Srs2 plays a critical role in reversible G2 arrest upon chronic and low doses of UV irradiation via two distinct homologous recombination-dependent mechanisms in postreplication repair-deficient cells

Abstract

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G(2) arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G(2) arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G(2) arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.

FIG. 1.

Deletion of SRS2 suppresses the sensitivity of rad18Δ cells to CLUV in a recombination-dependent manner. (A) The plating efficiencies for WT, rad18Δ, srs2Δ, and rad18Δ srs2Δ strains during exposure to CLUV irradiation. Asynchronized log-phase cells were grown in YPAD under CLUV irradiation, and samples were taken every 3 h to determine plating efficiency. Cell viability is represented as relative CFU (= 1 at time zero). Data were obtained from at least three independent experiments. Error bars indicate standard deviation. (B) Flow cytometry of WT, rad18Δ, srs2Δ, and rad18Δ srs2Δ cells exposed to CLUV. Asynchronous cells were grown under CLUV, and samples were collected for FACS analysis for DNA content. (C) Spot assays were performed using 10-fold serial dilutions of exponential-phase cultures of the indicated strains. DNA damage was induced by CLUV exposure for 2 days.

FIG. 2.

A deficiency of PCNA sumoylation suppresses the sensitivity of rad18Δ cells to CLUV. (A) Suppression of sensitivity of rad18Δ cells to CLUV by mutation of the SUMO accepter lysines K127 and/or K164. Cells were exposed to CLUV for 2 days. (B) Cells that carried the indicated SRS2 alleles on a plasmid were grown and spotted onto synthetic complete medium without leucine (SC-Leu) plates at 10-fold serial dilutions. The plates were incubated in the presence or absence of CLUV for 3days. (C) CLUV sensitivities of rad18Δ pol30^K127R/K164R and rad18Δ pol30^K127R/K164R srs2Δ cells expressing the indicated PCNA or SUMO-fused PCNA alleles on a plasmid. For quantitative assay, 10-fold serial dilutions of cells grown in SC-Leu were spotted on SC-Leu plates and incubated under CLUV conditions for 3 days.

FIG. 3.

Srs2 is required for checkpoint activation in CLUV-exposed rad18Δ cells. (A) CLUV-induced Rad53 phosphorylation. Protein extracts from cells exposed to CLUV for the indicated times were prepared and analyzed by 6% SDS-PAGE followed by Western blotting using an anti-Rad53 antibody. (B) Rad53 phosphorylation in CLUV-exposed PRR-deficient cells. Protein extracts from cells exposed to CLUV for 3 h in were prepared and analyzed by Western blotting with anti-Rad53 antibody.

FIG. 4.

srs2Δ mutation suppresses ssDNA production in CLUV-exposed rad18Δ cells. (A and C) Asynchronous cultures of the indicated strains were treated with CLUV for 3 h and examined by fluorescence microscopy. The percentage of cells with RPA-YFP (A) or Ddc2-YFP (C) foci is shown. The results represent the means of three independent measurements. The error bars indicate the standard deviations. (B) Representative RPA-YFP foci are shown. (D) Asynchronous cultures were treated with CLUV for the indicated times or with 0.1% MMS for 30 min. Chromosomal DNA was separated by PFGE and detected by staining with SYBR gold. The percentage of cells with RPA-YFP foci is indicated at the bottom. (E) Cell viability was measured as described for Fig. 1A. The error bars indicate the standard deviations. All strains contain a deletion of SML1, which suppresses mec1Δ lethality without suppressing the checkpoint defect. (F) Deletion of SRS2 does not affect CLUV-induced RPA foci in rad18Δ cells when HR function is impaired. Cells grown to logarithmic phase were treated with CLUV and examined by fluorescence microscopy as with panel A. The results represent the means of at least three independent measurements. The error bars indicate the standard deviations.

FIG. 5.

Srs2 is required to maintain the DNA damage checkpoint in CLUV-exposed rad18Δ cells. (A) Degron-mediated proteolysis of Srs2. Wild-type or srs2-td strains were grown in YP (Raf) at 25°C. The cells were transferred to YP (Gal) to induce UBR1 expression and then cultured at 25 or 37°C for 3 h. Srs2 was detected by Western blotting using an anti-Srs2 antibody. α-Tubulin was used as a loading control. (B) The indicated strains were cultured in YP (Raf) at 25°C. Tenfold serial dilutions of cells were spotted onto YP (Gal) plates and incubated at 25 or 37°C under CLUV conditions. (C) rad18Δ and rad18Δ srs2-td cells were grown at 25°C for 3 h under CLUV conditions to arrest the cell cycle at G₂ phase and then transferred to the restrictive conditions to degrade Srs2-td. Samples were collected at the indicated times for immunoblotting with anti-Rad53 and anti-Srs2. (D) Cells were treated with CLUV for the indicated times as for panel C and analyzed by flow cytometry. (E, F, and G) Cells arrested in G₂ under CLUV exposure as for panel C and were transferred to restrictive conditions (time = 0). At the indicated time points, cells were analyzed to determine the percentage of cells with RPA foci (E) or for plating efficiency (G). The results represent the averages of at least three independent measurements. The error bars indicate the standard deviations. Representative RPA-YFP foci observed after transfer to the restrictive conditions are shown (F).

FIG. 6.

Phosphorylation of Srs2 is required for HR-mediated recovery after release from CLUV exposure in rad18Δ cells. (A) The indicated strains were grown in the presence or absence of CLUV for 3 h, and the phosphorylation status of Srs2 and Rad53 was analyzed by immunoblotting. (B) Same experiment as in panel A, except that samples were subjected to FACS analysis. (C) Cell survival after CLUV irradiation was determined as for Fig. 1A. (D and E) Cells were treated with CLUV to activate the checkpoint and then removed from CLUV exposure to allow recovery. Aliquots were taken at the indicated times and processed by flow cytometry (D) or immunoblotting with anti-Rad53 (E). (F and G) rad18Δ and rad18Δ srs2-7AV mutants were analyzed for the presence of Rad52-YFP (F) or RPA-YFP (G) foci at the indicated time points after release from CLUV exposure. The error bars indicate the standard deviations.

FIG. 7.

A model summarizing CLUV damage tolerance in yeast. See Discussion for details. Red triangles represent UV lesions.