1. Telomerase and Tel1p Preferentially Associate with Short Telomeres in S. cerevisiae 
Michelle Sabourin, Creighton T. Tuzon, Virginia A. Zakian 
Molecular Cell 
Volume 27, Issue 4, Pages (August 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Generating a Single Short Telomere
(A) The endogenous telomere VII-L was modified as described for Lev220 (Marcand et al., 1999). The Flp1p recombinase is induced by addition of galactose. Its action removes the DNA between the two FRT sites and releases it as a nonreplicating circle, leaving behind a single FRT site and a telomere that contains only ∼100 bp of telomeric C1-3A/TG1-3 DNA. By restreaking postgalactose cells and screening for those that have lost the ability to grow in the absence of uracil we generated a control strain (B) for each experimental strain (A). Relevant restriction enzyme sites are indicated (X, XhoI; S, StuI; V, EcoRV; P, PstI; H, HindIII; S, StuI; and V, EcoRV). Drawing not to scale.
(B) Schematic of experimental protocol.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Binding of Est2p and Est1p Is Increased at Short Telomere VII-L in a TEL1-Dependent Manner
After α factor arrest and induction of telomere shortening, cells expressing either Myc-tagged Est2p (A–C) or Myc-tagged Est1p (D–F) were released into the cell cycle and samples were taken at multiple time points throughout the subsequent synchronous cell cycle for ChIP. In each panel, open symbols are binding in strain A and closed symbols are binding in strain B. Error bars indicate standard deviations and are provided for every data point. The sequence being PCR amplified is indicated in bold in the upper right corner of each panel.
(A) Est2p binding to short (open squares) differed from WT (closed squares) telomere VII-L in a statistically significant manner from 52.5 to 90 min (p values = –0.045). The large standard deviations at the 52.5 and 60 min time points are due to the fact that the exact timing of the peak of Est2p binding varied between independent synchronies. In the four different synchronies analyzed here, the average enrichment of Est2p at short telomere VII-L at its S phase peak was 3.1× over binding to WT telomere VII-L at the same time point, with a range of 2–4.4×.
(B) Est2p binding to unmodified telomere VI-R (triangles) was statistically indistinguishable in strains A and B.
(C) Est2p binding to telomere VII-L in tel1Δ cells. Without TEL1, Est2p binding to short telomere VII-L (open squares) was statistically indistinguishable from binding to WT telomere VII-L (closed squares), except at 90 min, when the p value was Inset, the 30–75 min time points are shown at a different scale versus no-tag control (circles).
(D) Est1p binding to short (open squares) differed from WT (closed squares) telomere VII-L in a statistically significant manner from 37.5 to 90 min (p values = –0.039). Error bars represent the standard deviations for the values above and below the mean calculated separately.
(E) Est1p binding to unmodified telomere VI-R (triangles) was statistically indistinguishable in strains A and B.
(F) Est1p binding to telomere VII-L in tel1Δ cells. Without TEL1, Est1p binding to short telomere VII-L (open squares) was statistically indistinguishable from binding to WT telomere VII-L (closed squares). Inset as in (C).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Telomere Binding of Ku80p, Cdc13p, and Rif1p Is Unaffected by Telomere Length
Experimental details are the same as in Figure 2 legend.
(A) Ku80p binding to short telomere VII-L (open squares) was statistically indistinguishable from binding to WT telomere VII-L (closed squares).
(B) Cdc13p binding to short telomere VII-L (open squares) was statistically indistinguishable from binding to WT telomere VII-L (closed squares).
(C) Rif1p binding to short telomere VII-L (open squares) was statistically indistinguishable from binding to WT-length telomere VII-L (closed squares), except at 0 min (p value = 0.023) and 30 min (p value = 0.029); other p values were 0.09–0.98.
(D–F) Binding of Ku80p (D), Cdc13p (E), and Rif1p (F) to unmodified telomere VI-R was statistically indistinguishable in strains A (open triangles) and B (closed triangles).
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5. Figure 4 Rif2p Binding Is Reduced at the Short VII-L Telomere
Error bars represent standard deviations and are provided for each data point.
(A) Rif2p binding to short telomere VII-L (open squares) was lower than binding to WT-length telomere VII-L (closed squares) (p values = –0.032), except at 90 min (p value = 0.23).
(B) Rif2p binding to unmodified telomere VI-R (triangles) was statistically indistinguishable in strains A and B, except at 30 min (p value = at 30 min; p values were 0.06–0.45 at other points).
(C) Rap1p binding to telomere VII-L (VII-L), telomere VI-R (VI-R), and the FLP circle product (URA3) was monitored in α factor-arrested cells after galactose induction of telomere shortening in strains A (open bars) and B (closed bars). Binding to short telomere VII-L was reduced ∼50% relative to WT VII-L in a statistically significant manner (p value < ). When the values of Rap1p binding to the FLP circle product were added to the values for the short telomere VII-L (hatched bars), they were statistically indistinguishable from the levels of Rap1p at WT telomere VII-L (closed bars; p value = ).
(D) When the values of Rif2p binding to the FLP circle product were added to the values for short telomere VII-L (open diamonds), they were statistically indistinguishable from the levels of Rif2p at WT telomere VII-L (closed squares; p values = 0.16–0.45).
(E) Binding of Est2p (open circles), Rif1p (open squares), and Rif2p (open triangles) to the FLP circle product. Because there is no circle in the control B strains, closed symbols show the background binding to ura3-1 in these strains.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Tel1p Preferentially Binds Short Telomeres
Error bars represent standard deviations and are provided for each data point.
(A) Tel1p bound preferentially to short (open squares) relative to WT (closed squares) telomere VII-L in a statistically significant manner throughout the cell cycle (p values = 5.6 × 10−6 to 0.031).
(B) Binding of Tel1p to unmodified telomere VI-R was statistically indistinguishable in strains A (open triangles) and B (closed triangles). Binding of Tel1p to VI-R (and WT telomere VII-L in A) at 52.5 and 60 min was significantly higher than binding to nontelomeric ARO1 DNA (p values ranged from 1.1 × 10−6 to 0.028). Inset is as in Figure 2C.
(C) Binding of Tel1p in strain A to short telomere VII-L (squares) and unmodified telomere VI-R (triangles) across two cell cycles (20 min time points from 0 to 180 min).
(D) Representative western blot for Clb2p using a polyclonal α-Clb2p antibody. Samples as in (C). Lane 1: A, asynchronous cells; lanes 2–11, 0–180 min after release from α factor arrest. Marker size is indicated at the right (kD).
(E) Representative RNR1 RNA quantitation using RT-PCR. Samples are as in (C) and (D).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Preferential Binding of Both Tel1p and Telomerase to Short Telomeres Is Dependent on the MRX Complex
Error bars represent standard deviations and are provided for each data point. Binding of Tel1p (A) and Est2p (B) to short telomere VII-L (open triangles) and WT telomere VI-R (closed triangles) in strain A cells with the xrs2-664 truncation is shown graphed versus the previously determined levels of binding at short (open squares) and WT (closed squares) telomere VII-L in XRS2 strains (dotted lines) from Figures 5A and 2A, respectively. Preferential binding of both Tel1p and Est2p to short telomere VII-L is lost when Tel1p can no longer interact with the MRX complex.
Molecular Cell  , DOI: ( /j.molcel )
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