Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597811
Title: Investigating and engineering the substrate specificity of DNA methyltransferases
Author: Cohen, H.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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Abstract:
DNA methyltransferases catalyse the transfer of methyl groups from the cofactor S-adenosyl-L-methionine to a target base (adenine or cytosine) within a cognate recognition sequence. I have studied cofactor binding, DNA specificity and the role of conserved amino acid motifs in the cytosine C5 methyltransferase M.HaeIII. By measuring the competitive inhibition of methylation by a series of cofactor analogues, each modified at a single position, the importance of each functional group for cofactor binding to M.HaeIII was probed. The functional significance of amino-acid residues in M.HaeIII was investigated using in vitro compartmentalisation (IVC), an activity-based selection method. IVC was used to obtain active variants of M.HaeIII from libraries diversified at conserved motifs in the catalytic and DNA binding domains. M.HaeIII modifies the central cytosine of the sequence (5’-GGCC-3’). Using bisulphite sequencing, cytosines in a variety of other sequence contexts were found to be methylated at lower levels by M.HaeIII both in vivo and in vitro. IVC was then used to select mutant enzymes with an improved ability to methylate the non-canonical site AGCC.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.597811  DOI: Not available
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