FIGURE 6:

Cdc10 phosphorylation after DNA damage is essential for viability. (A) Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or treated (0.1% MMS, 1 h at 30ºC) cultures of WT Cdc10 or the mutants indicated at the bottom. (B) Phosphorylation of S720 and S732 after ionizing radiation (IR) induces the release of Cdc10 from chromatin. Loading of Cdc10 on cdc22 and cdc18 promoters was measured by ChIP analysis of chromatin extracts isolated from untreated or IR (100 Gy) cultures of WT and Cdc10.2A cells. Average of three individual experiments (±SD). (C) Total RNA was prepared from untreated or MMS-treated (increasing doses) cultures of a cdc10.2AΔyox1Δnrm1 strain and analyzed by hybridization with the probes indicated on the left. rRNA is shown as loading control. (D) RNA was prepared from wild-type (Cdc10) or Cdc10.2A cells exponentially growing or treated with 0.1% MMS for 1 h. cdc18, cdt2, cdc22, and mik1 were quantitated by RT-qPCR. Results are shown as fold induction over untreated wild-type cells as the average of three individual experiments (±SD). (E) Survival was performed by spotting 10–10⁵ cells of the indicated strains (in a Δyox1Δnrm1 background) onto YE5S plates in the absence or presence of MMS or HU. Plates were incubated at 30°C for 3–4 d.