Studies on the mechanism of mos1 transposition
Transposable elements are mobile pieces of DNA that move inside the genome of their host and, can invade new genomes through horizontal transfer. They account for a major fraction of DNA in the genome of every organism including humans. Due to their mobility, transposable elements are major evolutio...
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University of Edinburgh
2005
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| Online Access: | http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.629585 |
| Summary: | Transposable elements are mobile pieces of DNA that move inside the genome of their host and, can invade new genomes through horizontal transfer. They account for a major fraction of DNA in the genome of every organism including humans. Due to their mobility, transposable elements are major evolutionary forces that can shape the genomes of their hosts. The Mosi element from Drosophila mauritiana is a member of the mariner family of transposable elements being one of the very few active mariners found to date. Mariner elements transpose through a cut-and-paste mechanism during which the element is excised from its original location and inserts into a new site. The same transposase enzyme catalyses both the excision and the integration steps. I have purified the Mosi transposase in a soluble form as a fusion to the Maltose Binding Protein of E. coli (MBP-Mosl). This fusion protein was used to characterise the steps leading to transposon excision with emphasis on the formation of a higher-order protein-DNA complex termed Paired End Complex. The results from the initial characterisation of MBP-MosI transposase are in good agreement with those obtained using the transposase purified through a denaturationrenaturation process. Next, the stoichiometry of Mosl Paired End Complex was studied using a mixture of MBP-Mosl fusion transposase and Mosl transposase. The results suggest a dimeric structure of Mosi Paired End Complex. Gel filtration experiments showed that Mosl transposase exists in solution as a mixture of monomers and dimers. The characterization of these forms showed that the monomer is the active form of Mosl transposase for DNA binding. The possible role of dimer formation in the regulation of Mosl transposition is also discussed. Transposition is a source of DNA double strand breaks, which are lethal to the host organism if they remain unrepaired. An assay to study the repair of these double-strand breaks is presented that takes advantage of Saccharomyces cerevisiae's ease of manipulation and wealth of knowledge regarding DNA repair processes. |
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