Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548212
Title: Novel antibiotics from DNA adenine methyltransferase inhibitors
Author: Maynard-Smith, Michael David
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2009
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Abstract:
DNA adenine methylation plays a role in several core bacterial processes, including DNA mismatch repair, the timing of DNA replication and gene expression. The dependence of bacterial virulence on the activity of Dam, an adenine methyltransferase, makes it an attractive target for novel antibiotics. Dam from Yersinia pestis, the plague causing bacteria, was expressed and purified by nickel affinity chromatography. A plasmid containing the methylation sensitive restriction endonuclease dpnI gene was assembled. DpnI was expressed and purified by nickel affinity chromatography. A Y. pestis Dam activity assay, which relied on the sensitivity of DpnI to DNA methylation, was developed. This continuous fluorescence based assay was used to determine several kinetic parameters of the enzyme. The assay was validated for use in a high throughput 96-well format and used to screen a library of one thousand compounds for Y. pestis Dam inhibitors. Several compounds of interest were identified. These compounds were synthesised, re-tested for activity against Dam, counter- screened against the restriction endonuclease DpnI and assayed for an ability to bind DNA. A plasmid encoding Dam from the hyperthermophile Pyrococcus horikoshii was assembled. Wild type P. horikoshii Dam was expressed and purified by nickel affinity chromatography. The resultant Dam was contaminated by a product of mis-initiation of translation at an internal methionine codon. Site directed mutagenesis was undertaken to replace the problematic codon in the dam gene. Mutant P. horikoshii Dam was expressed and purified by nickel affinity chromatography. This hyperthermophilic enzyme was used in the development of a direct and continuous fluorescence based assay for Dam activity. Several kinetic parameters of P. horikoshii Dam were determined with the direct assay
Supervisor: Roach, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.548212  DOI: Not available
Keywords: QD Chemistry
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