ten1-105 is synthetic lethal with cdc13-1 and est2-Δ, and progressively elongates telomere length independent of homologous recombination. (A) Telomere length increases as strains proliferate at 23°. The initial culture for each strain (1×) was inoculated from a colony arising on SD −Trp 5-FOA plates. To obtain successive generations of the ten1-ts strains, colonies from the SD −Trp 5-FOA plates were propagated by successive streak-outs on SD −Trp plates. To obtain cultures of later generations, cells were inoculated from colonies arising on these SD −Trp plates. Strains were cultured in liquid SD −Trp media at 23° for 3 days. Genomic DNA was digested with XhoI and the Southern blot was probed with ³²P[dGT/CA]. WT (wild-type, hc160), ten1-101 (hc1862), ten1-103 (hc1863), ten1-105 (hc1864), and ten1-106 (hc1865). (B) Telomere length defect is recessive in ten1Δ/pTEN1, pten1-105 strains. Southern blot showing ten1Δ strains bearing the following plasmids: lane 1, pCN250 (TEN1) + pCN284 (TEN1); lane 2, pCN250 (TEN1) + pCN358 (ten1-105); lane 3, pCN284 (TEN1) + pCN416 (vector). Strains were grown at 23° for 4 days, and the Southern blot was performed as in A. (C) Southern blot comparing telomere length in ten1-101 and ten1-101 rad52Δ strains. Cell cultures were incubated at 23° for 4 days. Genomic DNA was digested with XhoI, and the blot was probed with ³²P[dGT/CA]. Wild type (WT, hc160), ten1-101 (hc1862), rad52Δ TEN1 (hc1722), rad52Δ ten1-101 (hc1848). (D) Plates showing growth of ten1-105 double mutant strains that were streaked simultaneously on SD −Trp and SD −Trp 5-FOA plates. All strains initially contained the pTEN1-URA3 plasmid (pCN250); only cells losing this plasmid will grow on the SD −Trp 5-FOA plates. Plates were incubated at 23° for 5 days. Strains: hc2026, hc2025, hc2027, hc2028, hc2030, hc2029, hc2031, and hc2032.

de novo telomere addition requires Ten1 in G2/M arrested cells. (A) Schematic diagram of the left arm of chromosome VII containing the HO endonuclease recognition site adjacent to 81 bp of telomeric repeats (Diede and Gottschling 1999). The ADE2 gene is located on the centromere proximal side of the inserted telomere repeat. The chromosome fragment from the HO site to the end of the chromosome does not contain any essential genes. A LYS2 marker is located in this region to retain this nonessential fragment. Note: this diagram is not to scale. (B) Telomere addition at 36°. Wild-type and ten1-105 strains containing the telomere healing cassette were grown in SD −Lys media at 23°, and then switched to YP-raffinose media containing nocodazole to arrest cells in G2/M. While maintaining the arrest, galactose was added to induce expression of the HO endonuclease, and the cultures were simultaneously shifted to 36°. Cells were collected at 0, 1, 3, and 5 hr following addition of galactose. Genomic DNA was isolated, digested with SpeI, and fragments were separated on a 1% agarose gel. The Southern blot was probed with a [³²P]-labeled fragment that hybridizes to the ADE2 gene (Diede and Gottschling 1999). Both the native ADE2 locus (INT) and the HO-adjacent ADE2 gene are recognized by this probe. The gel was exposed on a Phosphorimager screen and the total amount of signal in the HO-adjacent ADE2 locus determined for each lane. The numbers below each lane represent the sum of the precut and cut fragments, normalized to the amount present at T = 0. PRE, fragment prior to HO digestion; INT, internal ADE2 control; arrow, fragment after HO digestion. WT (wild-type, hc1943), ten1-105 (hc1946). (C and D) Ade⁺Lys⁻ colony formation as a means to assess the ability of ten1-105 strains to heal the DSB if released from the G2/M arrest and plated at 23°. In C, cells were incubated at 23° while HO was induced; in D, cells were shifted to 36° during the induction. Colonies on SD −Ade were replica-plated to SD −Ade −Lys (and SD −Ade) after 5 days at 23°. The lines on the graphs show the proportion of Ade⁺ colonies that are also Lys⁻ at each time point [(Ade⁺ Lys⁻)/(Ade⁺)]. TEN1, ten1-105. The bars on the graphs show the percentage of Ade⁺ Lys⁻ colonies relative to the number of Ade⁺ colonies at T = 0; cells unable to heal the DSB are not expected to form colonies. Lighted shaded bars, TEN1; shaded bars, ten1-105 solid bars, ten1-105 .

ssTG_1-3 generation in ten1-101 and ten1-105 is cell cycle and Cdk1 dependent. (A) In-gel hybridization showing cell cycle dependency of single-strand generation in ten1-105. Asynchronous cell cultures growing at 23° were divided, with equivalent portions arrested at 23° in G1 with α-factor, in S phase with hydroxyurea (HU), or in G2/M with nocodazole. Cultures were then split and shifted to the indicated temperature for 4 hr while maintaining the arrested state. One culture was maintained in logarithmic growth prior to the temperature shift (Asyn). Genomic DNA was then isolated and split, with half treated with ExoI prior to XhoI digestion. The upper panel shows the gel probed with a [³²P]-CA oligo under native conditions, and the lower panel shows hybridization with the [³²P]-CA oligo following denaturation. WT (wild-type, hc160), yku80Δ (hc18), and ten1-105 (hc1864). (B) In-gel hybridization showing cell cycle dependency of single-strand generation in ten1-101. Asynchronous cell cultures were divided and either allowed to continue growth at 23°, or arrested at 23° in G1 with α-factor or in G2/M with nocodozole. Cultures were then split and incubated at the indicated temperature for 4 hr while maintaining the arrested state. Genomic DNA was then isolated and split, with half treated with ExoI prior to XhoI digestion. The agarose gel was probed with a [³²P]-CA oligo under native conditions. Only the native gel is shown; the total DNA loaded in each lane is shown in the ethidium bromide-stained gel slice. WT (wild-type, hc160), yku80Δ (hc18), and ten1-101 (hc1862). (C) In-gel hybridization showing the creation of ssTG_1-3 is dependent on functional Cdk1. Cells were arrested at 23° in G2/M with nocodazole, and then split, with half shifted to 36° for 4 hr. To inhibit the Cdc28-as1 activity, 1-NMPP1 was added to the cultures 1 hr prior to the temperature shift. The cells were arrested in nocodazole throughout the experiment. Treatment of the genomic DNA and gel probing were as in A, with the upper panel showing the native gel and the lower panel showing the denatured gel. Wild-type (WT, hc160), yku80Δ (hc18), cdc13-1 (hc1997), cdc13-1 cdc28-as1 (hc1998), ten1-105 (hc1864), and ten1-105 cdc28-as1 (hc2005) strains are shown.

Suppression of ten1-ts temperature sensitivity and TG_1-3 resection by exo1Δ. (A) Temperature sensitivity of ten1-101 and ten1-105 are suppressed by exo1Δ and not affected by rad52Δ. Serial 10-fold dilutions were plated on YPD and incubated at indicated temperatures. Wild-type (hc160), ten1-101 (hc1862), ten1-101 rad52Δ (hc1848), ten1-101 exo1Δ (hc1970), ten1-105 (hc1864), ten1-105 rad52Δ (hc1841), ten1-105 exo1Δ (hc1972), rad52Δ (hc2009), and exo1Δ (hc579). (B) exo1Δ reduces single-stranded TG_1-3 generation in ten1-105 and cdc13-1 strains. Cultures were grown at 23°, then split, with half shifted to 36°. Incubation continued at 23° and 36° for 4 hr. Genomic DNA was then isolated and half was treated with ExoI prior to XhoI digestion. The upper panel shows the gel probed with a [³²P]-CA oligo under native conditions, and the lower panel shows hybridization with the [³²P]-CA oligo after gel denaturation. The number indicated below is the ratio of signal in the lane on the native gel/denatured gel, minus the ratio determined from the wild-type lanes. Strains: wild-type (hc160), yku80Δ (hc18), ten1-105 (hc1864), ten1-105 exo1Δ (hc1972), cdc13-1 (hc1997), and cdc13-1 exo1Δ (hc1989).

ten1-ts alleles show progressive telomere elongation at permissive temperature. (A) Diagram of mutations in PCR-generated ten1 alleles. (B) Growth phenotype of ten1-ts alleles. Serial 10-fold dilutions were prepared from equivalent cell concentrations and stamped onto YPD plates. The plates were incubated at the indicated temperatures for 3–4 days. WT (wild-type, hc160), ten1Δ/pTEN1 (hc1832), ten1-101 (hc1862), ten1-103 (hc1863), ten1-105 (hc1864), and ten1-106 (hc1865). (C) Western blot showing expression of Ten1-ts proteins. The Ten1-ts two-hybrid plasmids were transformed into wild-type cells (hc160) and incubated at 30° overnight until the OD₆₀₀ reached 0.8–1.0. One hundred micrograms of each cell lysate was run on a 10% SDS–PAGE gel, and each HA-Gal4_DBD-Ten1-ts fusion protein was detected by anti-HA antibody (12CA5). A cross-reacting band present in the lysates is marked with an asterisk. Strains: No tag (hc160), Ten1 (pCN124), Ten1-101 (pCN445), Ten1-103 (pCN452), Ten1-105 (pCN441), and Ten1-106 (pCN447). (D) Yeast two-hybrid analysis of interaction of Ten1-ts with Stn1 and Cdc13. Ten1 and Ten1-ts proteins were expressed as a Gal4 DNA binding domain fusion (pCN124, pCN441, pCN445, pCN447, and pCN452). STN1 and CDC13 were expressed as Gal4 activation domain fusion proteins (S, pCN181; C, pVL835; V, vector, pACT2.2). An in-frame deletion removes a portion of the Cdc13 DNA binding domain in pVL835 (CDC13^Δ585-677). Tenfold serial dilutions of cultures were stamped on SD −Leu −Trp, SD −Leu −Trp −Ade, and SD −Leu −Trp −His +1 mM 3AT plates to examine the expression of the ADE2 and HIS3 reporter genes.

Cdc13-myc_18x telomere association is not diminished in ten1-101. (A) Chromatin immunoprecipitation of Cdc13myc_18x. Two hundred milliliters of wild-type untagged (hC160), CDC13myc₍₁₈₎ (hC1985) and ten1-101 CDC13myc_18x (hC2035) strains were grown in YPD or SD −His media to an OD₆₀₀ ∼1, and split into four 50-ml cultures. Two cultures were treated with nocodazole for 4 hr at 23° and 36° (Nz), and two cultures were diluted to an OD₆₀₀ = 0.4 and incubated at 23° or 36° for 4 hr (asynchronous cultures, A). Immunoprecipitations from cell lysates were carried out with an anti-myc antibody (12CA5). The percentage of the total telomeric DNA present in each IP was determined by dot blot analysis, hybridizing with a [³²P]d(GT/CA) probe, and exposing the blot on a Phosphorimager screen. To normalize the data, the percentage of telomere DNA recovered in each IP from Cdc13myc_18x strains was divided by the percentage of telomere DNA recovered in the IP from the untagged Cdc13 strain. The averages of four independent experiments are graphed, and the error bars represent the standard deviation. (B) Dot blot of ChIP DNA samples from strains grown either asynchronously or arrested in G2 with nocodazole probed with [³²P]d(GT/CA), as described in A. Shown are 10-fold dilutions of the immunoprecipitate and the input DNA for each strain.

ten1-103 and ten1-105 show conditional defects in telomere end protection. (A) Single-stranded telomeric DNA in ten1-105 at 23°, 30°, and 36°. Cultures were grown at 23° overnight and then shifted to the indicated temperature for 4 hr. Genomic DNA was isolated under nondenaturing condition and split, with half treated with 40 units ExoI (NEB) for 2.5 hr prior to digestion with XhoI. The gel was probed with a [³²P]-labeled CA oligo that hybridizes to the G-rich telomere strand. The upper panel shows the gel under native conditions. After exposure on a Phosphorimager, the gel was denatured under alkaline conditions and hybridized with the [³²P]-CA oligo to measure total TG_1-3 levels. The lower panel shows the denatured gel. The number indicated below is the ratio of signal in the lane on the native gel/denatured gel, minus the ratio determined from the wild-type lanes. Strains: WT (wild-type, hc160), yku80Δ (hc18), and ten1-105 (hc1864). (B) Single-stranded telomeric DNA in ten1-103 strains at 23°, 30°, and 36°. The strains were grown, and DNA was isolated and processed as described in A. The native gel is shown in full; a representative slice from the denatured gel is shown in the lower panel. WT (wild-type, hc160), yku80Δ (hc18), and ten1-103 (hc1863).

Rad52-YFP foci form in large-budded ten1-ts cells at semi-permissive and restrictive temperatures. (A) The percentage of nocodazole-arrested cells with Rad52-YFP foci. Cells were grown at 23°, incubated in nocodazole until arrested in G2/M (3 hr), and then split, with half of the cells shifted to 36° for 4 hr while maintaining the arrest. One hundred large-budded cells per strain were scored, and the average and standard deviation from four replicates are shown. (B) Micrographs of TEN1 RAD52-YFP and ten1-105 RAD52-YFP cells were taken following overnight incubation of strains in liquid media at 30° and observed by fluorescence microscopy (Nikon Eclipse 800). (C) The percentage of large-budded cells with Rad52-YFP foci following overnight incubation in liquid media at 30°. One hundred or more cells with large buds were scored from each culture, with at least 3 cultures scored for each strain. The average number of cells with foci is graphed, with error bars showing the standard deviations. Strains: TEN1 RAD52-YFP (hC1720), ten1-101 RAD52-YFP (hc1847), ten1-103 RAD52-YFP (hc1857), ten1-105 RAD52-YFP (hc1840), and ten1-106 RAD52-YFP (hc1853).

cdc17-1 significantly enhances the growth defect in ten1-ts, but not cdc13-1 strains. (A) Growth of single and double mutants. Ten-fold serial dilutions of strains were incubated on YPD at the indicated temperatures. cdc13-1 cdc17-1 strains were obtained by tetrad dissection. cdc17-1 ten1-ts strains were obtained by plasmid shuffle from parental strain hC1678, using plasmids pCN284, pCN309, pCN311, pCN358, and pCN359. The ten1-ts single mutant plates were incubated at the same time as the double mutants. (WT is hC160). (B) In-gel hybridization analysis of ssTG_1-3 in G2/M arrested cells. Strains were synchronized in nocodazole at 23°, and then split and incubated 3 more hr in nocodazole at 23° or 36°. DNA was isolated under native conditions; equivalent DNA concentrations were digested with XhoI and analyzed by in-gel hybridization. The total DNA loaded in each lane is similar, as visualized by ethidium bromide staining, shown below denatured gel.