As the scientific community continues to push the boundaries of molecular biology with technological advances in the field of proteomics, there is a constant need for new methodologies in order to effectively utilize these advances. To truly understand genetic regulation, there is a need to study the genomic landscape on a single locus basis. To properly understand protein regulation in a situational context, post-translational modification must be acknowledged. The research described herein is based upon these principles and the goal is to develop new methods and utilize existing technologies toward these ends.
The first major component of this dissertation describes the identification of a post-translational modification that occurs on the Saccharomyces cerevisiae helicase Pif1 during replication stress. Experiments describing the identification of a phosphorylation site near the N-terminus and expression of a phosphorylation-deficient mutant to further characterize its function are detailed. Expression of this mutant in the absence of another helicase involved in DNA damage repair causes impaired growth during exposure to hydroxyurea-induced replication stress. This phenotype is found to be dependent upon the helicase activity of Pif1.
The second major component of this dissertation describes the development and improvement of a technique called CRISPR-ChAP-MS for the isolation and proteomic analysis of a single genomic locus. This technique involves the use of the Cas9 and guide RNA components of the CRISPR genome editing technology to specifically target a single genetic element for affinity purification and subsequent downstream processing by tandem mass spectrometry to identify associated proteins. The initial development and validation of the technique at the GAL1 locus in S. cerevisiae is described first. Then the subsequent optimization experiments leading to an increase in the relative enrichment of a targeted locus is detailed.
|Advisor:||Raney, Kevin D., Tackett, Alan J.|
|Commitee:||Edmondson, Ricky D., Eoff, Robert L., Raney, Kevin D., Tackett, Alan J., Wahls, Wayne P.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 78/12(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biochemistry|
|Keywords:||CRISPR, Mass spectrometry, Methodology, Pif1, Proteomics|
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