Figure 4.

Mutation of the Est1 TPR does not compromise viability in S. cerevisiae but alters telomere length homeostasis. Heterozygous diploid est1Δ rad52Δ yeast were transformed with pRS426(URA3) plasmids expressing ScEst1-FLAG wild-type or indicated TPR mutants. Haploid est1Δ rad52Δ (A) and rad52Δ (B) spores were isolated and passaged every two days on plates containing synthetic dropout media lacking uracil. Refer to Additional file 1, Figure S5 for summary of spore growth. Telomere DNA Southern blots in haploid est1Δ rad52Δ (C) and rad52Δ (D) strains expressing empty vector (pRS426; vector), ScEst1-FLAG TPR mutants or wild-type Est1-FLAG, as indicated. Genomic DNA isolated from cells at the indicated passages (P; represented in A, B) was digested with XhoI. Telomeres were analyzed by Southern blotting using a (CACACCCA)₂CC DNA probe. In (C), two blots are represented by lanes 1-18, and 19-36; in (D), two blots are represented by lanes 1-16, and 17-31. Gaps represent removal of redundant samples, or slight changes between contrast enhancement. DNA ladders are shown (kbp) at right.