Synthetic interaction of stn1-186t with est2-Δ. (A) A dissection plate showing five tetrads that were dissected from a diploid created by mating stn1-186t with est2-Δ (dCN325). The segregation pattern for stn1-186t and stn1-186t est2-Δ is indicated; a square surrounds stn1-186t spores and a circle surrounds the double-mutant spores. (B) Each spore from tetrad 1 shown in (A) was streaked out for single colonies and grown at 30° for 3 days.

The interaction between Cdc13p and Stn1p contributes to telomere length regulation. For both Southern blots, the genomic DNA was digested with XhoI and the membrane was probed with [³²P]d(GT/CA). (A) Southern blot of strains overexpressing the stn1-t alleles (ADH promoter, 2μ) in a stn1-Δ strain (hC567, hC568, hC569). Duplicates are shown for each strain. (B) Southern blot of the integrated stn1-281t and stn1-186t strains, derived from dissection of dCN187 and dCN188.

Loss of RAD52 impairs growth, but not telomere length in the stn1-186t strain. (A) Southern blot of stn1-281t, stn1-186t, and stn1-186t rad52-Δ strains (hC671, hC672, hC1110). Genomic DNA was prepared from strains grown in liquid media to saturation, and digested overnight with AluI, HaeIII, HinfI, and MspI. The membrane was probed with [³²P]d(GT/CA). (B) Slower growth phenotype of stn1-186t, stn1-281t and stn1-186t rad52-Δ. Strains of the indicated genotypes were streaked out on YPD. The plate was photographed after 2 and 3 days of growth at 30°.

stn1-t alleles show unusual telomere integrity defect when expressed from the endogenous locus. In-gel hybridization assay to test for presence of ssTG_1–3 DNA in stn1-186t and stn1-281t strains, or in stn1-Δ strains overexpressing STN1 residues 1–186, 1–281, or 1–371 (hC567, hC568, and hC569). Wild-type and yku80-Δ strains are shown as controls. Genomic DNA was prepared from cells that were grown at 30° to an OD₆₀₀ ∼ 1 and arrested in G1 by α-factor. Samples were split and mock treated or digested with either ExoI or mung bean nuclease. All samples were then digested with XhoI and analyzed by in-gel hybridization as previously described (Bertuch and Lundblad 2003). The top gel (native) shows the detection of single-stranded TG_1–3 by the labeled telomere CA probe. The total TG_1–3 signal is detected in the bottom gel (denatured), following denaturation and reprobing of the native gel.

The interaction between Cdc13p and Stn1p is not required for viability. (A) Assay of ability of stn1 truncation alleles to complement the stn1 null. Single colonies of hC276 strains [stn1-Δ/pVL1046 (STN1 on a CEN-URA3 plasmid)] that had been transformed with the STN1 two-hybrid plasmids (pADH, 2μ, and LEU2) were inoculated into liquid media and grown for 4 days at 23°. Serial 10-fold dilutions of these cultures were plated on Leu⁻ or Leu⁻ 5-FOA plates and incubated at 23°, 30°, and 36°. (30° plates not shown). Cells that grow on 5-FOA have lost the pVL1046 plasmid encoding full-length STN1. Complementation of stn1-Δ is determined by the ability of the cells to grow on Leu⁻ 5-FOA. (B) Western blot comparing protein expression from the STN1 two-hybrid plasmids in the hc276 stn1-Δ strain. Protein extracts were prepared in buffer A from strains that grew on Leu⁻ 5-FOA in (A) and 400 μg of each protein lysate was run on a 10% polyacrylamide gel. “Wild-type” strains are hC572, which express untagged STN1 (pCN1).

Separable domains of Stn1p interact with Ten1p and Cdc13p. (A) Two-hybrid assay with pACT2.2-STN1 full-length and truncation constructs vs. pAS1-TEN1 (pCN124) or pAS1-CDC13^Δ585–677 (pVL705). The “empty” pACT2.2 plasmid was used as a vector control. Three reporters for the interaction were tested by assessing growth on Ade⁻Leu⁻Trp⁻ plates, growth on His⁻Leu⁻Trp⁻ + 1.5 mm 3AT plates, and β-Gal production in X-GAL overlays on Leu⁻Trp⁻ plates. The top row in each set shows growth on Leu⁻Trp⁻ plates. (B) Diagram of STN1 constructs used in the two-hybrid assay and summary of interactions. The Stn1p amino acids translated from each STN1 construct are indicated in superscript. A + or − indicates whether the construct is competent for interaction with pCN124 (Ten1p) or pVL705 (Cdc13^Δ585–677). (C) Western blot showing relative levels of Stn1p produced from the two-hybrid plasmids in a wild-type strain (hc160). The vector control plasmid is pACT2.2. Strains were grown at 30°, protein was extracted in SUMEB buffer, and 50 μl of each protein lysate was run on a 10% polyacrylamide gel. The Western blot was probed with α-HA antibody (12CA5).

Strains expressing stn1 residues 1–186 or 1–281 are more viable than strains expressing stn1 residues 1–371. (A) Test for complementation of stn1-Δ by pstn1^1–281 and pstn1^1–186 when expressed from the STN1 promoter on 2μ plasmids. Shown are stn1-Δ haploids covered by the indicated plasmids, obtained by dissection of the dCN157 diploid strain (stn1-Δ/STN1) that was transformed with the pVL1066, pCN319, or pCN320 plasmids. Cells were grown to similar densities at 23° in Leu⁻ liquid media. Serial 10-fold dilutions were stamped on Leu⁻ plates and incubated at 23°, 30°, and 36°. (B) pstn1^1–371 shows variability in its extent of stn1-Δ complementation when expressed from the STN1 promoter on a 2μ plasmid. The stn1-Δ/pVL1046 (pSTN1 on a URA3 plasmid) strain (hc35) that had been transformed with either the pCN318 or pCN1066 plasmids was grown in liquid culture at 30° for 3 days. Serial 10-fold dilutions of these cultures were plated on Leu⁻ or Leu⁻ 5-FOA and incubated at 30°. Cells that grow on 5-FOA have lost the pVL1046 plasmid encoding STN1. Two independent pstn1^1–371 transformants are shown. (C) Single-colony streakouts of strains in A, grown 5 days at 23° and 3 days at 36°. WT, wild-type strain (hC160). No significant deviation from wild-type growth was observed. (D) Growth phenotype of integrated stn1-186t and stn1-281t alleles on YPD. Shown are serial 10-fold dilutions of cultures of stn1-186t and stn1-281t colonies (two each) that were obtained from dissection of dCN187 and dCN188, compared with a wild-type haploid. The alleles are integrated at the chromosomal STN1 locus, truncating the wild-type STN1 gene.

Diploids inherit elongated telomeres through mating with stn1-186t strains. (A) Southern blot showing telomere lengths in diploids created by mating stn1-186t with est2-Δ (three dCN325 diploid strains shown on the left). The telomere lengths in haploids derived from one of these diploids are shown on the right. The “wild-type” strain is hC160, and was not a progeny of these diploids. Genomic DNA was isolated from cells grown for ∼25 generations and digested with XhoI and probed with [³²P]d(GT/CA). (B) Southern blot analysis of telomere length through progressive generations of growth. Haploid est2-Δ, stn1-186t est2-Δ, and wild-type strains that were isolated from the diploids in A were grown for ∼25, 35, and 45 generations prior to genomic DNA isolation. DNA was digested with XhoI and the blot was probed with [³²P]d(GT/CA). (C) Analysis of the terminal-telomere fragment from chromosome VI-R in dCN325 diploids (mated) and in haploid progeny from this diploid. The wild-type strains on each end of the blot are hC160. dCN325 has been propagated for ∼30 additional generations in this figure, as compared with the diploids in A. The haploids were propagated for ∼65 generations prior to isolation of DNA. The genomic DNA was digested with XhoI, and the blot was probed with a labeled PCR fragment that hybridizes to a unique telomere proximal sequence on chromosome VI-R (Zubko and Lydall 2006) (left) or with [³²P]d(GT/CA) (right). (D) Telomere length in diploids that were created by directly integrating the stn1-186t or stn1-281t allele in an est2-Δ/EST2 diploid (dCN187 and dCN188). Two independent diploids are shown for each strain. DNA was digested with XhoI and probed with [³²P]d(GT/CA).

The DNA damage checkpoint is required for stn1-t viability. (A) Rad53p phosphorylation in stn1-186t and stn1-281t strains. Asynchronously growing strains were split in half, with one portion treated with 0.1% MMS for 2.5 hr. All samples were lysed in 20% TCA, and equal amounts of each lysate were loaded and separated on a 10% 30:0.39 acrylamide:bisacrylamide gel. The Western blot was probed with an anti-Rad53p antibody. (B) FACS analysis of a synchronized cell cycle in wild-type, stn1-186t rad52-Δ, and stn1-281t strains. Cell-cycle progression was measured for 150 min following release from a G1 arrest, with the cells rearresting in G1 due to addition of α-factor during the release period. (C) Viability of the overexpressed stn1-t alleles (ADH, 2μ) in the stn1-Δ rad9-Δ background. A stn1-Δ rad9-Δ/pVL1046 (pSTN1 on a CEN-URA3 plasmid) strain (hC1577) was transformed with the pCN181, pCN234, pCN235, pCN249, pACT2.2, or pCN1 plasmids; liquid cultures of these transformants were grown at 30°. Serial 10-fold dilutions of the cultures were plated on −Leu or −Leu 5-FOA, and incubated at 30° for 3 days. (D) Viability of the stn1 truncations expressed from the STN1 promoter on 2μ plasmids in the stn1-Δ rad9-Δ background (plasmids pCN318, pCN319, pCN320, pVL1066, and YEplac181, transformed into hC1577). Strains were manipulated as in C.