The role of DNA gyrase in illegitimate recombination

Bush, Natassja (2017) The role of DNA gyrase in illegitimate recombination. Doctoral thesis, University of East Anglia.

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DNA, due to its double-helical structure, is subject to changes in topology due to the nature of transcription and replication. To overcome this, cells have processes and enzymes that ameliorate these changes. One such group of enzymes are the DNA topoisomerases, which are responsible for the maintenance of DNA topology. Despite this important role, these enzymes participate in illegitimate recombination (IR), which is genetic recombination between regions of DNA that share little or no homology. This can result in chromosomal rearrangements and is often a consequence of DNA-damaging agents. A consequence of topoisomerase-induced IR is thought to be therapy-related acute myeloid leukaemia (tAML). Analogously, there is evidence that exposure to sublethal concentrations of ciprofloxacin, a topoisomerase inhibitor, can cause resistance to non-quinolone antibiotics. This may work by a similar mechanism as that proposed for t-AML. This project centres around the examination of DNA gyrase-mediated IR focussing on the proposed subunit-exchange model. Using Blue-Native PAGE, I set up an assay to examine subunit exchange in topoisomerases. I have also characterised previously identified gyrase hyper-recombination mutations, known to increase the frequency of IR. Furthermore, I have investigated quinolone-induced antibiotic resistance and what the mechanism is. Here, I show that DNA gyrase can undergo subunit exchange, and that this seems to occur within higherorder oligomers of the enzyme, which have not been investigated before. Biochemical characterisation of the hyper-recombination mutations shows that they impair DNA gyrase activity which, in vivo, may have downstream consequences that may lead to IR. Using an in vivo assay where E. coli is treated with subinhibitory levels of quinolones, I have seen resistance to other non-quinolone antibiotics. This is not seen when other antibiotics, including other topoisomerase inhibitors, are tested. Whole genome sequencing has revealed point mutations that explain the resistances seen, however other larger chromosomal modifications have been observed as well.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Megan Ruddock
Date Deposited: 23 Mar 2018 15:21
Last Modified: 23 Mar 2018 15:21


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