Identification of Novel RPA-Protein Interactions Using the Yeast Two Hybrid Assay and Identification of Regions Important for Interaction Between RPA and Rad24

Replication Protein A (RPA) [Replication Factor A (RFA) in yeast] is an ssDNA binding protein composed of Rpa1, Rpa2, and Rpa3 and involved in numerous DNA processing pathways such as Replication, Recombination, and Repair. It participates in such diverse pathways by its ability to interact with numerous proteins. The goal of my project was to find novel RPA-protein interactions using the yeast two hybrid assay. Using this method, we identified several known and unknown proteins that interact with Rfa1 and showed that these interactions were dependent on the phosphorylation state of Rfa2. Next, we determine the region important for interaction between Rfa1 and Rad24. Rad24 is a checkpoint protein important for initiation of the DNA damage checkpoint signaling. By using the ?- galactosidase assay, we determined the N-terminal region of Rfa1 (DBD-F) and the C-terminal region of Rad24 (460-660 aa) to be necessary for their interaction