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Title: Strand passage in DNA gyrase.
Authors: Tingey, Andrew Philip.
Award date: 1996
Presented at: University of Leicester
Abstract: DNA gyrase, a type II topoisomerase, catalyses the introduction of negative supercoils into closed-circular DNA, using the energy from ATP hydrolysis. The reaction mechanism involves the breakage of one DNA double strand (the DNA gate) and the passing of another DNA strand (the passage helix) through that break and finally the re-sealing of the DNA gate. The strand-passage reaction was studied by the use of novel DNA substrates and by site-directed mutagenesis of one of the gyrase proteins. The DNA substrates were used to attempt to define the DNA segments used by the enzyme as the DNA gate and passage helix in a catenation reaction. This was achieved by using oligonucleotides to form partial duplex regions in single-stranded DNA. A high-affinity gyrase cleavage site from the plasmid pBR322 was cloned into M13mpl8 and generated both the single and double-stranded circular forms of the molecule (MAT1). It was shown that gyrase could form a specific DNA gate in a short duplex region in single-stranded MAT1 when quinolone drugs were present. This DNA gate was much smaller than that normally utilised by the enzyme. The catenation and decatenation reactions were examined in detail with normal duplex substrates; reactions using a non-hydrolysable ATP analogue gave different results to those previously reported for the eukaryotic homologue of gyrase, indicating a possible mechanistic difference between the enzymes. Conditions under which the partial duplex substrates would be catenated were not found. Site-directed mutagenesis was used to alter arginine residues thought to interact with the passage helix during the reaction cycle. Assays of the mutant protein revealed that supercoiling activity was markedly reduced, but that partial activities of gyrase, such as the ATPase and DNA cleavage reactions, were close to wild-type levels.
Level: Doctoral
Qualification: Ph.D.
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Theses, Dept. of Biochemistry
Leicester Theses

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