Phosphorylation of Replication Factor A Promotes Checkpoint Adaptation in Adaptation‐Deficient Phosphatase Deletion Strains
Authors: Baumgartner Trevor A , Haring Stuart J 
Published: November 2023

The cell cycle has long been studied and characterized. Major events (G1, S, G2, and M) occurring within the cycle are well understood; however, the molecular mechanisms controlling how the cell cycle is regulated are still being deciphered. Since the events of the cell cycle must occur in a proper temporal order, the progression of each step in the cell cycle must be carefully monitored. In the event that a particular step does not occur properly or that the cell is not ready to move to the next step, arrest of the cell cycle occurs (through the activation of a “checkpoint”). In the case of the introduction of double‐stranded DNA breaks (DSBs), cells initiate a checkpoint, arresting cells prior to the completion of mitosis (G2/M checkpoint). Processing of a DSB requires initial DNA resection resulting in single‐stranded DNA (ssDNA), which is bound by the ssDNA‐binding complex Replication Factor A (RFA). This leads to the eventual phosphorylation on the N‐terminus (NT) of the second subunit of RFA (Rfa2).

The signaling of the G2/M checkpoint involves several kinases, many of which also require phosphorylation to promote their activity. The phosphorylation/dephosphorylation of Rad53 (yeast Chk2) has long been used as a marker for checkpoint activation/exit. Previous results from our laboratory indicate that Rfa2 NT phosphorylation may play a key role in exit of the checkpoint through dephosphorylation of Rad53. Checkpoint exit can occur after complete repair of damaged DNA (checkpoint recovery), and it can also occur without the repair of difficult‐to‐repair DNA damage (checkpoint adaptation). Both checkpoint recovery and checkpoint adaptation require the dephosphorylation of Rad53 by a combination of phosphatases. Three phosphatases that have been shown to act on Rad53 previously include Pph3‐Psy2 (PP4), Ptc2, and Ptc3.

To determine if Rfa2 phosphorylation acts through/promotes phosphatases to enable checkpoint adaptation, several phosphatase knockout (KO) strains were created. In combination with the phosphatase KO's, Rfa2 NT mutant forms that either mimic Rfa2 hyper‐phosphorylation or prevent Rfa2 phosphorylation were used to replace wild‐type Rfa2. Strain sensitivity to several DNA damaging agents was characterized, and some phosphatase KO's enhanced the sensitivities of particular Rfa2 mutants. Phosphatase KO's display an inability to exit from the G2/M checkpoint in our system (i.e., a checkpoint adaptation deficiency); however, the Rfa2 NT mutant mimicking phosphorylation or a hybrid Rfa2 form that can be hyper‐phosphorylated suppressed the adaptation deficiency in pph3Δ and psy2Δ cells, both microscopically and biochemically. Since suppression occurs in single pph3Δ and psy2Δ mutants, it is also important to determine if suppression is mediated through other phosphatases, Ptc2 and/or Ptc3 (currently ongoing). To date, the only mutations reported to suppress checkpoint adaptation deficiencies all lie within RFA, suggesting that RFA is ultimately what determines checkpoint exit. We further submit that the state of the Rfa2 N‐terminus plays a key role in driving checkpoint exit.

Support or Funding Information

This work was supported by award NSF‐CAREER‐1253723 from the National Science Foundation to SJH.