Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA.

Abstract

ABSTRACr Two active components a and P of Micrococcus luteus DNA gyrase, of peptide weights of 115,000 and 97,000, respectively, have been purified. Each individual component exhibits litte DNA gyrase activity; the ATP-dependent negative supercoiling of a covalently closed circular DNA duplex is catalyzed by a combination of the two. Covalent closure by Escherichia coli ligase of a circular DNA containing single-chain scissions, when carried out in the presence of a combination of the DNA gyrase components a and f, gives a positively supercoiled DNA upon removal of the bound protein molecules. ATP was not present during the ligase treatment; therefore the positive supercoiling of DNA observed is a result of the binding of gyrase molecules, presumably as multi-subunit oligomers, during the ligation step. This is in contrast to the negative supercoiling of DNA catalyzed by gyrase in the presence of ATP. A model in which negative supercoiling of DNA is achieved by ATP-modulated repetitive wrapping of the DNA around gyrase is described. The model also suggests a plausible mode of action by which translocation of a DNA along its helix axis can be actively driven by an ATPase.A novel Escherichia coil enzyme, termed DNA gyrase, has been discovered recently (1). This enzyme converts a covalently closed circular DNA without superhelical turns to a highly negatively supercoiled form. In other words, the enzyme causes a reduction of the linking number (2) of the covalently closed circular duplex. The reaction requires ATP, the hydrolysis of which presumably provides the driving force for the otherwise energetically unfavorable reaction. The ATP-dependent negative supercoiling of DNA promoted by gyrase is phenomenologically opposite to the ATP-independent removal of negative superhelical turns by E. coli w protein [Eco DNA topoisomerase I (3)].* In vitro and in vivo studies indicate that in E. colt two widely separate genetic loci are involved in gyrase activity. These are the locus cou, which determines resistance to two related antibiotics (coumermycin A and novobiocin), and the locus nalA, which determines resistance to another two related drugs (nalidixic and oxolinic acids) (4-6). Gyrase purified from cells sensitive to these antibacterial agents is inhibited by them in its supercoiling of DNA, whereas the same enzyme purified from mutant cells resistant to these agents is not (4-6). Because in vivo these drugs block DNA replication (7-9) and the supercoiling of superinfected X DNA (4-6), the identification of gyrase as the target of these drugs in turn indicates that gyrase is involved in DNA replication and supercoiling in E. coli (1,4,6).E. coli gyrase has been purified by a number of groups (1,5,6,10). The nalA gene product has been purified to homogeneity (5). It appears to have a DNA topoisomerase activity distinct from Eco DNA topoisomerase 1 (5).In this communication, we report our studies on two active components, a and fl, of Micrococcus luteus gyrase. The possibility that negative superhelical turns in a DNA...

FIS modulates growth phase‐dependent topological transitions of DNA in Escherichia coli

“…The ability of FIS to prevent the supercoiling of DNA by DNA gyrase was unexpected but is consistent with the proposed mechanism. Although we cannot exclude a direct interaction of FIS with gyrase, the stabilization of negative writhe by FIS could antagonize the initial positive wrapping of DNA around the gyrase (Liu and Wang, 1978). We, therefore, suggest that FIS stabilizes the topoisomers of intermediate superhelical density primarily by constraint but also by indirect modification of the activity of topoisomerase I and DNA gyrase.…”

Mechanochemical analysis of DNA gyrase using rotor bead tracking

“…The equilibrium properties of DNA wrapped around gyrase or its subdomain have been studied extensively 4,5,7,8,16,17 , but the dynamics of DNA wrapping remain largely uncharacterized.…”

“…The equilibrium properties of DNA wrapped around gyrase or its subdomain have been studied extensively 4,5,7,8,16,17 , but the dynamics of DNA wrapping remain largely uncharacterized. Other poorly understood aspects of gyrase dynamics include the mechanism of processivity (by which gyrase is able to perform multiple successive strand passages without releasing the DNA substrate), the location of the rate-limiting step for the overall reaction cycle, and the coupling between ATP consumption and supercoil introduction.…”

Potassium Ions Are Required for Nucleotide-induced Closure of Gyrase N-gate

“…K ϩ increases the thermodynamic stability of GyrB by stabilizing the ATPase domain (18). It was shown previously that Micrococcus luteus gyrase requires K ϩ ions for DNA supercoiling (19). We have noted that K ϩ ions are also required for DNA supercoiling by Bacillus subtilis gyrase (7).…”