Exploring the genetic interactome of a Saccharomyces cerevisiae separase mutant

• Main Author: Ho, Krystina
• Language: English
• Published: University of British Columbia 2013
• Online Access: http://hdl.handle.net/2429/44279

The budding yeast Saccharomyces cerevisiae is used as a model organism for understanding cellular dynamics such as cell cycle regulation. Separase endopeptidases are essential to these events, being responsible for cleavage of cohesin – the molecular glue holding sister chromatids together. To examine the function of separases in a systematic fashion, a temperature sensitive mutant of the yeast homologue, Esp1, was subjected to a high-throughput technique known as synthetic genetic array to identify genetic interactions. Examination of the list of alleles confirmed to cause a synthetic lethal or synthetic dosage lethal phenotype in the query esp1-1 mutant (“hits”) established the legitimacy of these screens, as many related to known Esp1 functions. Surprisingly, categorization of these hits by biological process revealed an enrichment for genes involved in RNA metabolic processes. Concurrent affinity immunoprecipitation of separase followed by mass spectrometry further showed a physical interaction between Esp1 and the integrase portion of Ty1 retrotransposons. Evidence that this interaction was indicative of a role for separase in retrotransposition was attained when the esp1-1 mutant was found to have transposition defects. Similar defects were also present in mutants of both the cohesin loader, SCC2, and the structural maintenance of chromosome, SMC3, genes. Interestingly, cohesin loads at loci transcribed by RNA polymerase III, while hotspots of Ty1 integration are upstream of the same sites. I propose that separase acts as a bridge to aid in targeting the Ty1 pre-integration complex to these hotspots. The aforementioned categorization of esp1-1 genetic interactors also indicated that separase may somehow be involved in mRNA biogenesis. As esp1-1 mutants have intact translational machinery, my data suggests that separase functions either directly in transcription or, more likely, post-transcriptionally. In all, screening for esp1-1 genetic interactions has revealed several new avenues for separase studies. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate