| Summary: | 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
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