Understanding the Effects of Continuous Rap1 Sumoylation on Cell Senescence in Saccharomyces cerevisiae

Abstract:

The goal of this study is to better understand the role of sumoylation in maintaining telomere length and governing cell senescence in the budding yeast Saccharomyces cerevisiae. Senescence serves as a major barrier in cancer cell proliferation and better understanding the mechanism in model organisms is essential for advancement in cancer biology research. The RAP1 gene in yeast is involved in the maintenance of telomeres, delaying senescence. However, when Rap1 becomes polysumoylated, this leaves the cell more susceptible to telomere fusion presumably due to the prolonged exposure of unprotected telomere ends. I hypothesize that the continuous sumoylation of Rap1 leads to senescence since this post-translational modification impairs Rap1’s function. Through this study, I also hope to better understand if telomere fusion also leads to senescence and if so, the timeline at which this process occurs. To create a condition in which Rap1 is continuously sumoylated, the gene that encodes the protein involved in degrading polysumoylated Rap1, ULS1, has been knocked out. The uls1∆ mutants will be compared to est1∆ mutants, because est1∆ mutants have been shown to undergo senescence earlier than wild-type cells. The senescence assay will consist of growing the appropriate strain in YPD media to saturation and diluting by 10⁵, followed by re-growing to saturation for up to five cycles. OD₆₀₀ readings which approximate cell density will be taken before and after each dilution to determine the generational time. Cells will be plated after each cycle and counted. Based on my hypothesis, I predict that the uls1∆ mutants will show a similar number of colonies after each cycle of dilution as the est1∆ mutants. In other words, the generation at which uls1∆ mutants undergo senescence will be comparable to that of est1∆ mutants.