Characterization of RPA‐protein interactions and their dependence on the phospho‐mimetic state of the N‐terminus of Saccharomyces cerevisiae Rfa2 (735.13)

Abstract

Replication protein A (RPA) is a heterotrimeric complex in all eukaryotic cells that is essential for DNA replication, repair/recombination, and cell cycle regulation. A common intermediate of each of these DNA processes is the formation of single‐stranded DNA (ssDNA) and the major biochemical function of RPA is to bind to ssDNA. Therefore, RPA not only acts as a sensor of incomplete duplex DNA, but also as a recruiter of factors necessary to process this intermediate. It is clear that RPA interacts with a number of proteins; however, many of these interactions have not been well characterized in yeast, making unclear the physiological role of each RPA‐protein interaction in cellular DNA metabolism.It is well known that in response to DNA damage, the N‐terminus of the human 32‐kDa subunit (Rpa2) becomes phosphorylated, and this has been demonstrated to modulate some RPA‐protein interactions. Although phosphorylation of the Rfa2 N‐terminus has not been definitively demonstrated during the DNA damage response, we have demonstrated in our lab that phospho‐mutant forms of the budding yeast Rfa2 N‐terminus show DNA damage‐dependent phenotypes. Therefore, we hypothesize that the phosphorylation state of Rpa2 (or Rfa2) coordinates the response to DNA damage through regulation of RPA‐protein interactions. The goals of this research were: 1) to identify yeast RPA‐protein interactions, 2) to map regions important for protein interaction, and 3) to characterize whether these protein interactions are dependent on the phosphorylation state of the Rfa2 N‐terminus We have identified a number of RPA‐protein interactions that are dependent on the state of the Rfa2 N‐terminus, and we have mapped the region(s) of RPA that are important for some of these interactions. This combined data suggests potential models for how the Rfa2 N‐terminal phosphorylation state may be regulating protein interactions to coordinate RPA cellular function. Grant Funding Source: Supported by NIH, NIJ, NSF