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Title: Investigation of de novo methylation activity in mutants of the EcoKI methyltransferase
Author: Winter, Markus
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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A group of mutants of the EcoKI R/M-system displaying de novo methylation activity have been isolated (Kelleher et al., 1991). The mutated genes were transferred into an overexpressing plasmid vector. Two of the over-expressed proteins were purified to near homogeneity from clones transformed with the plasmids. Cofactor binding activities of wild-type and the two mutant enzymes were compared by 1,8-anilino-napthalene sulphonic acid fluorescence displacement experiments. A DNA methylation assay based upon the transfer of a tritiated methyl group from the cofactor AdoMet to the substrate DNA was established and used to examine the dependency of the reaction on cofactor, substrate, and enzyme concentration. In addition the stability of the trimeric enzyme at different protein concentrations was followed by HPLC gel filtration. Sequence alignments, secondary structure predictions, and tertiary structural modelling were used to show the similarity of the Type I system EcoKI with methyltransferases from other classes (especially Type II methyltransferases), thereby establishing a structural and suggesting an evolutionary link between the different methyltransferase classes. The information obtained by these comparisons enabled the subsequent modelling of a more refined model of the EcoKI structure. A model is proposed to explain the different activities observed in wild-type and mutant enzymes based on the biochemical and structural data obtained during these investigations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available