Figure 6.—
ssTG1-3 generation in ten1-101 and ten1-105 is cell cycle and Cdk1 dependent. (A) In-gel hybridization showing cell cycle dependency of single-strand generation in ten1-105. Asynchronous cell cultures growing at 23° were divided, with equivalent portions arrested at 23° in G1 with α-factor, in S phase with hydroxyurea (HU), or in G2/M with nocodazole. Cultures were then split and shifted to the indicated temperature for 4 hr while maintaining the arrested state. One culture was maintained in logarithmic growth prior to the temperature shift (Asyn). Genomic DNA was then isolated and split, with half treated with ExoI prior to XhoI digestion. The upper panel shows the gel probed with a [32P]-CA oligo under native conditions, and the lower panel shows hybridization with the [32P]-CA oligo following denaturation. WT (wild-type, hc160), yku80Δ (hc18), and ten1-105 (hc1864). (B) In-gel hybridization showing cell cycle dependency of single-strand generation in ten1-101. Asynchronous cell cultures were divided and either allowed to continue growth at 23°, or arrested at 23° in G1 with α-factor or in G2/M with nocodozole. Cultures were then split and incubated at the indicated temperature for 4 hr while maintaining the arrested state. Genomic DNA was then isolated and split, with half treated with ExoI prior to XhoI digestion. The agarose gel was probed with a [32P]-CA oligo under native conditions. Only the native gel is shown; the total DNA loaded in each lane is shown in the ethidium bromide-stained gel slice. WT (wild-type, hc160), yku80Δ (hc18), and ten1-101 (hc1862). (C) In-gel hybridization showing the creation of ssTG1-3 is dependent on functional Cdk1. Cells were arrested at 23° in G2/M with nocodazole, and then split, with half shifted to 36° for 4 hr. To inhibit the Cdc28-as1 activity, 1-NMPP1 was added to the cultures 1 hr prior to the temperature shift. The cells were arrested in nocodazole throughout the experiment. Treatment of the genomic DNA and gel probing were as in A, with the upper panel showing the native gel and the lower panel showing the denatured gel. Wild-type (WT, hc160), yku80Δ (hc18), cdc13-1 (hc1997), cdc13-1 cdc28-as1 (hc1998), ten1-105 (hc1864), and ten1-105 cdc28-as1 (hc2005) strains are shown.