Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae

Abstract

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2-Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3⌬ cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair.[Keywords: DNA replication fork; DNA damage; DNA repair; cell cycle checkpoint; BrdU; phosphatase; microarray] Supplemental material is available at http://www.genesdev.org.

Dephosphorylation of γH2A by Glc7/Protein Phosphatase 1 Promotes Recovery from Inhibition of DNA Replication

“…Furthermore, Pph3/PP4 is required for ␥H2A dephosphorylation, which contributes to recovery from a DSB-induced checkpoint (4,17,25). Finally, Pph3 and Ptc2 have been implicated in Rad53 dephosphorylation and hence deactivation during recovery from MMS exposure (26,32).…”

“…This process is known as recovery and is characterized by the disappearance of hyperphosphorylated Rad53 (reviewed in reference 16). Rad53 deactivation is also required for replication restart following transient exposure to methylmethane sulfonate (MMS) during early S phase (26,32). To date, the S. cerevisiae Ser/Thr phosphatases Ptc2 and Ptc3, as well as the protein phosphatase 4 (PP4)-like protein phosphatase Pph3, have been found to be important for checkpoint recovery after a single DSB (17,18).…”

“…Furthermore, Pph3/PP4 is required for ␥H2A dephosphorylation, which contributes to recovery from a DSB-induced checkpoint (4,17,25). Finally, Pph3 and Ptc2 have been implicated in Rad53 dephosphorylation and hence deactivation during recovery from MMS exposure (26,32).Notably, none of the above phosphatases is required for Rad53 deactivation after inhibition of DNA replication by deoxynucleoside triphosphate (dNTP) depletion with hydroxyurea (HU), as pph3⌬ ptc2⌬ ptc3⌬ triple mutant cells are not defective in recovering from HU-induced S phase arrest and do not show hypersensitivity to HU treatment (32,35). Thus, a yet-unidentified phosphatase might be responsible for checkpoint termination under these conditions.…”

Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

“…The best-characterized mechanism of checkpoint deactivation is via Rad53 phosphatases Pph3 and Ptc2. Deletion of Pph3 and Ptc2 results in elevated sensitivity to replication inhibitors and complete replication fork arrest (Szyjka et al 2008). This result demonstrates that proper control of the amplitude and inactivation of the S-phase checkpoint is also essential for DNA replication control in the presence of replication stress.…”

“…Other than Ptc2p, no outliers have been described previously to have a function in the S-phase checkpoint. No other Rad53 phosphatases were found to have different protein abundances in the SILAC data, and because of the redundancy of the Rad53 phosphatases, Ptc2 alone plays only a minor role in checkpoint recovery after MMS treatment (Szyjka et al 2008). Furthermore, we have shown previously that Isw2 and Ino80 clearly function independent of the Rad53 phosphatases (Au et al 2011).…”