Fig. 4. (A) Rad53p in situ autophosphorylation of strains grown at 25 or 37°C. (B) Rad53p in situ autophosphorylation of cdc13-1 mutant strains containing YEP13 (2µ), YEP13–EST2, –TLC1 or –YKU80, and grown at either 25 or 37°C.

Fig. 5. (A) Southern blot analysis of telomeres from various yeast strains. (B) Top, Rad53p in situ autophosphorylation of strains grown at 25 or 37°C. Bottom, Western blot analysis of Rad53p was performed on 12% polyacrylamide gels. (C) Strains were spotted in 10-fold serial dilutions onto YPAD plates and grown at 30, 36 or 37°C.

Fig. 3. (A) Autoradiograph of the Rad53p in situ autophosphorylation assays. Wild-type or yku80 mutant strains were grown in the presence of 0.02% MMS at 30°C for 2 h, or at 25, 30 or 37°C for 5 h. (B) yku80 mutant strains containing YEP13 (2µ), YEP13–EST2, –TLC1 or –YKU80 were grown at 25, 30 or 37°C for 5 h. (C) Strains were grown at 25 or 37°C, or with 0.005% MMS (M) or 20 µg/ml phleomycin (P), and Rad53p autophosphorylation was assayed as above.

Fig. 2. (A) Strains were spotted in 5-fold serial dilutions onto plates without (left and middle) or with 0.005% MMS (right) and grown at 30°C (left and right) or 37°C (middle) for 3 days. (B) Exponentially growing cells were exposed to 0.05% MMS for the times indicated and then plated onto YPD. Percentage cell survival was plotted against time in MMS. (C) Plasmid repair assay was performed by transforming either supercoiled or XbaI-linearized pRS413 into various yeast strains (described in Figure 1A). The number of HIS⁺ colonies obtained with linear versus supercoiled DNA was taken as a measure of NHEJ efficiency.

Fig. 1. (A) Southern blot of XhoI-digested genomic DNA probed with Y′-specific sequences of yeast telomeres. Genomic DNA was prepared from strains grown at 30°C (left) or 37°C (right) for 18 h, from wild-type (lane 1) or yku80 mutant strains containing either the YEP13 (2µ) plasmid (lanes 2, 6), YEP13–EST2 (lanes 3, 7), –TLC1 (lanes 4, 8) or –YKU80 (lanes 5, 9). Identical results (with the exception of yku80 with YEP13) were obtained with strains grown for >100 generations. Telomeric restriction fragments are indicated by a square bracket. (B) Telomere PCR. Genomic DNA was isolated, denatured and tailed with dCTP using TdT. The G-rich strand is specifically amplified with a primer complementary to the dC tail and a primer specific for a sequence distal of the subtelomeric ADE2 gene. The bottom panel shows PCR products analysed on an ethidium bromide-stained agarose gel. (C) In-gel hybridization of XhoI-digested genomic DNA probed with a radiolabelled oligonucleotide corresponding to the CA-repeat sequence of yeast telomeres, in native or denaturing conditions. Quantitation of the single-strand telomeric regions in yku80 mutants with or without EST2 or TLC1 overexpression showed no reproducible differences. (D) TPE was tested using a strain containing a telomere-proximal URA3 gene. Yeast strains were spotted in 10-fold serial dilutions on plates with (right) or without (left) FOA, and grown at 30°C (top) or 37°C (bottom) for 3 days.