Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase.

Abstract

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...

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“…This design is vital for the cell because the torsional tension of supercoiled DNA is an important reservoir of free energy, which helps to drive all processes requiring the melting of DNA strands, such as transcription [Lim et al, 2003;Peter et al, 2004], replication [Funnell et al, 1986], and recombination [Nash, 1990]. The relevance of limiting the spread of relaxation is underlined by the fact that even slight changes in the overall superhelicity of the chromosome are lethal [Gellert et al, 1976[Gellert et al, , 1977. Short distances between domain boundaries might, in addition, facilitate the repair of double-strand breaks because they restrict the movement of the two ends to be joined.…”