A Novel Role For RPA Phosphorylation In The Regulation Of G2 Exit In The Face Of Persistent DNA Damage

Abstract

BackgroundReplication Protein A (RPA), a heterotrimeric complex with critical roles in DNA metabolism and cell cycle progression, is observed to be hyper‐phosphorylated in response to genotoxic stress. Hyper‐phosphorylation of human RPA (hRPA) occurs primarily on the N‐terminus (NT) of the 32 kDa Rpa2 (hRpa2) subunit. In contrast, phosphorylation of yeast RPA (yRPA) on the Rpa2 (yRpa2) NT is not detectable upon exposure to commonly used genotoxic agents (e.g., methyl methanesulfonate, hydroxyurea). In fact, the yRpa2 NT has never been identified as a major site of phosphorylation upon DNA damage treatment. However, the examination of yRpa2 NT phospho‐mutants suggests that the phospho‐state of yRpa2 NT may play a key role in cell cycle progression despite the continued presence of DNA damage (checkpoint adaptation). In some instances checkpoint adaptation might allow for the positive cellular consequence of continued cell growth. This comes with a potential negative organismal cost, as checkpoint adaptation is often detrimental to cell survival and is also associated with increased genomic instability, which can ultimately lead to mutations that cause cellular disease.Study ObjectiveTo elucidate the importance of the yeast Rpa2 NT in regulating exit from the G2/M checkpoint.MethodWe examined yRpa2 phosphorylation and the cell cycle progression of yRpa2 NT mutants in an inducible HO endonuclease system that generates a single double‐strand break (DSB) that cannot be repaired by homologous recombination (HR).Results and ConclusionsWe have demonstrated that the presence of the yRpa2 NT is necessary for checkpoint adaptation. Furthermore, we have recently discovered that in the presence of a persistent lesion, yRpa2 NT hyper‐phosphorylation is now readily detected. This is consistent with our published data demonstrating that checkpoint adaptation is affected in yRpa2 phospho‐mutant cells. Because N‐terminal phosphorylation is now detectable under this condition, we have employed alanine scanning mutagenesis to identify which sites on the yRpa2 NT are phosphorylated and important for promoting checkpoint adaptation. We have also examined the kinases responsible for phosphorylating the yRpa2 NT during checkpoint adaptation. In addition, we have evidence that phosphorylation of the yRpa2 NT occurs primarily upon prolonged exposure to other agents that specifically result in DSB formation. Finally, we propose a model that suggests that Rad53 deactivation and Rfa2 phosphorylation may be linked to coordinate checkpoint adaptation in yeast. Understanding how yRPA phospho‐states influence cellular division in the presence of persistent/prolonged genotoxic stress may help provide the framework for deciphering the molecular mechanism(s) cells employ to maintain their genome.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH.