The intrinsic ATPase of DNA gyrase.

Escherichia coli DNA gyrase introduces negative superhelical turns into DNA in the presence of ATP.It is composed of two subunits, termed A and B. We conclude that gyrase contains an intrinsic, DNA-dependent, ATPase because: 1) the supercoiling and ATPase activities of native gyrase co-purified; 2) using an improved procedure, gyrase subunit B was purified to homogeneity and the activities of subunit B in reconstituting ATPase and supercoiling co-purified; 3) reconstitution of ATPase activity required subunits A and B at the same ratio that reconstituted supercoiling activity; and 4) the supercoiling and ATPase activities shared many common properties.The products of ATP hydrolysis were ADP and Pi.The reaction required a divalent metal ion, had a broad pH optimum, and was inhibited by high ionic strength.Like supercoiling activity, hydrolytic activity is highly specific for ATP.However, several ATP analogues and the reaction product ADP were good inhibitors competitive with ATP.Duplex DNA was about 12 times better as an effector than single-stranded DNA.The good ATPase activity in the presence of several nucleic acid homopolymer pairs and an alternating co-polymer implies that there is very little sequence specificity for the effector.Tertiary structure of the DNA does influence the reaction because negatively supercoiled circular DNA was a poorer effector than relaxed or nicked circular DNA.ATP hydrolysis is not necessarily coupled to net supertwist formation because ATP hydrolysis continued after maximum coiling of a DNA effector and many ATPase effectors cannot be supercoiled.With an initially relaxed circular duplex DNA effector, the hydrolysis of ATP molecules was roughly equivalent to the number of superhelical turns introduced.ATP hydrolysis is relatively insensitive to oxolinic acid, a good inhibitor of supercoiling, whereas novobiocin inhibits both processes equally.