fig. 3). We used this assay previously to purify Pnal (6), which is identical to subunit A. (iv) A temperature-sensitive mutation in nalA caused loss of gyrase activity in extracts which was restored specifically by addition of subunit A (K. N. Kreuzer and N. R Cozzarelli, unpublished data). conferred Oxo

by

nalAy

extracts

a

The B subunit is controlled

product has

been

implicated

by as

the

cots

gene:

(i) The

cots

gene

the target for novobiocin (4, 9),

and minute levels of novobiocin

identically inhibited gyrase, purified in the associated form and gyrase, reconstituted from purified subunits (Fig. 5). (ii) The B subunit from a cous? mutant reconstituted

a

novobiocin-resistant enzyme which is made

drug-sensitive by addition of wild-type subunit B (Fig. 4B). It is likely that subunit B contains a 95,000-dalton protomer. This is the only prominent polypeptide in subunit B preparations and is one of two polypeptides discernable in DNA gyrase, (Fig. 1). Subunit B activity was proportional to the concentration of the 95,000-dalton polypeptide in the preparations analyzed. The

1. Wang, J. C. & Liu, L. F. (1978) in Molecular Genetics, ed. Taylor, J. H. (Academic Press, New York), Part III, in press. 2. Gellert, M., Mizuuchi, K., O'Dea, M. H. & Nash, H. A. (1976) Proc. Natl. Acad. Sci. USA 73, 3872-3876. 3. Marians, K. J., Ikeda, J., Schlagman, S. & Hurwitz, J. (1977) Proc.

Nati. Acad. Sci. USA 74, 1965-1968. 4. Cozzarelli, N. R. (1977) Annu. Rev. Biochem. 46,641-668. 5. Gellert, M., O'Dea, M. H., Itoh, T. & Tomizawa, J. (1976) Proc.

Natl. Acad. Sci. USA 73,4474-4478. 6. Sugino, A., Peebles, C. L., Kreuzer, K. N. & Cozzarelli, N. R. (1977) Proc. Natl. Acad. Sci. USA 74,47674771. 7. Gellert, M., Mizuuchi, K., O'Dea, M. H., Itoh, T. & Tomizawa, J. (1977) Proc. Natl. Acad. Sci. USA 74,4772-4776. 8. Bourguignon, G. J., Levitt, M. & Sternglanz, R. (1973) Antimicrob. Agents Chemother. 4,479-486. 9. Ryan, M. J. (1976) Biochemistry 15,3769-3777. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 11. Wang, J. C. (1971) J. Mol. Biol. 55,523-53. 12. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta 112, 346-362. 13. Liu, L. F. & Wang, J. C. (1978) Proc. Natl. Acad. Sci. USA 75, in press. 14. McHenry, C. & Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484. 15. Wickner, S. (1976) Proc. Natl. Acad. Sci. USA 73,3511-3515. 16. Germond, J. E., Hirt, B., Oudet, P., Gross-Bellard, M. & Chambon, P. (1975) Proc. Natl. Acad. Sci. USA 72, 1843-1847.