Mec1-HAp associates with telomeres as cells escape the cell cycle arrest that occurs due to telomere shortening. a Telomere shortening in Mec1-HAp cells lacking the telomerase catalytic subunit Est2p. Cells lacking telomerase were generated by deleting the genomic copy of EST2, covering the deletion with a plasmid bearing the EST2 gene, and then isolating cells that had lost the plasmid. Cells lacking telomerase were then cultured for the number of population doublings (PD) shown, where the 0 PD sample contains cells that retain the EST2 plasmid and the other PD samples contain cells that lack telomerase. Genomic DNA from each PD was digested with EcoR V and analyzed by Southern blotting using the VI-R PCR product from the primers shown in panel b as probe. Telomeres gradually shortened with increasing PD, allowing the isolation of cells with different telomere lengths. The black arrow designates a background band that hybridizes to probe. The white arrowheads designate rearranged telomeres in PD 70 that are characteristic of the survivor cells that escape the short telomere cell cycle arrest. b A schematic of the loci and primers used to monitor telomere association. The telomeres from the right arm of chromosome VI (VI-R) and two internal loci from separate chromosomes (GAL10 and ARO1) were PCR amplified in the ChIP experiments in this study. c Analysis of the Mec1-HAp immunoprecipitates using the PCR primers indicated in panel b. Telomere enrichment values are calculated as the ratio of the intensities of the VI-R telomere band to the control ARO1 chromosomal band as described in the “Materials and methods.” The values shown are averages of multiple determinations. The input dilution is the input DNA that was used to determine the linear range of PCR amplification (see “Materials and methods”). d Comparison of cell doubling times and Mec1-HAp telomere enrichments. Mec1-HAp association increases at PD 70 just as survivors are beginning to arise in the culture, as indicated by the simultaneous decrease in doubling time and appearance of rearranged telomeres (panel a)

The VI-R telomere sequences isolated from the Mec1-HAp ChIP are capped by short stretches of TG_1–3 sequences. a The telomere PCR method (adapted from the method of Förstemann et al. (2000)). The Mec1-HAp ChIP is heated to reverse the formaldehyde cross-links, and DNA is subsequently purified and treated with dCTP and lambda terminal transferase to add an oligo-dC 3' extension. PCR is then performed with a specific primer to the VI-R telomere (TELVIR-88) and an oligo-dG primer (dG₁₈-BamHI) to amplify a fragment containing the TG_1–3 sequences (represented by the red box). PCR products were then cloned and sequenced. b Telomere sequences from the Mec1-HAp ChIP from population doubling 70 (Fig. 1). The VI-R reference sequence is the published genomic sequence that starts at chromosome VI base number 270,089 (Engel et al. 2010)

Mec1-HAp and Tel1-HAp telomere association during induced telomere dysfunction in yku70Δ cells. a Telomere dysfunction is inducible in yku70Δ cells. A schematic of chromosomal ends where the telomere repeats are shown as curved lines. At 25°C, yku70Δ cell telomere repeats are maintained at a constant length. Previous work in the W303 strain, the same strain used here, showed that following prolonged incubation at 37°C, the 5' strand of the chromosome is degraded and a DNA damage checkpoint-mediated arrest ensues (Maringele and Lydall 2002). b A Southern blot for VI-R telomere lengths of cells grown at 25°C or shifted to 37°C and grown for the times indicated. The blot was probed and processed as in Fig. 1a. The 0-h time point corresponds to cells grown at 25°C. WT indicates samples from MEC1-HA cells or TEL1-HA cells with a wild-type YKU70 gene. This Southern analysis showed no evidence of survivor formation in the cells grown for 24 h at 37°C. c Representative Mec1-HAp ChIP results for wild-type and yku70Δ cells grown under the conditions shown in panel b. The ChIP was performed and analyzed as in Fig. 1c. The levels of VI-R telomere enrichment show the results of two independent ChIP experiments. d Culture doubling times of yku70Δ Mec1-HAp cells are plotted along with the VI-R enrichment values from panel c. The 70-h doubling time between the 12- and 24-h time points is indistinguishable from cessation of cell growth. e Representative Tel1-HAp ChIP results for wild-type and yku70Δ cells grown under the conditions shown in panel b. The levels of VI-R telomere enrichment show the results of two independent ChIP experiments. f Culture doubling times of yku70Δ Tel1-HAp cells are plotted along with the VI-R enrichment values from panel e. The telomere lengths and ChIP enrichments for the Tel1-HAp wild-type and yku70Δ samples at 0 and 8 h in panels b, e, and f have been published previously ((Hector et al. 2007) used with permission) and are shown as part of the larger time course to allow a direct comparison of Mec1-HAp telomere association assayed under the same conditions

Suppressing telomere dysfunction in yku70Δ cells abrogates Mec1-HAp telomere association. a Temperature sensitive growth is suppressed in yku70Δ exo1Δ cells. Aliquots (5 μl) of growing cells were spotted onto plates pre-warmed to 25°C or 37°C and then grown for 24 h at the indicated temperatures. b A Southern blot for VI-R telomere lengths of cells grown as indicated, where the 0-h time point corresponds to cells grown continuously at 25°C, probed and processed as in Fig. 1a. c Representative Mec1-HAp ChIP results for cells grown under the conditions shown in panel b. The ChIP was performed and analyzed as in Fig. 1c. The levels of VI-R telomere enrichment show the results of two independent ChIP experiments. The wild-type and yku70Δ cell samples from Fig. 3c were processed alongside the other samples for comparison. d Representative Tel1-HAp ChIP results for cells grown under the conditions shown in panel b, where the enrichment values are the results of two independent experiments. The wild-type and yku70Δ cell samples from Fig. 3e were processed alongside the other samples for comparison. e Graphical summary of the ChIP results of different Mec1-HAp and Tel1-HAp cells grown at 25°C or shifted to 37°C for 24 h. The Mec1-HAp telomere association observed in yku70Δ cells at 37°C is absent in yku70Δ exo1Δ cells

Mec1-HAp and Tel1-HAp association with the XV-L telomere parallels the results with the VI-R telomere. a A schematic showing the loci and primers used to monitor Mec1-HAp association with the XV-L telomere and the negative control internal locus ARO1, with the PCR primers indicated by arrows. b Representative Mec1-HAp ChIP results from the same strains analyzed for VI-R telomere association in Fig. 4c for cells grown continuously at 25°C or shifted to 37°C for 24 h. The quantitations shown are the results of two independent ChIP experiments. c Representative Tel1-HAp ChIP results for the same strains analyzed in Fig. 4d. The quantitations shown are the results of two independent ChIP experiments. d Graphical summary of the ChIP results. The magnitude of Tel1-HAp association with the XV-L and VI-R (Fig. 4) telomeres in yku70Δ cells was similar at 37°C but different at 25°C, possibly due to the different DNA sequences adjacent to the TG_1–3 sequences at each locus

Cell cycle analysis of Mec1-HAp telomere association in yku70Δ cells (a–c) and wild-type cells (d–f). Cells were grown at the permissive temperature (20°C), arrested with α-factor and released into the cell cycle until all of the cells were in G2/M phase, and samples were taken for FACS and ChIP. Both strains bear the bar1Δ mutation, which eliminates a secreted protein that degrades α-factor and facilitates the synchronization and release. a FACS analysis showing DNA content of the yku70Δ cells analyzed. b Representative ChIP results for the yku70Δ VI-R telomere. Values below each lane are the quantitations of all enrichment determinations, each analyzed in triplicate. c Graphical presentation of the fold enrichments in panel b, where the error bars represent the standard deviations of the analysis of each time point. d FACS analysis showing DNA content of the wild-type cells analyzed. This synchrony was allowed to proceed until some of the cells began entering the next G1 phase. e Representative ChIP results for the wild-type VI-R telomere. Quantitations shown below the gel are the average of two separate experiments, where each ChIP experiment was analyzed in triplicate. f Graphical presentation of a representative cell cycle synchrony, where the error bars represent the standard deviations of the analysis of each sample. The cell cycle phases from panel d are indicated, including the portion of cells that re-entered G1 phase. The entire experiment was done twice