DNA gyrase is an enzyme that introduces superhelical turns into DNA. It was purified from Escherichia coli cells and was shown to convert relaxed closed-circular DNA into negatively supercoiled DNA. The reaction requires ATP and Mg²⁺ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin E1, phage λ, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellular supercoiled DNA. The study was conducted to investigate the integrative recombination of phage λ DNA in a cell-free system from E. coli. It was found that the process required a negatively supercoiled DNA substrate. This substrate could be replaced by relaxed closed-circular DNA only if the latter was incubated with an E. coli cell fraction and ATP. The simplest interpretation of these results was that the E. coli extract contains an ATP-dependent activity capable of converting relaxed closed-circular DNA to the supercoiled form. This enzyme, called DNA gyrase, was purified and characterized. The purification process involved several steps, including treatment with lysozyme, precipitation with polymin P, and chromatography on DEAE-cellulose and phosphocellulose. The final purified enzyme, fraction IV, represented about 1/2000 of the total starting protein of the extract, with an apparent recovery of activity well above 100%. The enzyme catalyzes supercoiling of relaxed Col E1, λ, and simian virus 40 DNA. The reaction is relatively specific for ATP, with half-maximal activity obtained with about 15 μM ATP. The reaction is stimulated by spermidine, although with purified DNA gyrase the requirement was not absolute. The enzyme is sensitive to N-ethylmaleimide and is 50% inactivated by incubation at 55°C for 5 min. The reaction mechanism is believed to involve the direct introduction of superhelical turns into closed-circular DNA in an ATP-dependent reaction. The enzyme is thought to function in DNA replication, in addition to its role in the recombination reaction which led to its purification. DNA gyrase is strongly inhibited by novobiocin, suggesting that it supplies an essential function for the replication of double-stranded circular DNA.DNA gyrase is an enzyme that introduces superhelical turns into DNA. It was purified from Escherichia coli cells and was shown to convert relaxed closed-circular DNA into negatively supercoiled DNA. The reaction requires ATP and Mg²⁺ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin E1, phage λ, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellular supercoiled DNA. The study was conducted to investigate the integrative recombination of phage λ DNA in a cell-free system from E. coli. It was found that the process required a negatively supercoiled DNA substrate. This substrate could be replaced by relaxed closed-circular DNA only if the latter was incubated with an E. coli cell fraction and ATP. The simplest interpretation of these results was that the E. coli extract contains an ATP-dependent activity capable of converting relaxed closed-circular DNA to the supercoiled form. This enzyme, called DNA gyrase, was purified and characterized. The purification process involved several steps, including treatment with lysozyme, precipitation with polymin P, and chromatography on DEAE-cellulose and phosphocellulose. The final purified enzyme, fraction IV, represented about 1/2000 of the total starting protein of the extract, with an apparent recovery of activity well above 100%. The enzyme catalyzes supercoiling of relaxed Col E1, λ, and simian virus 40 DNA. The reaction is relatively specific for ATP, with half-maximal activity obtained with about 15 μM ATP. The reaction is stimulated by spermidine, although with purified DNA gyrase the requirement was not absolute. The enzyme is sensitive to N-ethylmaleimide and is 50% inactivated by incubation at 55°C for 5 min. The reaction mechanism is believed to involve the direct introduction of superhelical turns into closed-circular DNA in an ATP-dependent reaction. The enzyme is thought to function in DNA replication, in addition to its role in the recombination reaction which led to its purification. DNA gyrase is strongly inhibited by novobiocin, suggesting that it supplies an essential function for the replication of double-stranded circular DNA.