Figure 4.

ago1⁺ and dcr1⁺ are both required for hyperphosphorylation of Cdc2 and enactment of the DNA damage and replication checkpoints. (A) Serial dilutions of TV294 and corresponding Δago1, Δdcr1, and Δrdp1 strains were spotted on YE agar or YE agar containing 3.5 mM HU and were cultured for 3 and 5 d at 30°C, respectively. (B) TV294, Δago1, Δdcr1, and Δrdp1 liquid cultures were grown at 30°C to an OD₅₉₅ = 0.5, and aliquots were removed before (-) or after (+) a 4-h HU treatment. Whole cell lysates were prepared and separated by SDS-PAGE before immunoblot analyses of total and phosphorylated Cdc2. The normalized levels of HU-induced Cdc2 phosphorylation are shown below the immunoblots in B and C. (C) Levels of total and phosphorylated Cdc2 were analyzed in strains transformed with vector alone or vector encoding Ago1 or Dcr1. (D) TV294, Δago1, Δdcr1 Δrdp1, and Δrad3 liquid cultures were grown at 30°C to an OD₅₉₅ = 0.8, spread at a density of 100-300 cells per plate and exposed to UV radiation (0, 75, 150, 225, or 300 J/M²). Cell survival is shown as a percentage of the nonirradiated control cell survival. (E) TV294, Δago1, Δdcr1, and Δrdp1 were subjected to mock (0 J/m²) treatment and UV irradiation (75 J/m²), and the septation index for each strain was determined every hour postirradiation (n = 3, 300 cells counted/time point). Similar results were obtained when these strains were subjected to a 100 J/m² dose (our unpublished data).