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Title: The role of DEAD box motifs in the restriction of DNA by EcoKI
Author: Webb, Julie Lynette
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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EcoKI cuts or modifies DNA according to the methylation states of specific adenine residues in its target recognition site. If these residues are unmethylated the DNA is cut, often thousands of base pairs away from the recognition site. Communication between EcoKI bound to the recognition site and the cleavage site occurs by DNA translocation, which is driven by ATP hydrolysis. The amino acid sequence of the subunit required for restriction contains seven motifs that are conserved in the DEAD box family of proteins. This family is a sub-group of the superfamily of DNA and RNA helicases. Previous studies on DEAD box proteins have found these motifs are involved in the ATPase and helicase activities of these proteins. To assess the importance of the DEAD box motifs in the restriction of DNA by EcoKI, amino acid residues in each of the seven motifs were changed and the effects of these substitutions on restriction were investigated. Eight proteins, each containing a different amino acid substitution, were purified and the DNA binding abilities, nuclease activities and ATPase activities of the proteins were studied. All changes had an effect on restriction except those changes in motif IV. Motif IV was defined prior to the discovery of a frame-shift in the hsdR DNA sequence and these results suggest it has been incorrectly identified. An A619G substitution in motif III slightly impaired restriction, but other substitutions at this position (A619D and A619V) abolished restriction. All other changes prevented any DNA restriction. The nuclease assays with the purified proteins confirmed the in vivo results. None of the available evidence indicates that the amino acid substitutions prevent interaction with ATP. The protein with a D577H substitution in motif II showed a reduced DNA binding ability, none of the other changes investigated affected DNA binding.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available