Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663449
Title: The sequence specific DNA binding activity of pogo transposase
Author: Wang, Hongmei
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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Abstract:
In this thesis, the two ORFs believed to code for pogo transposase were joined in frame by PCR and cloned into a pGEX vector. The pogo transposase was fused to the C-termini of glutathione-S-transferase (GST) and expressed in E. coli. The DNA binding activity of pogo transposase was tested by gel retardation assays in the presence of specific and non-specific competitors. pogo transposase was shown to be able to bind specifically to the end sequences of the element. The transposase binding sites within the pogo element were identified by testing the transposase binding ability of deleted end sequences of the element. A 12 base pair consensus sequence was found and shown to be responsible for binding to the transposase. There are several copies of the 12 bp transposase binding site located near each end, in the 5' subterminal region, and in the middle of the element respectively. The binding sites at the ends might be involved in forming the synaptic DNA-protein complex and others in regulating transposition. The DNA binding domain of pogo transposase was identified by expressing different regions of the transposase and determining their sequence specific DNA binding ability. The DNA binding domain has been shown to be located in the N-terminal 75 amino acid region. Site direct mutagenesis was used to study the role of the predicted helix-turn-helix (HTH) motif located within the DNA binding domain. Substitution of the positively charged basic amino acids within the recognition helix by alanine abolished the DNA binding activity of the protein. Substitutions introducing prolines into the first or second helices to disrupt their structures also greatly reduced the DNA binding activity of the protein. These data support the idea that the HTH motif in the DNA binding domain is responsible for the specific DNA binding activity of the transposase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.663449  DOI: Not available
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