Date of Award
Doctor of Philosophy
Zoology and Genetics
Bioinformatics and Computational Biology; Genetics;
Daniel F. Voytas
Ty5 is a retrotransposon in Saccharomyces cerevisiae. In wild type yeast strains, over 90% of Ty5 insertions occur at the telomeres or HM loci. These regions are bound in silent chromatin, which is analogous to heterochromatin of higher eukaryotes. Our laboratory previously described a targeting mutant of Ty5 that has an amino acid substitution near the C-terminus of integrase. Targeting to silent chromatin is reduced more than 20-fold in the mutant. We further defined the Ty5 targeting domain (TD) by saturation mutagenesis. All of the targeting mutations mapped to a stretch of six amino acids (LDSSPP). We tethered TD to a crippled HMR-E silencer by fusing it to the GAL4 DNA binding domain (GBD-TD). The GBD-TD fusion could nucleate the assembly of silent chromatin, as measured by the transcriptional status of an adjacent marker gene. This silencing was dependent upon the silent chromatin components Sir2p, Sir3p, and Sir4p. When over-expressed, GBD-TD was found to disrupt silencing at the telomeres. These results support our model that Ty5 target specificity results from a protein-protein interaction, which recruits the Ty5 pre-integration complex to silent chromatin. We further determined that TD interacts with Sir4p, a structural component of silent chromatin. This interaction between TD and Sir4p C-terminus was demonstrated by two-hybrid assays and in vitro affinity binding. We noticed that TD is able to bind Sir4p in vitro when it is expressed and purified from yeast cells but not from E. coli cells. In addition, protein phosphatase treatment of TD disrupts this binding. When we replaced the serines of TD with threonine or glutamate, TD is partially functional, and mutant Ty5 elements are able to target effectively. We inferred that TD is phosphorylated by kinases and that TD function depends on this modification. A collection of kinase knockout strains was used to identify the kinases that modify TD. Dun1p, the DNA damage checkpoint kinase, is the most likely kinase involved in Ty5 targeting and TD modification. This finding sheds light on how Ty5 targeting is regulated by the host and further defines the close relationship between Ty5 and its host.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Xie, Weiwu, "The mechanism of integration preference to heterochromatin of yeast retrotransposon Ty5 " (2003). Retrospective Theses and Dissertations. 1475.