MEC1/TEL1-dependent phosphorylation of Cdc13p in vivo. (A) Cdc13p phosphorylation was analyzed by western blot analysis. Lysates from different strains were TCA precipitated for western blot analysis to detect Cdc13p by a Myc antibody. Pgk1p is the loading control. (B) Phosphatase treatment of wild-type Cdc13p. Immunoprecipitated Cdc13p was incubated in PPase buffer, PPase buffer with PPase, or PPase buffer containing PPase and PPase inhibitors (PI). Western blot analysis was performed as in (A).

2D gel electrophoresis analysis of Cdc13p. (A) Western blot analysis of the 2D gel electrophoresis pattern of Cdc13p from wild-type or mec1 tel1 cells. Immunoprecipitated Cdc13p was subjected to 2D gel electrophoresis and western blot analysis. The membrane was detected using a Myc antibody. Area of Cdc13p was enlarged at the bottom. As a merged control, equal amount of precipitated Cdc13p from wild-type and mec1 tel1 cells were combined prior to protein separation. The different Cdc13p forms were numbered according to their position relative to the scale of isoelectric point shifts. (B) Western blot analysis of the 2D gel electrophoresis pattern of Cdc13p from untreated or PPase-treated wild-type cells. The assay was performed and presented as in (A).

Cdc13p is directly phosphorylated by Mec1p and Tel1p in vitro. (A) Phosphorylation of the telomerase recruitment domain of Cdc13p. Schematic diagram of Cdc13p illustrates its domain structure and potential S/T phosphorylation sites. GST-fusion proteins to the telomerase recruitment (RD) and DNA binding (DBD) domains of Cdc13p were purified. Strains KSC1333 (MEC1-HA sml1::HIS3), KSC1752 (mec1-HA(KD) sml1::HIS3), LSS93 (HA-TEL1) and LSS233 (HA-Tel1(KD)) were lysed, and extracts were immunoprecipitated with anti-HA antibodies. Immunoprecipitated kinases were analyzed by SDS–PAGE and western blotted with anti-HA antibody (shown at the bottom). IP-kinase assay was performed with GST-Cdc13p(185–332) or GST-Cdc13p(570–676) and [γ-³²P]ATP. Proteins were resolved by SDS–PAGE and the phosphorylated proteins were detected by autoradiography (shown on top). The kinase reactions were also Coomassie stained (shown below) to confirm that lanes were equally loaded. (B) Mec1p/Tel1p phosphorylation sites in the telomerase recruitment domain of Cdc13p. The kinase assay was performed as in (A) except that purified GST–Cdc13p(185–275) or GST–Cdc13p(276–332) was included in the kinase reaction. IP-kinase assays were performed as in (A) using strains KSC1333 (MEC1-HA sml1::HIS3) and LSS93 (HA-TEL1). Schematic diagram of Cdc13p illustrates the positions of four SQ motifs in the telomerase recruitment domain. Amino acid positions are numbered at the top. Recombinant wild-type and mutant GST–Cdc13p containing the S to A mutations were used as substrates in the Mec1-HA kinase assay (left) and Tel1-HA kinase assay (right). Asterisks indicate the complete abolishment of phosphorylation. (C) Summary of the Mec1p and Tel1p phosphorylation sites in the Cdc13p telomerase recruitment domain between amino acids 129 and 332.

Telomere analysis of cdc13-S mutants. (A) A cdc13 strain was constructed that also carried a CDC13 URA3 plasmid. Wild-type or cdc13 mutants on a CEN TRP1 plasmid were introduced into this strain. Since the drug 5-fluoro orotic acid (5-FOA) kills cells expressing Ura3p, only cells that lost the CDC13 URA3 plasmid will grow on FOA plates. (B) Mec1p/Tel1p phosphorylation sites were mutated from S to alanine (A), aspartic acid (D) and glutamic acid (E). The telomere lengths were monitored in these cdc13-S mutant strains. DNA from each strain was digested with XhoI, separated in a 1% agarose gel, transferred to a nylon membrane, and hybridized with a Y′ probe. DNA from the wild-type YPH499 was loaded as a control. Collected yeast cells have lost the CDC13 URA3 plasmid for at least 50 generations.

Telomere analysis of cdc13-S mutants following cell divisions. Assays were conducted as described in Figure 4. The telomere lengths were monitored in these cdc13-SA and cdc13-SE mutant strains. Genomic DNA from serial liquid dilutions was digested with XhoI, separated in a 1% agarose gel, transferred to a nylon membrane, and hybridized with a Y′ probe. DNA from the wild-type YPH499 was loaded as a control.

Growth of cdc13-S mutants. Spores of each strain were obtained from tetradissection. Each strain was repeatedly streaked on solid YEPD plates and grown for 3 days at 30°C. Cells from the first four restreaks are shown. It was noticeable that cdc13AA and cdc13AAA spores senesced fast, and S249/255DD or EE mutations suppressed this phenotype. This suppression was maintained at additional restreaks (data not shown).

Lack of the in vivo telomere addition in cdc13-SA mutants. UCC5706 cells were introduced with wild-type or cdc13-S mutants. Resulting strains were arrested using nocodazole. When cells were arrested, galactose was added to induce HO endonuclease expression. DNA was isolated after 0, 1, 2, 3 and 4 h, and digested with SpeI and analyzed with a fragment that recognizes sequence distal to the HO recognition site. Telomere addition (*) was observed in the wild-type, but not in the cdc13AA or EE strain.

Telomere lengthening by targeted Est1p in cdc13 cells. Telomere length was examined in strains of the indicated genotypes carrying a CDC13-EST1 (lanes 1 and 5), cdc13AA-EST1 (lanes 2 and 6), cdc13EE-EST1 (lane 7), CDC13 (lane 3) or cdc13AA (lane 4) plasmid. These strains were obtained as described in Figure 3. DNA was prepared at ∼60 cell divisions after the loss of the CDC13 URA3 plasmid. DNA from each strain was digested with XhoI, separated in a 1% agarose gel, transferred on to a nylon membrane, and hybridized with a Y′ probe.

Interaction of Cdc13p-AA with Pol1p. (A) Yeast cells Y190/pAS2-1, Y190/pAS2-1-CDC13 or Y190/pAS2-1-cdc13-AA carrying plasmid pACT2 or pACT2-POL1 were grown on SC medium lacking leucine and tryptophan at 30°C. Cells were then analyzed by their ability to grow in the absence of histidine. Ten-fold serial dilution of cells were spotted on glucose plates either without leucine and tryptophan or without leucine, tryptophan and histidine and incubated at 30°C until colonies formed. Photographs of the plates are shown. (B) Extracts were produced from strains expressing both bait and prey vectors as indicated below. Data are the average of three independent β-galactosidase measurements. Error bars, SD.