title: Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein.
abstract: In a previous report (Reece, R. J., and Maxwell, A. (1989) J. Biol. Chem. 264, 19648-19653) we showed that treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two stable fragments. The N-terminal 64-kDa fragment supports DNA supercoiling, while the C-terminal 33-kDa fragment shows no enzymic activity. We proposed that the 64-kDa fragment represents the DNA breakage-reunion domain of the A protein. We have now engineered the gyrA gene such that the 64-kDa protein is generated as a gene product. The properties of this protein confirm the findings of the experiments with the 64-kDa tryptic fragment. We have also generated a series of deletions of the gyrA gene such that C-terminal and N-terminal truncated versions of the A protein are produced. The smallest of the N-terminal fragments found to be able to carry out the DNA breakage-reunion reaction is GyrA(1-523). The cleavage reaction mediated by this protein occurs with equal efficacy as that performed by the intact GyrA protein. Deletion of the N-terminal 6 amino acids from either the A protein or these deletion derivatives has no effect on enzymic activity, while deletion of the N-terminal 69 amino acids completely abolishes the DNA breakage-reunion reaction. Therefore the smallest GyrA protein we have found that will perform DNA breakage and reunion is GyrA(7-523). A model is proposed for the domain organization of the gyrase A protein.
authors.affiliation: Department of Biochemistry, University of Leicester, United Kingdom.
authors.forename: R J
authors.initials: RJ
authors.lastname: Reece
authors.forename: A
authors.initials: A
authors.lastname: Maxwell
mesh_terms: D003001:Cloning, Molecular; D004249:DNA Damage; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D009154:Mutation; D010957:Plasmids; D015202:Protein Engineering; D015363:Quinolones
chemical_list: D015363:Quinolones; D004250:DNA Topoisomerases, Type II