Replication Protein A (RPA) is a heterotrimeric, single-stranded DNA binding complex with critical roles in DNA metabolism and cell cycle progression. While human RPA is observed to be hyper-phosphorylated in response to genotoxic stress, primarily on the N-terminus (NT) of the 32 kDa subunit (Rpa2), the Rfa2 NT of yeast Replication Factor A (RFA) had not been identified as a major site of phosphorylation upon DNA damage induction. Despite this, examination of Rfa2 NT phospho-mutants strongly suggested that the phospho-state of the Rfa2 NT plays a key role in cell cycle progression, especially when DNA damage persists. This phenomenon is termed checkpoint adaptation, and although it provides the cell the chance to survive and grow, it comes with the negative consequence of increased genomic instability that can ultimately lead to cellular disease.
Using Saccharomyces cerevisiae, it was demonstrated that in the presence of persistent DNA damage, Rfa2 NT hyper-phosphorylation is readily detected, and that the Rfa2 NT is required for checkpoint adaptation to occur. Phosphorylation of the Rfa2 NT is also observed upon prolonged exposure to an array of genotoxic agents. Using checkpoint kinase deletion strains, some kinases responsible for phosphorylation of the Rfa2 NT during genotoxic stress have been identified.
Additionally, a chimeric yeast Rfa2 containing the human Rpa2 NT was examined. This chimeric protein was previously demonstrated to be hyper-phosphorylated in response to an array of DNA damaging agents. The yeast Rfa2 containing the human Rpa2 NT displayed earlier and more robust phosphorylation after stress than cells containing wild-type (WT) Rfa2. Furthermore, this robust phosphorylation of the chimeric Rfa2 promoted dephosphorylation of Rad53 and even earlier and more efficient checkpoint adaptation than in WT Rfa2-containing yeast cells.
Taken together, the data suggest a model where Rad53 deactivation (necessary for checkpoint bypass) and Rfa2 NT phosphorylation are linked to coordinate checkpoint adaptation in yeast. Specifically, Rfa2 NT phosphorylation drives Rad53 dephosphorylation to promote bypass of the checkpoint despite the continued presence of DNA lesions. Understanding how the Rfa2 phospho-state influences cell cycle progression in the presence of genotoxic stress will assist in deciphering the molecular signaling mechanisms cells employ to maintain the integrity of their genomes.