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Title: DNA binding activity and subunit interactions of the mariner transposase
Author: Zhang, Lei
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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The functional mariner element Mos1 is 1286 bp long with 28 bp imperfect terminal repeats and contains a single open reading frame encoding a 345 amino acid transposase. In this thesis, the N-terminal domain of mariner transposase was found to bind specificity to inverted repeats at both ends. The minimal DNA-binding domain was identified to be located between amino acid 1 and 120. This contains a helix-turn-helix (HTH) motif. Site-directed mutagenesis indicates that the HTH is required for sequence-specific recognition of the terminal inverted repeats by Mos1 transposase. The transposase has 5 to 6 times higher affinity for the right end sequence of Mos1 than for the left end. Yeast two-hybrid assays were performed to identify protein-protein interactions of Mos1 transposase. Deletion mutants suggest that residues required for the interaction of transposase monomers are distributed along the length of the protein. Twelve single point mutations which reduce subunit interactions have been isolated. The L124S mutation is located in neither the DNA-binding nor the catalytic domain. The purified Mos1(L124S) mutant reduces transposase activity in both excision and transposition assay, indicating that subunit interactions are involved in the transposition reaction. A change in target site selection other than TA dinucleotide observed with this mutant may reflect a change in the conformation of the transpososome. The promoter activity of Mos1 was detected in transgenic flies by P element-mediated germline transformation. The promoter of Mos1 lies within nucleotides 1-171 which shows low activity. There is an enhancer-like element in the region of nucleotides 172-516 that stimulates reporter gene expression. No autoregulatory inhibition of Mos1 transposase on its own promoter was observed. This suggests that "overproduction inhibition" may not act at the level of transcription through autorepression of the Mos1 promoter.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available