Functional Characterization of a Novel Role for Rfa2 N‐terminal Hyper‐phosphorylation During Checkpoint Adaptation

Abstract

Replication Protein A (RPA) is an essential heterotrimeric complex found in all eukaryotic organisms and has critical roles in DNA metabolism and cell cycle progression. In human cells, RPA is known to be hyper‐phosphorylated, primarily on the N‐terminus of the second subunit (Rpa2), in response to genotoxic stress. The significance and function of Rpa2 N‐terminal (NT) phosphorylation has been the subject of over 25 years of research; however, the physiological function of this modification has yet to be clearly determined. In budding yeast, Rpa2 N‐terminal phosphorylation is detectable upon prolonged exposure to commonly used clastogens or upon generation of an irreparable break at the MAT locus using HO endonuclease. Upon cleavage of the MAT locus, the master checkpoint kinase Rad53 becomes hyper‐phosphorylated, leading to signaling events that arrest yeast cells at the G2/M checkpoint to allow adequate time for DNA repair.During checkpoint adaptation, cell cycle progression restarts, despite the presence of unrepaired DNA. Checkpoint adaptation affords cells the ability to bypass an established checkpoint, with the potential negative consequence of cellular division with broken chromosomes, leading to mutations and/or chromosome loss. Through the use of functional and phenotypic analysis of yeast containing Rpa2 NT mutations, we have determined that the phospho‐state of the Rpa2 NT may play a key role in mediating checkpoint adaptation. We demonstrate here that the Rpa2 NT is necessary for, and readily hyper‐phosphorylated during, checkpoint adaptation. We have determined the subregions of the Rpa2 NT that promote checkpoint adaptation when phosphorylated, as well as some of the conserved kinases that appear to phosphorylate the Rpa2 NT during checkpoint adaptation. Finally, we propose a model by which Rfa2 NT hyper‐phosphorylation directly leads to dephosphorylation/deactivation of the master checkpoint signaling kinase Rad53 in order to coordinate checkpoint adaptation in yeast. Understanding the mechanistic role the Rpa2 NT plays in coordinated checkpoint exit in the face of persistent damage may shed light on events that lead to deregulation of checkpoint signaling, and subsequently, mechanisms that lead to aberrant cell growth.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH