Cdc28 does not have a replication checkpoint function. (A) Cell survival after HU- or MMS-induced DNA damage does not depend on Cdc28 activity. WT cells or cdc28-as1 or rad53 mutants were treated with 10 µM 1-NM-PP1, 200 mM HU, or 0.05% MMS for 2 h, washed, and plated on YPD as described in Materials and methods. Cell survival was calculated as a percentage of untreated WT cells. (B) Cell survival after phleomycin (Phleo)-induced DNA damage does not depend on Cdc28 activity. WT cells or cdc28-as1 mutants were treated as in A with 1-NM-PP1 and increasing concentrations of phleomycin, and the percentage of cell survival was determined. (A and B) Error bars represent standard deviation. CFU, colony-forming unit.

Cdc28 cooperates with Mre11 to maintain genome stability. Cdc28 cooperates with Mre11 to prevent mitotic catastrophe after DNA replication arrest. (A–D) WT cells (A), cdc28-5M mutants (B), mre11Δ mutants (C), and mre11Δ cdc28-5M double mutants (D) were arrested in HU for 3 h before being released into YPD supplemented with α factor at 30°C. Samples were taken at the indicated time intervals and analyzed using FACS analysis. (E) Cdc28 prevents chromosome fragmentation. WT cells and mre11Δ, cdc28-5M, and mre11Δ cdc28-5M mutants were treated as indicated, and whole chromosomes were analyzed by PFGE. The relative amount of chromosome fragmentation (at 30°C because incubation at 37°C did not further increase fragmentation) was calculated as described in Materials and methods. Error bars represent the standard error of the mean.

Cdc28 functions in the DNA damage response. 10-fold dilutions of log-phase cultures were spotted on YPD supplemented with increasing concentrations of 1-NM-PP1 and fixed concentrations of 10 µg/ml CPT, 0.05% MMS, 100 mM HU, and 1 µg/ml phleomycin (Phleo) or irradiated with 100 J/m² UV, as indicated.

Cdc28 has no major role in DNA damage–induced checkpoint activation. (A) DNA damage–induced formation of Ddc2 foci is independent of Cdc28. Cells were arrested with α factor and released into YPD or YPD containing 10 µM 1-NM-PP1 and the indicated DNA-damaging agents for 2 or 4 h, as indicated (see Materials and methods). Ddc2-GFP foci were visualized using fluorescence microscopy, and the percentage of cells with foci relative to the total cell population of that specific sample was calculated. Phleo, phleomycin. (B) Checkpoint activation upon endogenous DNA damage does not depend on Cdc28 activity. WT cells and cdc28-as1, mre11Δ, and cdc28-as1 mre11Δ mutants were arrested in α factor before being released into YPD supplemented with 10 µM 1-NM-PP1 for 2 h, and the percentage of cells with Ddc2-GFP foci were determined as described in A. Error bars represent standard deviation. (C) MMS-induced phosphorylation of Rad53 is independent of Cdc28. WT cells and cdc28-as1 mutants were arrested in α factor (α) and released into YPD for 30 min to allow cells to enter S phase. Then, either DMSO or 1-NM-PP1 was added to inactivate Cdc28-as1 (time point 0) followed by treatment with MMS for 30, 60, or 120 min. Cell lysates were analyzed by Western blotting using Flag antibodies to detect Rad53. (D) Phleomycin-induced phosphorylation of Rad53 is partially dependent on Cdc28. WT cells and cdc28-as1 mutants were arrested in α factor and released into YPD for 30 min to allow cells to enter S phase. Then, either DMSO or 1-NM-PP1 was added to inactivate Cdc28-as1 (time point 0) followed by treatment with phleomycin for 60, 120, or 180 min. Cell lysates were analyzed by Western blotting using Flag antibodies to detect Rad53. (E) Cdc28 is not required for MMS-induced S-phase arrest. WT cells and cdc28-5M and rad53Δ mutants were grown into log phase (log) or arrested in α factor before being released into YPD supplemented with 0.05% MMS. Samples were taken at the indicated time points and analyzed by FACS. (F) Checkpoint inactivation after replication arrest does not require Cdc28 activity. WT cells and cdc28-as1 mutants were arrested in HU for 3 h before HU was washed away, and cells were resuspended in YPD supplemented with either DMSO or 10 µM 1-NM-PP1. Samples were taken at the indicated time intervals, and Rad53 phosphorylation was analyzed as in C. P-Rad53, phospho-Rad53.

CDC28 genetically interacts with pathways involved in maintenance of genome stability. (A) Synthetic lethal interaction between cdc28-5M and rad6Δ. CDC28/cdc25-5M RAD6/rad6Δ diploids were sporulated, and tetrads were dissected. All combinations except the rad6 cdc28-5M double mutant were observed. (B) Expression of rad6-149 partially rescues the synthetic lethality of the cdc28-5M rad6Δ mutant. 10-fold dilutions of log-phase cell cultures were spotted on YPD plates and incubated until colonies were visible. (C) CDC28 genetic interaction map.