The Saccharomyces cerevisiae F-box protein Dia2 is a mediator of S-phase checkpoint recovery from DNA damage

Abstract

Cell-cycle progression is monitored by checkpoint pathways that pause the cell cycle when stress arises to threaten the integrity of the genome. Although activation of checkpoint pathways has been extensively studied, our understanding of how cells resume the cell cycle when the stress is resolved is relatively limited. In this study, we identify the Saccharomyces cerevisiae F-box protein Dia2 as a novel player in the S-phase checkpoint recovery pathway. Dia2 is required for robust deactivation of the Rad53 checkpoint kinase and timely completion of DNA replication during recovery from DNA damage induced by methyl methanesulfonate (MMS). Aiming to identify the substrate of SCF(Dia2) (Skp1/Cul1/F-box Dia2) in checkpoint recovery, we performed a genetic screen to identify suppressors of dia2Δ cells. The screen identified a new checkpoint-defective allele of MRC1 truncated at the C terminus. We found that checkpoint-defective mrc1 alleles suppress the MMS sensitivity and the checkpoint recovery defect of dia2Δ cells. In addition, Dia2 contributes to Mrc1 degradation during S-phase checkpoint recovery. Furthermore, induced degradation of checkpoint-functional Mrc1 partially rescues the checkpoint recovery defect of dia2Δ cells. We propose a model in which Dia2 mediates Mrc1 degradation to help cells resume the cell cycle during recovery from MMS-induced DNA damage in S-phase.

Figure 1

Dia2 is required for checkpoint recovery from MMS-induced DNA damage in S-phase. (A) Cells were arrested in late G1 by α-factor (αF), released into rich media (YPD) + 0.033% MMS for 40 min, and then released into YPD. Samples were analyzed at the indicated time points by flow cytometry. 1C and 2C indicate DNA content. Percentage of cells with 2C DNA content is indicated on the right of selected profiles. (B) Cells were arrested in late G1 by αF and then released into YPD at 30°. Samples were analyzed by flow cytometry. (C and D) DIA2 genetically interacts with Rad53-phosphatase PPH3 in response to MMS. (C) Tenfold serial dilutions of the indicated strains were spotted on YPD or YPD + 0.007% MMS and incubated at 30°. (D) Equal numbers of cells were plated on media containing the indicated amounts of MMS, and colony-forming units were counted after 4 days at 30°. Error bars represent standard deviations from three independent experiments. (E and F) Dia2 functions in parallel to Pph3 for S-phase checkpoint recovery. Samples were prepared and analyzed as described in A and B.

Figure 2

A genetic screen identified a checkpoint-defective allele of mrc1 that suppresses the MMS sensitivity of dia2Δ. (A) Structural schematic of the Mrc1 protein. Arrowheads indicate S/TQ Mec1-directed phosphosites. Mutations used in these studies are marked. (B) mrc1_1–971 suppresses the MMS sensitivity of dia2Δ. The indicated strains were spotted using 10-fold serial dilutions on rich media with or without 0.007% MMS and incubated at 30°. (C) mrc1_1–971 is functional in DNA replication. Cells were arrested in G1 by α-factor and released into YPD at 30°. The indicated time points were analyzed by flow cytometry. 1C and 2C indicate DNA content. (D) The mrc1_1–971 allele exhibits negative genetic interactions with other S-phase checkpoint mediator mutants. Tenfold serial dilutions of the indicated strains were spotted on YPD or YPD + 0.007% MMS and incubated at 30°. (E) Checkpoint activation of Rad53 is compromised in mrc1_1–971. Cells were arrested in G1 by α-factor and released into YPD + 0.033% MMS at 30°. Protein samples were taken at the indicated time points. Pgk1 was used as a loading control. The checkpoint activation of Rad53 was measured using the intensity of Rad53 phosphorylation shift. (F) The mrc1_1–971 allele bypasses checkpoint-activated slowing of DNA replication. Samples were prepared as described in E and analyzed at the indicated time points by flow cytometry.

Figure 3

Checkpoint-defective alleles of mrc1 suppress dia2Δ MMS sensitivity and checkpoint recovery defects. (A) mrc1 mutant alleles suppress dia2Δ MMS sensitivity. Tenfold serial dilutions of the indicated strains were spotted on YPD or YPD + 0.007% MMS and incubated at 30°. (B) Checkpoint-defective mrc1 alleles enhance viability of dia2Δ in MMS. Equal numbers of cells were plated on media containing the indicated amounts of MMS, and colony-forming units were counted after 4 days at 30°. Error bars represent standard deviations from three independent experiments. (C) mrc1_1–971 does not suppress pph3Δ MMS sensitivity. Tenfold serial dilutions of the indicated strains were spotted on YPD or YPD + 0.01% MMS and incubated at 30°. (D and E) Checkpoint-defective mrc1 alleles accelerate dia2Δ checkpoint recovery. Cells were arrested in late G1 by α-factor, (D) released into YPD + 0.033% MMS for 40 min, and then released into YPD or, (E) released into YPD at 30°. 1C and 2C indicate DNA content. (F) mrc1_1–971 and mrc1_AQ accelerate Rad53 deactivation of dia2Δ. Cells were arrested in G1 by α-factor, released in YPD + 0.009% MMS for 1 hr, and then released into YPD + 15 μg/ml nocodazole. Protein samples were taken as indicated. Rad53-P and Rad53 represent phosphorylated and unphosphorylated Rad53 proteins, respectively. The very top modified band of Rad53 was quantified using ImageJ and the percentage of that in each time point relative to time zero is shown in the graph.

Figure 4

Rad53 mediator mutants suppress dia2Δ MMS sensitivity and checkpoint recovery defects. (A) rad9Δ, tof1Δ, and csm3Δ mutants suppress dia2Δ MMS sensitivity. Tenfold serial dilutions of the indicated strains were spotted on YPD or YPD + 0.007% MMS and incubated at 30°. (B) rad9Δ, tof1Δ, and csm3Δ mutants accelerate dia2Δ checkpoint recovery. Cells were arrested in G1 by α-factor, released into YPD + 0.033% MMS for 40 min, and then released into YPD at 30°. 1C and 2C indicate DNA content.

Figure 5

Dia2 is required for the degradation of Mrc1 during checkpoint recovery. (A) Mrc1 is degraded in a Dia2-dependent manner. Cells were arrested in G1 by α-factor, released into YPD + 0.033% MMS for 40 min, and then released into YPD + 200 μg/ml CHX. Protein samples were taken at indicated times. Mrc1-P and Mrc1 represent phosphorylated and unphosphorylated Mrc1 proteins, respectively. Pgk1 serves as a loading control. The graph shows the quantification of three independent experiments. Error bars indicate standard deviations. (B) Rad9, Csm3, and Tof1 are not degraded in a Dia2-dependent manner during recovery from an MMS-induced checkpoint. The experiment was performed as in A. Pgk1 serves as a loading control. (C) S/TQ phosphosites play a role in the degradation of Mrc1 during checkpoint recovery. Cells were treated as described in A. Mrc1-P and Mrc1 represent phosphorylated and unphosphorylated Mrc1 proteins, respectively. Pgk1 serves as a loading control. Stability of total Mrc1 protein was quantified from three independent experiments. Error bars were derived from standard deviations of the three experiments. (D) Degradation of phosphorylated Mrc1 is proteasome dependent. Wild-type cells were subjected to the same arrest and release treatment as described in A, except that during checkpoint recovery one set of cells was released into YPD + 200 μg/ml CHX and another set into YPD + 200 μg/ml CHX + 50 μM MG-132. Mrc1-P and Mrc1 represent phosphorylated and unphosphorylated Mrc1 proteins, respectively. Pgk1 serves as a loading control.

Figure 6

Induced degradation of checkpoint-functional Mrc1 contributes to Dia2-mediated checkpoint recovery. Cells were arrested in G1 by α-factor, released into YPD + 0.033% MMS for 1 hr, and then released into either YPD + ethanol (vehicle) or YPD + 1.5 mM IAA (auxin) at 30°. Results for wild type are shown in A and dia2Δ in B. (C) The Mrc1_1–971 protein was degraded in a dia2Δ strain. Left: Cell-cycle progression was monitored by flow cytometry. 1C and 2C indicate DNA content. Arrows mark significant difference between –IAA and +IAA samples in the dia2Δ strain. Top right: Mrc1 was rapidly degraded upon IAA treatment. Protein samples were taken at indicated times with or without IAA treatment. Pgk1 serves as a loading control. Bottom right: Quantification of 2C DNA content from at least three replicates of each experiment. Error bars indicate standard deviations. P-values calculated using paired Student’s t-test analysis (n = 4).

Figure 7

Model for the role of Dia2 in S-phase checkpoint recovery. (Left; Checkpoint activation) Dia2 is stabilized by the activation of the S-phase checkpoint (Kile and Koepp 2010). (Right; Checkpoint recovery) During checkpoint recovery, Dia2 targets checkpoint-activated Mrc1 for degradation to downregulate the checkpoint activation of Rad53. Rad53 phosphatase Pph3 removes phosphate groups from Rad53 to allow DNA replication to resume in S-phase (O’Neill et al. 2007; Szyjka et al. 2008). Ub, ubiquitin; P, phosphorylation.