FIG. 7.

Checkpoint proteins inhibit Dia2 turnover in response to replication stress. (A) Dia2 proteolysis is cell cycle regulated. Cells were arrested as described in Materials and Methods and taken at the indicated time points (lanes 1 to 6) after addition of CHX. The 9MYC-DIA2 cdc15-2 strain was shifted to the nonpermissive temperature for 2 h prior to the addition of CHX. The 0-min time point of each arrest was monitored by flow cytometry. Quantitation of results from three independent experiments is shown on the graph. Error bars indicate standard deviations. (B) Checkpoint pathway mutants fail to stabilize Dia2 in response to HU treatment. 9MYC-DIA2 alleles were generated in the indicated checkpoint mutants and arrested with HU as described above. Dia2 protein turnover was monitored for the indicated times after the addition of CHX, and immunoblotting was with anti-myc and anti-Pgk1antibodies (upper panel). Quantitation of results from three independent experiments is shown on the graph. (C) During G₁, Dia2 shows turnover rates in checkpoint mutants comparable to those in the wild type. Stability assays were performed as for panel B except that cells were arrested using α-factor. Quantitation of results from a representative experiment is shown on the graph. (D) rad53-21 mutants fail to stabilize Dia2 during replication stress caused by MMS. 9MYC-DIA2 or 9MYC-DIA2 rad53-21 cultures were arrested in G₁ with α-factor for 2 h. Cultures were washed and released into fresh medium containing 0.033% MMS. Protein synthesis was halted after 60 min by the addition of CHX, and cells were collected at the indicated time points. Dia2 was monitored with anti-myc antibodies, and anti-Pgk1 antibodies were used as a loading control. (E) dia2Δ and checkpoint mutants show synthetic growth defects in response to HU. The specified strains were spotted in 10-fold serial dilutions on YPD plates containing the indicated amount of HU and grown at room temperature.