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 A2B2 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 A2B2 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.
KeywordsColumn Volume Micrococcus Luteus Enzyme Buffer Coupling Buffer A2B2 Complex
- 2.Wigley, D. B. (1995) Structure and mechanism of DNA gyrase, in Nucleic Acids and Molecular Biology (Eckstein, F. and Lilley, D. M. J., eds.), Springer-Verlag, Berlin, pp. 165–176.Google Scholar