PMID:1847377
| Citation |
Reece, RJ and Maxwell, A (1991) Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein. J. Biol. Chem. 266:3540-6 |
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| 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. |
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| Keywords |
Cloning, Molecular; DNA Damage; DNA Topoisomerases, Type II/genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Mutation; Plasmids; Protein Engineering; Quinolones |
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