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Evaluating the Impact of Replication Fork Pausing on Epigenetic Conversions in Saccharomyces cerevisiae: A Novel Approach and First Analysis

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Title: Evaluating the Impact of Replication Fork Pausing on Epigenetic Conversions in Saccharomyces cerevisiae: A Novel Approach and First Analysis
Author: Cheng, Ashley
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Yankulov, Joseph
Abstract: The subtelomeric regions of Saccharomyces cerevisiae undergo infrequent conversions in the expression state of a gene. The switch in gene expression is thought to be a consequence of a change in the epigenetic information encoded by post-translational modifications on histone proteins. Previous assays used to study epigenetic conversions involved the use of the counter selection drug, 5-FOA, but recent evidence has shown that 5-FOA can lead to unreliable conclusions. Using a new approach involving cellular fluorescence, I developed an assay that circumvents the need for 5-FOA. With flow cytometry, I analyzed the effects of a replication fork pause site on the frequency of epigenetic conversions. The results were cross-validated with microscopy and with the previous assay. However, the data was inconclusive. It is plausible that the design of the reporter protein may be responsible for skewing the results. I have included recommendations for further optimization. Furthermore, I discovered that a combination of serine to alanine point mutations to CDC7 and CDC28 phosphorylation sites, S94A-S238A and S238A-S515A, on Chromatin Assembly Factor I (CAF-I) can significantly alter the morphology of the cell. This morphology is consistent with arrest in the G1 phase of the cell cycle; therefore, this novel observation demonstrates that the deregulation of histone chaperone CAF-1 may cause deregulation of the cell cycle.
URI: http://hdl.handle.net/10214/13936
Date: 2018-05


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