Probing the structure and mechanism of DNA gyrase.

Abstract

DNA gyrase is the enzyme from bacteria which is unique among type II topoisomerases in its ability to introduce negative supercoils into DNA. The enzyme acts as an A2B2 tetramer of molecular weight 374 kDa. The supercoiling reaction of gyrase involves wrapping of DNA around the enzyme and the coupling of ATP binding and hydrolysis to the passage of a DNA segment through a transient double strand break stabilised by gyrase, although its details are undefined. This reaction of gyrase is inhibited by two classes of anti-bacterial compounds, the quinolones and the coumarins. Hydroxyl radical footprinting was used to probe the complex between gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. Gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl,Y-imidodiphosphate (ADPNP) or a quinolone drag causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. This is thought to represent an intermediate in the supercoiling cycle of gyrase. Protein-DNA crosslinking using the photoactivatable thymine analogue 4-thiothymidine was used in an attempt to identify regions of gyrase involved in DNA binding. Complexes containing gyrase and 4-thiothymidine-substituted DNA were irradiated with long-wave UV light to activate the photoreactive reagent. However, no protein-DNA crosslinks were detected. An attempt to radiolabel DNA-binding lysine residues of gyrase was also unsuccessful due to the dissociation of the gyrase-DNA complex upon modification of its lysines. Irradiation of gyrase DNA complexes with short-wave laser-UV light results in the formation of covalent protein-DNA complexes at an efficiency of ~1%. Primer extension analysis was used to tentatively assign the crosslinks to two positions along the 147 bp DNA fragment used. The 43 kDa N-terminal domain of the gyrase B protein is responsible for ATP hydrolysis and also interacts with the coumarin class of gyrase inhibitors. To gain insight into the nature of the ATP-induced conformational change in the gyrase A2B2 tetramer, the effect of the non-hydrolysable analogue ADPNP on the conformation and oligomeric state of the 43 kDa domain was examined. Protein crosslinking studies suggest that the protein exists as a monomer but dimerises in the presence of ADPNP. Limited trypsin proteolysis in the presence of ADPNP results in the protection of a 33 kDa N-terminal fragment of the protein (residues 2-307), consistent with an altered conformation of the 43 kDa domain in the presence of the nucleotide. The effects of the coumarin drugs novobiocin and coumermycin A1 on the 43 kDa domain were also investigated. Novobiocin does not cause oligomerisation of the 43 kDa protein but coumermycin A1 induces dimerisation when present at molar concentrations approaching half that of the 43 kDa protein. Limited trypsin digestion in the presence of either drug results in the protection of a 16 kDa proteolytic fragment from digestion (residues 111-247). Moreover, the products of trypsin digestion in the presence of novobiocin can stably bind novobiocin when denatured and renatured. The 16 kDa fragment was cloned and overexpressed as a direct gene product but was found to be incapable of stable novobiocin binding