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Title: The molecular basis of quinolone drug action on DNA gyrase.
Authors: Critchlow, Susan Elizabeth.
Award date: 1996
Presented at: University of Leicester
Abstract: Quinolones are a clinically-useful class of antibacterial agents known to target DNA gyrase, a bacterial type II topoisomerase. Gyrase is unique among topoisomerases in its ability to introduce negative supercoils into DNA using the energy derived from ATP hydrolysis. The active enzyme is composed of two GyrA and two GyrB subunits, forming an A2B2 tetramer of molecular weight 374 kDa. The mechanism of supercoiling by gyrase involves the ATP-driven passage of one segment of DNA through a gyrase-stabilised double-stranded break in another. Tyrosine 122 of E. coli GyrA becomes covalently attached to DNA when gyrase breaks the phosphodiester bonds of DNA during supercoiling. When this residue is mutated to serine or phenylalanine, gyrase can no longer cleave or supercoil DNA, but can bind DNA normally. Rapid-gel filtration experiments have shown that quinolones can still bind to proteins bearing these mutations, suggesting that DNA cleavage by gyrase is not required for quinolone binding. Transcription by T7 and E. coli RNA polymerases is blocked by the presence of a gyrase-quinolone-DNA complex. Mapping of the transcription termination sites in the presence of gyrase and quinolones shows that blocking occurs about 10 to 20 base-pairs upstream of the gyrase cleavage site. Blocking of transcription by T7 RNA polymerase by a gyrase-quinolone complex on DNA does not occur when the active-site tyrosine of gyrase is mutated to serine, which indicates that the polymerase blocking requires DNA cleavage. Analysis of transcription in the absence of drug suggest that RNA polymerase does not displace gyrase from the template. DNA gyrase is also the target of the CcdB protein which is encoded by the F plasmid. When its action is not prevented by CcdA protein, CcdB is a potent cytotoxin. Using in vitro transcription by T7 RNA polymerase, it has been shown that CcdB complexed with gyrase can block transcription in a similar manner to the gyrase-quinolone complex. Furthermore, in the presence of CcdA, CcdB can no longer induce gyrase to block transcription.
Type: Thesis
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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