Proc. Natl. Acad. Sci. USA Vol. 73, No. 12, pp. 4474-4478, December 1976 Biochemistry Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase (Escherichia coli/colicin El DNA replication/phage A DNA) MARTIN GELLERT, MARY H. O'DEA, TATEO ITOH, AND JUN-ICHI TOMIZAWA Laboratory of Molecular Biology, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Gary Felsenfeld, October 6, 1976 ABSTRACT Novobiocin and coumermycin are known to Among several spontaneous coumermycin-resistant mutants inhibit the replication of DNA in Escherichia coli. We show that of NI708 which were tested, all gave resistant extracts for the these drugs inhibit the supercoiling of DNA catalyzed by E. coli DNA gyrase, a recently discovered enzyme that introduces in vitro Col El DNA replication system. One of these mutants, negative superhelical turns into covalently circular DNA. The N1741, was chosen for further work because of its good growth. activity of DNA gyrase purified from a coumermycin-resistant This strain apparently has a partial reversion of the permeability mutant strain is resistant to both drugs. The inhibition by no- mutation of N1708. The couR mutation of NI741 was trans- vobiocin of colicin El plasmid DNA replication in a cell-free ferred, by phage P1 cotransduction with dnaA+, to strain system is partially relieved by adding resistant DNA gyrase. CRT46 dnaA (10). The resulting strain, N1748, was used as a Both in the case of colicin El DNA in E. coli extracts and of phage X DNA in whole cells, DNA molecules which are con- source for purification of drug-resistant DNA gyrase. DNA verted to the covalently circular form in the presence of coum- gyrase sensitive to both novobiocin and coumermycin was ermycin remain relaxed, instead of achieving their normal su- isolated from N99 recB21 (7). Strain N1071(Xind-) (11) was percoiled conformation. We conclude that DNA gyrase controls used for experiments of superinfection by phage X, and strain the supercoiling of DNA in E. coli. YSl (8) for testing supercoiling of Col El DNA in extracts. Of these strains, the coumermycin-resistant isolates NI741 Novobiocin and the related drug coumermycin are preferential and N1748 are able to grow in liquid culture containing 60 inhibitors of DNA replication in intact Escherichia coli cells gg/ml of coumermycin; growth of the other strains is blocked (1, 2). In toluenized cells of E. coli, these drugs inhibit repli- by 15 gg/ml of the drug. cative DNA synthesis but not repair synthesis (3, 4). They are Chemicals. Novobiocin was obtained from Sigma Chemical also effective inhibitors in cell-free systems for the replication Co. Samples of coumermycin A1 (referred to as coumermycin of colicin El (Col El) DNA (5) and of phage OX174 replicative throughout this paper) were gifts from W. F. Minor (Bristol form DNA (6), but they do not inhibit the synthesis of the Laboratories) and J. Davies (University of Wisconsin). Sources complementary strand of OX174 single-stranded DNA (6). of other materials have been described previously (7, 8, 12). Coumermycin-resistant mutants of E. coli have been isolated, Methods. Procedures for the assay of Col El DNA replica- and the mutation has been mapped near the dnaA locus (2). tion (8, 12, 13), and for the purification and assay of DNA gy- An enzyme, DNA gyrase, that introduces negative su- rase (7), have been described before. DNA gyrase from the perhelical turns into double-stranded closed circular DNA, has coumermycin-resistant strain NI748 was purified by the same recently been purified from E. coli (7). In this paper, we show method. Fraction IV enzyme was used for all experiments. that DNA gyrase activity in vitro as well as in vivo is specifically DNA Preparations. Relaxed and intracellularly supercoiled inhibited by coumermycin and novobiocin. Col El DNA samples were prepared as described (7, 8). Relaxed covalently-circular phage X DNA was prepared by sealing with MATERIALS AND METHODS DNA ligase as described (7). Bacterial Strains. Coumermycin-resistant E. coli mutants used for the present study were isolated by a two-step proce- RESULTS dure. The starting strain, E. coli NT525 pnp rns end strA, was constructed by recombination between EB5004 end (8) and a Inhibition of DNA gyrase activity by novobiocin and thy derivative of SK107 pnp rns strA polA+, the parental strain coumermycin of SK108 polA (9), provided by S. Kushner. Strain NT525, like For visualizing the activity of DNA gyrase, we have made use most other E. coli K12 strains, is quite resistant to novobiocin of the difference in mobility between relaxed and supercoiled (grows on tryptone agar containing 600 ,g/ml of novobiocin) closed-circular Col El DNA on agarose gel electrophoresis (7, because of low permeability to the drug (W. G. Coleman, 14). As is shown in Fig. 1, intracellularly supercoiled Col El personal communication). Strain NI708, with increased sensi- DNA (Fig. la) has roughly twice the mobility of relaxed tivity to novobiocin, was constructed by mating NT525 with closed-circular Col El DNA (Fig. lb). When relaxed closed- strain CL2, an HfrH derivative which carries a mutation con- circular Col El DNA was incubated with DNA gyrase purified ferring increased permeability to a number of drugs (W. G. from the coumermycin-sensitive strain N99 recB, most of the Coleman, personal communication). Mutants of NI708 with DNA was converted to a form with the increased mobility of various extents of increased resistance to novobiocin were iso- supercoiled Col El DNA (Fig. 1c). This reaction was progres- lated but all the isolates tested turned out to be resistant due to sively inhibited by increasing concentrations of novobiocin (Fig. reduced permeability. The selection was therefore repeated ld-h). At low drug concentrations (0.3-1.0 ,ug/ml), interme- with coumermycin, which is apparently indifferent to the diate DNA species were seen which had lesser degrees of su- permeability barrier for novobiocin (unpublished results). percoiling (15); at 3 ,ug/mi of novobiocin the reaction was totally inhibited. By contrast, the activity of DNA gyrase purified from Abbreviation: Col El, colicin El. the coumermycin-resistant strain N1748 was unaffected by all 4474 Downloaded by guest on October 2, 2021 Acad. Sci. USA 73 (1976) 4475 Biochemistry: Gellert et al. Proc. Natl. Table 1. Inhibition of Col El DNA replication by i j k I m n o p a b c d e f g h novobiocin and complementation by resistant DNA gyrase dTMP Source incor- of porated extract Additions (pmol) NT525 None 3.8 Sensitive DNA gyrase 3.9 Resistant DNA gyrase 3.0 Novobiocin 0.4 Novobiocin + sensitive DNA gyrase 0.3 Novobiocin + resistant DNA gyrase 1.1 N1741 None 3.4 FIG. 1. Novobiocin sensitivity of DNA gyrase from sensitive and Sensitive DNA gyrase 3.4 resistant E. coli strains. Channel (a) intracellularly supercoiled Col Resistant DNA gyrase 3.7 El DNA; (b) relaxed Col El DNA; (c-h) relaxed Col El DNA incu- 3.1 and Novobiocin bated with DNA gyrase purified from strain N99 recB21 (0.65 ,g) + DNA gyrase 2.0 of novobiocin; (i-n) relaxed Col El DNA in- Novobiocin sensitive varying concentrations Novobiocin + resistant DNA gyrase 3.7 cubated with DNA gyrase purified from strain NI748 (0.40 Mg) and varying concentrations of novobiocin. Novobiocin concentrations (f) Extracts were prepared as described from strain NT525 (coumer- were: (c) and (i), none; (d) and U), 0.3 Mg/ml; (e) and (k), 1 Mg/ml; and mycin-sensitive) and from strain NI741 (coumermycin-resistant) and (1), 3 Mg/ml; (g) and (m), 10 Mg/ml; (h) and (n), 30 Mg/ml. In (o)' The procedure for N99 recB and 0.20 Mg of DNA (8), except that the cells were cultured at 300. (p), 0.33 ,g of DNA gyrase from strain DNA replication was essentially as described were mixed and assayed in the absence (o) the assay of Col El gyrase from strain NI748 (12). The reaction mixtures (30 Il) contained 10 ,g/ml of intracel- and presence (p) of 3 ,g/ml novobiocin. lularly supercoiled Col El DNA, 25 MM each of four deoxyribonu- cleoside triphosphates (dNTPs), 400 ,M ATP, 200 AM each of other 1 NAD, 41 mM potas- Fig. lo and p show that ribonucleoside triphosphates (rNTPs), mM these levels of novobiocin (Fig. li-n). 33 mM KCl, 25 mM (NH4)2SO4, 8 mM novobiocin resistance on sium phosphate at pH 7.1, the resistant enzyme cannot confer MgCl2, 60MVM dithiothreitol, 60MgM Na3EDTA, 330 Mg/ml of bovine sensitive DNA gyrase; a mixture of the two enzymes showed plasma albumin, 10% (vol/vol) glycerol, and 2 mM spermidine. The more than 50% inhibition by novobiocin. Coum- reaction mixture contained 10 ,ul (100 Ag of protein) of extract. evidence of was 500 cpm/pmol. DNA ermycin also inhibited DNA gyrase activity (data not shown); Specific radioactivity of [a-32P]dTTP gyrase (fraction IV) was concentrated by precipitation with am- this drug was effective at roughly 10-fold lower concentrations a concentration of 1-2 in- monium sulfate and was resuspended at than novobiocin. DNA gyrase purified from N99 recB was mg/ml in 50% (vol/vol) glycerol, 0.15 M potassium phosphate at hibited more than 90% by 0.3 ,ug/ml of coumermycin; DNA pH 6.8, 2.5 mg/ml of bovine plasma albumin, 0.5 mM Na3EDTA, from the resistant mutant was unaffected by concen- and 0.5 mM dithiothreitol. Novobiocin-sensitive DNA gyrase (from gyrase DNA gyrase (from trations of the drug up to 1 gg/ml. strain N99 recB2l) and novobiocin-resistant strain N1748) were added in the amounts of 5.3 Mg and 6.3 Mg per Inhibition of in vitro Col El DNA replication by assay, respectively.