Saccharomyces cerevisiae as a Tool to Examine Rpa2 N‐Terminal Phosphorylation Across Common Eukarya

Abstract

BackgroundReplication Protein A (RPA) is a heterotrimeric complex that serves critical roles in eukaryotic genome duplication and maintenance. In human cells, the N‐terminus of the 32‐kDa subunit of RPA (Rpa2) is hyper‐phosphorylated by multiple kinases in response to DNA damage. We demonstrated previously in the budding yeast Saccharomyces cerevisiae that Rpa2 N‐terminal phosphorylation does not occur under genotoxic conditions, yet this domain is vital for the damage response.Study Objective: Determine why there is a difference between yeast and human Rpa2 phosphorylation in response to genomic insult and its importance for the DNA damage response.Methods: We utilized chimeric Rpa2 proteins, yeast Rpa2 antibodies, and human Rpa2 phospho‐specific antibodies to examine the potential for human and other eukaryotic Rpa2 N‐termini to be phosphorylated and their contribution to the DNA damage response in yeast.Results andConclusionsWe determined that the human Rpa2 N‐terminal domain is phosphorylated in yeast. Furthermore, hyper‐phosphorylation of chimeric Rpa2 in yeast occurs in a manner analogous to that of Rpa2 in human cells. We also determined the yeast kinases responsible for phosphorylating specific residues within the human N‐terminus of chimeric Rpa2. Finally, we identified other eukaryotic Rpa2 N‐termini that have the potential to be phosphorylated. Identification and characterization of phosphorylation of eukaryotic Rpa2 N‐termini in yeast allow for an understanding of why yeast Rpa2 phosphorylation does not occur as well as insight into the evolutionary significance of higher eukaryotic Rpa2 phosphorylation.Funding: This research was supported by NSF‐CAREER 1253723 to SJH.