Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage

Abstract

Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3-Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. In the absence of Pph3-Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.

Fig. 1.

Pph3–Psy2 contributes to MMS resistance. (A) Whole-cell extracts were prepared from yeast strains expressing Psy2-TAP, Pph3–13Myc, and/or Ybl046w-13Myc and incubated with IgG-Sepharose. Whole-cell extracts (W, 10 μg of protein) and precipitated proteins (P, 5% of precipitated fraction) were analyzed by Western blotting with the indicated antibodies. (B–D) Survival assays of yeast containing wild-type or null alleles of PSY2, PPH3, and/or YBL046W. (B and D) Colony forming units (cfu) were counted 3 days after deposition onto YPD-agar media containing the indicated concentration of MMS. Strains are as listed in SI Table 2 and are isogenic with BY4741. (C) Fivefold serial dilutions of wild-type (BY4741) or pph3Δ (FR1046) cells harboring YEplac195 or YEplac195 carrying a wild-type or mutant allele of PPH3 were deposited on SC-URA plates containing MMS.

Fig. 2.

PSY2 and PPH3 genetically interact with cell cycle checkpoint components. Survival assays of yeast containing wild-type or null alleles of PSY2, PPH3, and/or YBL046W in combination with wild-type or null alleles of cell cycle checkpoint components. Colony forming units (cfu) were counted 3 days after deposition onto YPD-agar media containing the indicated concentration of MMS. Strains are as listed in SI Table 2 and are isogenic with BY4741 (A and D) or RDKY3615 (B, C, and E).

Fig. 3.

Pph3–Psy2 regulates the phosphorylation state of Rad53. (A) Yeast two-hybrid analysis of Psy2 interaction with Rad53. pGBT9 expressing the Gal4BD-Psy2 fusion protein was introduced into the Y190 tester strain along with pACTIIst expressing the Gal4AD fused to full-length Rad53 or fragments encompassing the Rad53 FHA1, kinase (KD) or FHA2 domains. (B–F) Western blot analysis of Rad53 phosphorylation state. (B and C) In vitro assay of Rad53 dephosphorylation by Pph3–Psy2. Incubation of Rad53 isolated from E. coli (B) or MMS-treat yeast (C) cells with immunoprecipitates from whole-cell lysates of the indicated yeast strains (isogenic with W1588–4C). (D–F) In vivo regulation of Rad53 by Pph3–Psy2. (D) Log phase cultures of the indicated strains (isogenic with BY4741 or RDKY3615) were treated with 0.1% MMS for 0, 1, and 2 h. Phosphoglycerate kinase (PGK) is a loading control. (E) Analysis of undamaged log phase cultures of the indicated strains (isogenic with BY4741). (F) The indicated strains (isogenic with BY4741) were synchronized in G₁ at 30°C, released into 0.033% MMS for 1 h, and then shifted to YPD. Rad53* denotes phosphorylated Rad53.

Fig. 4.

Pph3–Psy2 promotes efficient replication restart during recovery from MMS exposure in S phase. (A and B) DNA content analysis by flow cytometry of wild-type (SSy187), pph3Δ (SSy188), and psy2Δ (SSy189) cells that were blocked in G₁ with α-factor and released into S-phase in YPD (A) or YPD with 0.033% MMS for 1 h, and then shifted to YPD (B). All steps were conducted at 30°C. (C) BrdU incorporation analysis of wild-type (SSy205) and pph3Δ (SSy210) cells treated as in B but grown at 23°C. Immunoprecipitated DNA sequences at ARS607 and at distances of 15, 33, 53, and 73 kb were detected by PCR amplification. (D and E) Two-dimensional gel analysis of samples collected in B that were digested with BamHI and NcoI. Blots were probed for ARS1 (D) or ARS603 (E). Filled arrowheads indicate large replication bubbles, open arrowheads indicate large replication forks, and caret indicates small and medium replication forks.