Figure 5.

 Activation of Cds1 kinase is S-phase dependent. (A) Septation of wild-type cells synchronized in G₂ by elutriation. The culture was divided into three and incubated at 30°. HU was added (20 mm) at either t = 0 (□) or t = 60 (•) min; control (▪). The cell-cycle stage of the culture was followed by septation index by Calcofluor and DAPI staining. Untreated cells show two peaks in septation. The second peak is missing in the HU-treated culture indicating the activation of the replication checkpoint. (B) Samples were taken from all three cultures and processed for Cds1 kinase assay. Cds1 kinase does not activate during G₂ despite the presence of 20 mm HU (30+, 60+). Cds1 kinase activity accumulates after 90 min when the majority of the cells have entered or are entering S phase. (C) Septation index of wild-type cells synchronized in G₂ by elutriation (30°C). (D) Cds1 kinase activity of irradiated or unirradiated cells removed at the times indicated and treated with 250 Gy of radiation. Cell extracts were prepared after 40 min of recovery incubation at 30°C. Cells irradiated in G₂ at t = 0 do not activate Cds1 kinase after irradiation, whereas cells entering S phase after irradiation develop significant activity. Consistent with the S-phase dependency of the kinase activation cells irradiated late in S phase/G₂ (t = 90,110) fail to generate significant kinase activity compared with the unirradiated control. (E) cdc10.V50 cells irradiated at the permissive temperature (26°C) show activation of Cds1 kinase whereas cells irradiated and held at the restrictive temperature (37°C) do not, thus showing that Cds1 kinase is not activated by DNA damage during G₁ phase. (F) Activation of Cds1 by temperature shift in various cdc mutants defective in DNA synthesis. Logarithmic cultures of wild-type, cdc6, cdc17, cdc20, cdc21, cdc22, and polα cells grown at 27°C were split into two samples, one was incubated for a further 2 hr, 30 min at 27°C, whereas the other was incubated at 37°C for 2 hr, 30 min before extracts were prepared and tested for IP kinase activity.