Browsing by Author "Ghospurkar, Padmaja Laxman"

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    Characterization of RPA2 N-terminal Function in the DNA Damage Response in Saccharomyces Cerevisiae
    (North Dakota State University, 2015) Ghospurkar, Padmaja Laxman
    In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. A key factor involved in the DNA damage response is the heterotrimeric protein complex Replication Protein A (RPA), which is not only essential for the repair of damaged DNA, but also is post-translationally modified on at least two of the three subunits in response to DNA damage by checkpoint kinases. Of particular interest is the 32-kDa subunit, called Rpa2, which is hyper-phosphorylated on its serine/threonine-rich N-terminus following DNA damage in human cells. This unstructured N-terminus is often referred to as the phosphorylation domain (PD) and is conserved amongst eukaryotic Rpa2 subunits, including Rfa2 in Saccharomyces cerevisiae. In this work we aim to characterize the function of Rfa2 N-terminus (Rfa2 NT) in DNA damage response and develop yeast as a tool to study human RPA. With the help of mutagenesis we developed Rfa2 NT extreme mutants, which showed that the phosphorylation of Rfa2 NT is dispensable in DNA damage response. However, the presence of Rfa2 NT is essential for cells to survive in stressed condition indicating an uncharacterized function. We further discovered seven S/T sites are responsible for the damage sensitive phenotype of Rfa2 NT extreme mutants. And the phosphorylation affects protein interaction of RFA complex. Although, the phosphorylation event of Rfa2 NT is dispensable in S. cerevisiae the cells have conserved the ability to phosphorylate Rfa2 N terminus. With the help Rfa2 NT fusion mutants we showed that S. cerevisiae could phosphorylate N terminus from seven different eukaryotic species. Hence, we successfully developed yeast as a tool to study Rpa2 phosphorylation amongst various eukaryotic species.