Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663799
Title: A mutational analysis of motifs in EcoKI common to adenine methyltransferases
Author: Willcock, Damion F.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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Abstract:
Type 1 restriction/modification systems are complex multifunctional enzymes comprising three polypeptides, Hsd R, M and S. All three polypeptides are required to form a restriction enzyme but M and S alone are sufficient to form an N6 adenine methyltransferase. The Hsd M polypeptide of the type I system Eco K contains motifs characteristic of N6 adenine methyltransferases (Asn/Asp Pro Pro Phe/Tyr) and of methyltransferases in general (Phe X Gly X Gly). These motifs may identify amino acid sequences critical to methyltransferase function. This work describes an in vivo and in vitro analysis of site directed mutations within these two motifs and is the first report of such changes within a type I system. Biochemical analysis identifies the Asn/Asp Pro Pro Phe/Tyr region as critical to catalysis and in close proximity to the S-adenosylmethionine binding site. Mutations which remain within the overall consensus do not necessarily retain activity and hence indicate a degree of stringency associated with this motif. A mutation within the Phe X Gly X Gly motif abolishes SAM binding but retains DNA binding activity. In addition this mutant is temperature sensitive.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.663799  DOI: Not available
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