Date of Award
Doctor of Philosophy
Zoology and Genetics
Molecular, Cellular, and Developmental Biology
Daniel F. Voytas
The nonrandom distribution of retrotransposons and retroviruses in their host genomes suggests that they actively select integration sites. The integration site specificity is particularly strong for retrotransposons, which rely on their host to persist and may use targeting to avoid disrupting important host sequences. This is the case for the yeast Ty5 retrotransposons, which preferentially integrate into domains of silent chromatin at telomeres and the HM loci. Loss of silent information regulator three or four (Sir3p or Sir4p)---components of silent chromatin---causes a greater than nine-fold decrease in Ty5 targeting to the HM loci and largely randomizes chromosomal integration patterns. Loss of Sir4p also causes a ten-fold increase in cDNA recombination, which is due to the expression of a/alpha mating-type information and the absence of Sir4p. It is known that in old yeast cells or in strains carrying the sir4-42 allele, the Sir complex relocalizes to the rDNA. About 26% of Ty5 insertions occur within the rDNA in sir4-42 strains compared to 3% in wild type. Ty5, therefore, is sensitive to changes in chromatin, indicating that retrotransposons may be useful for understanding chromatin dynamics during developmental programs such as aging. Based on the evidence that silent chromatin and Sir proteins are important for Ty5 target specificity, two-hybrid assays were used to test the interaction between integrase and Sir proteins. Interaction was detected between the Ty5 integrase C-terminus and the Sir4p C-terminus. This interaction was greatly reduced by mutations that abolish target specificity. Furthermore, the targeting domain by itself is sufficient for the interaction, although it occurs at reduced levels. The Sir4p interaction domain was narrowed down to amino acids 971 to 1237 through serial deletions. Two amino acid residues, tryptophan 974 and arginine 975, were identified as critical for the interaction. Tethering the SIR4 C-terminus to non-silent chromatin creates Ty5 integration hotspots, suggesting that the interaction between integrase and Sir4p is the driving force behind Ty5 target specificity. These findings may make it possible to do site-specific genome manipulation and to develop better gene therapy vectors, whose integration sites can be controlled.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Zhu, Yunxia, "Dissecting the targeting mechanism of the Saccharomyces retrotransposon Ty5 " (2001). Retrospective Theses and Dissertations. 1095.