Coulthard, Steven John (2025). Engineering methyltransferase-cofactor binding interactions: towards therapeutic and diagnostic applications. University of Birmingham. Ph.D.
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Coulthard2025PhD.pdf
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Abstract
DNA methylation is an important epigenetic modification that regulates gene expression and maintains cellular function. The main cofactor for this process, S-adenosylmethionine (SAM), provides the methyl group necessary for DNA methyltransferases (DNMTs), enzymes that catalyse the addition of methyl groups to cytosine residues. While SAM is essential for maintaining normal DNA methylation patterns, its limitations—such as poor bioavailability, lack of tissue specificity, and off-target effects—present challenges for therapeutic applications that target DNA methylation in diseases like cancer, neurological disorders, and developmental diseases. To address these challenges, the development of SAM analogues with improved stability, selectivity, and targeted delivery could enhance the precision and efficacy of epigenetic therapies. Furthermore, the exploration of mutant DNMTs, engineered to recognize and modify specific DNA sequences or epigenetic marks, offers a promising strategy to enhance the specificity of DNA methylation-based interventions.
This thesis presents the synthesis of two novel analogues of SAM using an efficient, protecting-group free synthesis to create N6-substitued analogues. The activity of these analogues was tested on DNA with two methyltransferases (M.TaqI and M.MpeI). The M.MpeI active site has been probed, and mutations to the active site were achieved and subsequently tested against multiple cofactors using a new in vitro transcription translation method. Mutants of interest were upscaled to discern the required concentrations of cofactor and MTase DNA activity and whether this was comparable to the wild type M.MpeI. This thesis reports identification of a new cofactor and a mutant MTase that are compatible with one another. However, this mutant MTase is incompatible with SAM which makes this mutant a more selective MTase that could be expanded upon for diagnostic and therapeutic purposes.
| Type of Work: | Thesis (Doctorates > Ph.D.) | |||||||||
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| Award Type: | Doctorates > Ph.D. | |||||||||
| Supervisor(s): |
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| Licence: | All rights reserved | |||||||||
| College/Faculty: | Colleges > College of Engineering & Physical Sciences | |||||||||
| School or Department: | School of Chemistry | |||||||||
| Funders: | Engineering and Physical Sciences Research Council | |||||||||
| Other Funders: | UK Research and Innovation | |||||||||
| Subjects: | Q Science > QD Chemistry | |||||||||
| URI: | http://etheses.bham.ac.uk/id/eprint/16326 |
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