Understanding and Exploiting the DNA Cleavage and Resealing Reactions of Bacterial Type II Topoisomerases

Baskerville, Victoria (2023) Understanding and Exploiting the DNA Cleavage and Resealing Reactions of Bacterial Type II Topoisomerases. Doctoral thesis, University of East Anglia.

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Abstract

Bacterial type II DNA topoisomerases resolve DNA topological problems by a DNA strand passage mechanism. Transient double-stranded covalent phosphotyrosine linkages form between the enzyme and DNA that are subsequently resealed are critical to the reaction. Exploitation of the reaction by antibiotics leads to accumulation of lethal double-stranded DNA breaks. However, antibiotic resistance is a problem. To address this, a novel series of antibiotics has been developed by Uppsala University: the quinazoliniums. Here, the compounds have been found to target DNA gyrase and DNA topoisomerase IV. The potency of the quinazoliniums has been assessed, and the binding site on DNA gyrase has been characterised using X-ray crystallography, which shows that the compounds bind at the DNA cleavage site without a water-metal ion bridge. The quinazoliniums have some cross-resistance to fluoroquinolone-resistant gyrase mutations, however, the lead quinazolinium is more active than ciprofloxacin at inhibiting DNA supercoiling in the quinolone-resistant mutant E. coli gyrase GyrA S83L.

Due to the importance of the DNA cleavage/resealing reaction to antibiotic action, this equilibrium has been investigated. A lysine residue the conserved YKGLG motif, and the Greek Key domain have been suggested to be involved in DNA resealing. Mutations in the lysine and Greek Key domain of Escherichia coli and Staphylococcus aureus gyrase leads to a reduced rate of reaction. Experiments investigating how the concentration of free metal ions influence the equilibrium between gyrase-DNA cleavage complexes and free DNA show the catalytic metal ions to be very dynamic. The DNA resealing and cleavage reactions have different metal ion concentration requirements. An adapted model for the metal-ion mechanism to catalyse the reaction, which takes into consideration the dynamic nature of the catalytic metal ion, is proposed.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	12 Oct 2023 10:24
Last Modified:	31 Mar 2024 01:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/93275
DOI:

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