Browsing by Subject "Budding Yeast"
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Item Investigation of the function and control of Dia2, a regulator of genomic stability in budding yeast.(2009-11) Kile, Andrew CraigMaintenance of genomic integrity can be particularly challenged during DNA replication, which is critical for cellular viability and proliferation. Cancer cells exhibit loss of genomic integrity, thus it is critical to understand the pathways involved in genome maintenance. We have identified the F-box protein Dia2 as a novel and previously unappreciated mediator of genome stability. F-box proteins are substrate specificity subunits of SCF ubiquitin ligases for ubiquitin mediated proteolysis, although most remain uncharacterized in their function or targets. Deletion of the DIA2 gene in Saccharomyces cerevisiae leads to genomic integrity defects, and the Dia2 protein associates with chromatin and origins of replication, indicating it performs a chromatin-associated role in DNA replication. Interestingly, the Yra1 protein was identified to physically interact with Dia2 and promotes Dia2 binding to replication origins yet is not a proteolytic substrate of SCF-Dia2. The Dia2 protein itself is subject to proteolysis, but is stabilized by the activation of the replication checkpoint and this suggests it plays a role during periods of replication stress and DNA damage during S phase. Surprisingly, Dia2 turnover is not controlled by an autocatalytic mechanism involving its F-box domain, but instead relies on a region upstream of its F-box that controls both its stability and nuclear localization. Replication checkpoint activation leads to inhibition of late-firing origins, stabilization of replication forks, as well as stabilization of the Dia2 protein. Our observations indicate that SCF-Dia2 activity performs ubiquitin ligase activity at one or both of these sites that are regulated by the checkpoint. These studies establish a novel link between DNA replication and genomic integrity to the SCF ubiquitin ligase via Dia2.Item A Quantitative Exploration of Tension Sensing at Metaphase in Budding Yeast(2018-07) Mukherjee, SoumyaDuring mitosis, motors associate with microtubules to exert forces that push spindle poles apart, thus establishing a mitotic spindle. These pushing forces in turn cause tension in the chromatin that connects oppositely attached sister chromosomes. This tension has been hypothesized to act as a mechanical signal that allows the cell to detect chromosome attachment errors during mitosis. However, the magnitude of changes in tension that could be detected by the cell to initiate an error correction response during metaphase has not been measured, and the underlying mechanics of tension based error detection and error correction remains unknown. In this study, we generated a gradient in tension over multiple isogenic budding yeast cell lines by genetically altering the magnitude of motor-based spindle forces. This allowed us, for the first time, to quantitatively elucidate the mechanics of tension based error detection pathway in mitosis. We found that a decreasing gradient in tension led to an increasing gradient in rates of kinetochore detachment and anaphase chromosome mis-segregration, with a corresponding gradient in metaphase times. Further, these tension-based cellular response gradients were abrogated in the absence of key error-correction pathway proteins. The underlying mechanism involves as increasing gradient in the degree of phosphorylation of proteins, comprising the load-bearing component of the kinetochore-microtubule interface, in response to a decreasing gradient in the magnitude of tension. We conclude that the cell is exquisitely tuned to the magnitude of tension as a signal to detect potential chromosome segregation errors during mitosis.