Overexpression and Purification of Bacterial DNA Gyrase

DNA gyrase is the bacterial type II topoisomerase that can introduce negative supercoils into DNA using the free energy of ATP hydrolysis (1 ,2 ). The enzyme from Escherichia coli consists of two proteins, A and B (termed GyrA and GyrB), of molecular masses 97 and 90 kDa, respectively; the active enzyme is an A₂ B₂ complex. All DNA topoisomerases are able to relax negatively supercoiled DNA, but only gyrase can also catalyze the introduction of negative supercoils, in a reaction coupled to ATP hydrolysis. Mechanistic studies have identified the steps involved in the supercoiling reaction. Briefly, this involves the wrapping of DNA around the A₂ B₂ complex, cleavage of this DNA in both strands (involving the formation of DNA–protein covalent bonds), and passage of another segment of DNA through this double-stranded break. Resealing of the break results in the introduction of two negative supercoils. Catalytic supercoiling requires the hydrolysis of ATP.