FIG. 6.

The 3′ single-stranded overhang of est2^LT mutants is normal. (A) Single-stranded telomeric DNA detected by nondenaturing gel electrophoresis. All strains are derived from YKF120 (est2::HIS3) and carry plasmids expressing wild-type or mutant protein A-tagged EST2. Genomic DNA from strains expressing ProA-EST2 (lanes 1 and 2), ProA-est2^E76K (lanes 5 and 6), ProA-est2^N95A (lanes 7 and 8), or ProA-est2^FCA720WCG (lanes 9 and 10) was digested with XhoI, incubated with a labeled 22-nucleotide telomeric probe, and subjected to nondenaturing gel electrophoresis. DNA from a ku70 deletion strain previously demonstrated to increase the 3′ telomeric overhang (17) was used as a positive control (lanes 3 and 4). Samples in lanes 2, 4, 6, 8, and 10 were treated with exonuclease I prior to XhoI cleavage to remove the 3′ single-stranded overhang. The bar indicates the approximate position of the terminal XhoI fragment generated from chromosomes that contain a subtelomeric Y′ element. Additional bands detected in lane 3 correspond to individual telomeres lacking the Y′ element. Lane M, end-labeled DNA size standards. (B) Total telomeric DNA detected by hybridization after denaturation. The same gel shown in panel A was denatured by treatment with 0.5 M NaOH and hybridized with the telomeric probe described in panel A. The bar indicates the approximate position of the terminal XhoI fragment generated from chromosomes that contain a subtelomeric Y′ element. Although fragments corresponding to the shortest telomeres of the yku70Δ strain were preferentially lost during the wash steps, relative loading can be assessed by comparison of the higher-molecular-weight bands.