1. Volume 27, Issue 5, Pages 851-858 (September 2007)
Tel1p Preferentially Associates with Short Telomeres to Stimulate Their Elongation 
Ronald E. Hector, Rebecca L. Shtofman, Alo Ray, Bo-Ruei Chen, Thihan Nyun, Kathleen L. Berkner, Kurt W. Runge 
Molecular Cell 
Volume 27, Issue 5, Pages (September 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Tel1-HAp Associates with Shortening Telomeres
(A) Tel1-HAp association with the VI-R telomere. Analysis of the Tel1-HAp immunoprecipitates using PCR primers that amplify sequences adjacent to the VI-R telomere repeats and two internal chromosomal loci (ARO1 and GAL10) as negative controls. The 0 population doubling cells are those that have telomerase activity. Enrichment values are calculated as the ratio of the VI-R telomere band to ARO1 chromosomal band intensities in the + antibody and − antibody lanes, and the values shown are the average of two PCR quantitations (Supplemental Data). Total chromatin shows dilutions of the crosslinked “input” DNA for the immunoprecipitation and was used to determine the fold enrichment of the VI-R band (Taggart et al., 2002). The generation of the est2Δ strains, the location of the PCR primers, the lengths of the telomeres at each population doubling, and the appearance of “survivor” telomeres are described in Figure S1.
(B) Tel1-HAp association with the XV-L telomere. The ChIP samples in (A) were analyzed for the enrichment of a sequence adjacent to XV-L, except that enrichment values were calculated from three PCR quantitations.
(C and D) Tel1-HAp telomere association peaks at senescence and falls as survivors overtake the culture. Tel1-HAp association (C) parallels the changes in doubling time (D) seen for cells that arrest due to telomere shortening (Enomoto et al., 2002; IJpma and Greider, 2003) and escape due to the production of survivor cells. The error bars in (C) are the ranges of telomere enrichment reported in (A) and (B).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Tel1-HAp Telomere Association with Short Telomeres in yku70Δ Cells
(A) A telomere-specific checkpoint is inducible in yku70Δ cells grown at 37°C. Shown is a schematic of chromosome ends where the telomere repeats are depicted as curved lines. At 25°C, yku70Δ cell telomere repeats are maintained at a constant short length. After incubation at 37°C, a telomere-specific checkpoint is induced and telomeres shorten slightly (Teo and Jackson, 2001).
(B) Southern analysis of VI-R telomeres of wild-type and yku70Δ cells expressing Tel1-HAp and grown continuously at 25°C or shifted to 37°C for 8 hr.
(C) Tel1-HAp associates with the VI-R telomere in yku70Δ cells grown at 25°C or shifted to 37°C for 8 hr. A representative gel and averaged data from duplicate cultures are shown. ChIP analyses were performed as described in Supplemental Data.
(D) Quantitation of the telomere enrichments from (C). Error bars are the ranges reported in (C).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Tel1-HAp Associates with the Shortest Telomeres and Stimulates Telomere Elongation
(A) The telomere PCR method to determine TG1-3 strand repeat length (Förstemann et al., 2000). C-tailed genomic, input, or ChIP DNA is amplified with a primer that hybridizes to a unique telomere and an oligoG primer.
(B) Preparation of DNA for ChIP does not alter telomere length. Lanes labeled “Genomic” were prepared by using standard procedures, whereas lanes labeled “Input” were the sheared, crosslinked chromatin samples prior to immunoprecipitation that were processed for PCR analysis. Both methods gave the same calculated TG1-3 tract sizes for a range of telomere sizes.
(C and D) Tel1-HAp associates with the shortest VI-R (C) and XV-L (D) telomeres. ChIP samples where Tel1-HAp association was clearly detectable after loss of the telomerase RNA gene (Figure S2, PD 40 and 50) have telomeres that are shorter than the majority of telomeres in the input chromatin.
(E and F) A high-copy plasmid expressing a GAL4(DNA binding domain)-TEL1 fusion results in association with VI-R (E) and XV-L (F) telomeres of all lengths and telomerase-dependent telomere elongation. Cells without telomerase were generated by loss of the gene for TLC1 using the method in Figure S1, and ChIP was performed with antibody against the Gal4p DNA binding domain. The rightmost lane labeled “wild type” in (F) is the 0 PD input sample from (D), which is shown for size comparison with telomeres from cells bearing the GAL4-TEL1 plasmid.
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5. Figure 4
Tel1-HAp Telomere Association Is Independent of Tel1p Kinase Activity and Is Enhanced by Mre11p
(A) The tel1-HA-kd allele causes cells to maintain short telomeres, and the mre11 mutation blocks GAL1-TEL1-HA-mediated telomere elongation. VI-R TG1-3 tract lengths determined by telomere PCR are shown for strains grown for 100 divisions under the condition shown to establish steady-state telomere lengths. Cells bearing the GAL1-TEL1-HA construct were grown on glucose (glc) or galactose (gal) to repress or induce TEL1-HA, respectively.
(B) The tel1-HA-kd protein associates with telomeres. A representative gel from triplicate ChIP assays with multiple PCR quantitations of each ChIP is shown.
(C) Tel1-HAp VI-R telomere enrichments for different strains with similar telomere lengths. Fold telomere enrichments for the tel1-HA-kd, the mre11 GAL1-TEL1-HA, and MRE11 GAL1-TEL1-HA (grown on galactose) strains in (A) and (B) and the yku70 strain grown at 25°C (Figure 2B) are shown. Telomere enrichments for the mre11 and MRE11 strains are from triplicate ChIP experiments. Error bars are the ranges and standard deviations of telomere enrichments from Figure 2B (yku70), (B) (tel1-kd), and the mre11 and MRE11 strain ChIP experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions