The 80-kD truncated Cdc13 protein retains DNA binding activity. DNA binding ability of wild-type Cdc13 (lanes 2,3) and the 80-kD truncated derivative of Cdc13-3 (lanes 4,5), as assessed by DNA gel shift assays with 5′ end-labeled d(TGT GTGGG)₃ oligomer at 1 nM and protein concentration at 10 or 1 nM, as indicated; lane 1, no protein added.

Cdc13, but not Cdc13-2, interacts with Stn1, by a two-hybrid test. Activation of a lacZ two-hybrid reporter gene was evaluated using X-gal overlay assays to detect β-galactosidase activity. Results are shown for three independent transformants each of CDC13 and STN1 (pVL705 and pVL859), cdc13-1 and STN1 (pVL667 and pVL859), cdc13-2 and STN1 (pVL854 and pVL859), and CDC13 and vector (pVL705 and pACT2). The ADE2 and HIS3 reporter genes present in this strain (pJ69-4A; James et al. 1996) gave results similar to the lacZ reporter (data not shown).

Cdc13 plays a dual role in regulation of G-strand synthesis by telomerase. The extended G strand, which is generated by a telomerase-independent mechanism (Wellinger et al. 1996), provides a substrate for binding by Cdc13. Once bound to the telomere, Cdc13 initiates synthesis of the G strand by recruiting telomerase through an interaction with the Est1 telomerase subunit. Binding of Est1 to Cdc13, and hence enzyme recruitment, is blocked by the cdc13-2 mutation. Elongation of the G strand by telomerase is subsequently followed by fill-in synthesis of the C strand by lagging strand synthesis machinery, which inhibits further synthesis of the G strand by telomerase. This inhibitory step is defective in the cdc13-5 mutant, allowing telomerase to further extend the G strand. We propose that binding of Stn1 to Cdc13, thereby displacing telomerase, is the mechanism by which unchecked telomerase synthesis is inhibited.

The cdc13-5 strain exhibits an altered end structure during S phase. (A) DNA from CDC13⁺, cdc13-3, and cdc13-5 strains were mock treated (lanes 3,5,7) or treated with ExoI (lanes 4,6,8), digested with XhoI, and analyzed by nondenaturing in-gel hybridization with a C_1–3A oligonucleotide probe (top); the same gel, denatured and rehybridized with the same oligonucleotide probe (bottom); lanes 1,2: pVL260 (with 80 bp of G_1–3T telomeric repeat DNA) heat denatured or nondenatured, respectively. Size markers: λ HindIII-digested DNA. (B) DNA from a cdc13-5 strain, prepared from time points collected every 20 min after release from an α factor–induced G₁ arrest, was examined by native in-gel hybridization with a C_1–3A oligonucleotide probe (top, left), denaturation and rehybridization with the same probe (bottom, left), and FACS analysis (right); lanes 1,2: pVL260, denatured or nondenatured, respectively.

Overexpression of STN1 suppresses the telomere elongation phenotypes of cdc13-5 and pol1 mutant strains. (A) Telomere length of a cdc13-Δ strain covered by a CDC13⁺ plasmid (pVL440; lanes 1–4) or a cdc13-5 plasmid (pVL1033; lanes 5–8) plus a plasmid overexpressing STN1 (pVL1035) or a control vector (pVL1036). (B) DNA prepared from a cdc13-Δ strain covered by a CDC13⁺ plasmid (pVL440; lanes 3,4) or a cdc13-5 plasmid (pVL1033; lanes 5,6) plus a plasmid overexpressing STN1 (pVL1035; lanes 4,6) or a control vector (pVL1036; lanes 3,5) was analyzed by nondenaturing in-gel hybridization with a C_1–3A oligonucleotide probe (left); denatured and rehybridized with a Y′ probe (middle); and denatured and rehybridized with a C_1–3A oligonucleotide probe (right); lanes 1,2: pVL260 heat denatured or nondenatured, respectively; lane 7: pJH354, containing a region of Y′ sequence. (C) Telomere length of the indicated pol1 mutant strains or the isogenic POL1⁺ strain (indicated as wt) transformed with either a plasmid overexpressing STN1 (pVL1034) or vector (YEplac112). (D) CDC13 (UCC3505, Singer and Gottschling 1994), cdc13-5 (TVL422) and sir3-Δ (TV318), each containing a telomere-proximal URA3 gene, were grown in rich media, and 10-fold serial dilutions were plated on rich media, on media lacking uracil, and on media containing 5-FOA and incubated for 48 h at 30°C.

The cdc13-5 mutation is not compromised for the essential role of CDC13. (A) 10-fold serial dilutions of CDC13⁺, cdc13-5, and cdc13-1 strains were plated on rich media and incubated at 26°C, 30°C, and 36°C. (B) Progression of individual cells to microcolonies, for CDC13, cdc13-1, and cdc13-5 strains, measured as described by Weinert and Hartwell 1993. Strains were grown to early log growth phase at 23°C and plated for individual cells on prewarmed plates at 30°C (a nonpermissive temperature for the cdc13-1 strain); for each genotype, ≥100 cells were monitored at 0, 4, and 8 h for the proportion of large budded cells. (C) Northern analysis monitoring the expression level of RNR genes. CDC13⁺ and cdc13-5 strains were grown to an OD₆₀₀ of 0.4 at 30°C, and an aliquot of CDC13⁺ cells was incubated in the presence of 200 mM hydroxyurea for 3 h; the cdc13-1 strain was grown at 23°C and an aliquot shifted to 30°C for 3 h. Relative RNR transcript levels are shown, normalized to the fold difference between the level of the RNR transcript relative to the U1 transcript levels in the wild-type strain.

Cdc13 is a negative regulator of telomere elongation. (A) Telomere length of CDC13⁺ (lane 1), cdc13-3 (lane 2), and cdc13-5 (lane 3). The cdc13-3 and cdc13-5 haploid strains were derived from DVL233 and DVL326, respectively, by tetrad dissection; DNA was prepared after ∼35 generations of growth. (B) Anti-HA immunoblot of anti-HA immunoprecipitates from equal amounts of whole cell extract prepared from a cdc13-Δ strain expressing HA3–Cdc13 (lane 1; pVL841), HA3–Cdc13-5 (lane 2; pVL903) and untagged Cdc13 (lane 3; pVL440); all plasmids were single-copy CEN vectors, with CDC13 expressed from its native promoter. Arrowheads indicate the full-length Cdc13 protein (∼116 kD) and the truncated Cdc13-5 protein (∼80 kD). (C) Telomere length of CDC13/cdc13-5 EST1/est1-Δ RAD52/rad52-Δ (lane 1), CDC13⁺ (lane 2), rad52-Δ (lane 3), cdc13-5 (lanes 4,5), cdc13-5 rad52-Δ (lanes 6,7), est1-Δ (lane 8), and cdc13-5 est1-Δ (lane 9). The haploid strains shown in lanes 2–9 were obtained from DVL328 (lane 1) by tetrad dissection; DNA was prepared after ∼35 generations of growth, except for lanes 5 and 7, which were grown for an additional 10 generations. (D) Telomere length of cdc13-5 (lane 1), cdc13-2,5 (lane 2), cdc13-2 (lane 3), and CDC13⁺ (lane 5); strains were generated by transforming cdc13-Δ/pVL438 with pVL1033 (cdc13-5), pVL1360 (cdc13-2,5), pVL690 (cdc13-2), and pVL440 (CDC13), followed by eviction of pVL438 on 5-FOA. Although a cdc13-2,5 strain exhibits a senescence phenotype characteristic of a telomere replication defect, telomere length of the cdc13-2,5 strain is reproducibly not quite as short as the cdc13-2 strain at any given time point (cf. lanes 2 and 3). This may be caused by residual recruitment activity of the Cdc13-2 mutant protein (which is consistent with the prior demonstration that the senescence phenotype of a cdc13-2 strain is somewhat delayed relative to telomerase null mutations; Lendvay et al. 1996), thereby allowing partial manifestation of the cdc13-5 defect.