In order to determine whether the mobility of transposable elements is affected by Ku, a genetic system using the Tyl retrotransposon in <I>Saccharomyces cerevisiae</I> was used. Phenotypic analysis of yeast strains containing a null mutation of either subunit of Ku showed that Tyl retrotransposition rates were markedly reduced compared to wild-type cells. This phenotype appears to be specific to mutations in Ku, as strains with mutations in other DNA repair proteins were analysed and no significant effect on retrotransposition was observed. Biochemical techniques were employed to determine the stage of the retrotransposon life-cycle at which Ku was acting to facilitate the process. It has been determined that transcription, translation, and reverse transcription of the retroelement and element-encoded proteins are unaffected by the absence of Ku. Furthermore, there is no defect in proteolytic processing of a precursor fusion protein, retrotransposon virus-like particle assembly, or reverse transcriptase activity in the absence of Ku. Examination of another retrotransposon shows that same phenotype in the absence of Ku, suggesting that the mechanism by which Ku is facilitating retrotransposition is a general one, and not likely to be via targeting the integration machinery to specific regions of the genome. Ku was demonstrated to co-fractionate with the virus-like particles during purification, and immunoprecipitation experiments using antibodies directed at one of the Ku subunits demonstrate an interaction with the retrotransposon cDNA and integrase, but not the reverse transcriptase. Ku has previously been shown to bind to DNA double-strand ends <I>in vitro</I>, so one model by which Ku could facilitate retrotransposition is by binding to the cDNA ends and protecting them from nuclease-mediated degradation. However, examination of purified cDNA from wild-type and <I>yku70</I> mutant strains shows no difference in the amount or integrity of DNA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:598628 |
| Date | January 1999 |
| Creators | Downs, J. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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