1. Delivery of Yeast Telomerase to a DNA Break Depends on the Recruitment Functions of Cdc13 and Est1 
Alessandro Bianchi, Simona Negrini, David Shore 
Molecular Cell 
Volume 16, Issue 1, Pages (October 2004)
DOI: /j.molcel

2. Figure 1
Full-Length Est1 and a Small N-Terminal Domain of Cdc13 Promote Telomere Healing when Tethered to a DSB
(A) Schematic representation of the modified subtelomeric region of Chromosome VII-L. The box marked “PCR” indicates an 83 bp mouse DNA sequence introduced as a target for quantitative PCR in chromatin immunoprecipitation experiments. The sequence distal to the HO cut site is dispensable for cell viability.
(B) A replica-plating assay for de novo telomere formation (telomere healing). Colonies grown on medium lacking tryptophan (left plates) were replica plated onto galactose-containing plates to induce HO cleavage (data not shown), allowed to grow overnight, and then replica plated again onto 5-fluoroorotic acid (FOA, which inhibits the growth of URA3+ cells) plates lacking adenine (right plates).
(C) Schematic representation of the domain organization of Cdc13 (top) and of the five GBD-Cdc13 fusion proteins recovered from the screen.
(D and E) A quantitative plating assay for telomere healing using counterselection of LYS2 on α-aminoadipate plates lacking adenine. In (D) and (E), approximately 6 × 104 and 2 × 105 (within ±10% in each plate), respectively, viable cells that received a cut at the HO site were spread on each plate, in the presence of the indicated GBD fusion proteins. In (E), a second copy of EST1, in either wild-type or mutant form, was present on a centromeric plasmid.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Tethered Est1, Est1-60, and Yku70 Recruit Est2 to an Internal Chromosomal Site
Quantification by ChIP of the binding of Myc-tagged Est2 to three UASg sites located in the subtelomeric region of Chromosome VII-L (Figure 1A) in the absence of HO cleavage and in the presence of GBD alone, GBD-Est1, GBD-Est1-60, and GBD-Yku70.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Est1- and Cdc13-Dependent Association of Est1 with the DSB
(A) Cleavage at the HO site in a wild-type strain expressing Myc-tagged Est1 was monitored by Southern blotting. Top panel: “I” indicates a band from the NMD5 locus that serves as an internal loading standard, “U” denotes the uncut fragment hybridizing to an ADE2 probe, and “C” indicates the band resulting from “U” after HO cleavage, which corresponds to the centromere-proximal fragment. Telomere formation at the DSB was detectable as a smeared band just above the “cut” (C) fragment. Bottom panel: a representative quantification of cleavage (from a cdc13-2 est1-60-Myc strain) achieved by measuring the amount of “uncut” (U) band relative to the internal control, normalized to the uninduced (t = 0) sample.
(B and C) Quantification by ChIP of binding of (B) Myc-tagged Est1 and (C) Cdc13 proteins to the DSB upon induction of cleavage at the HO site.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Est1- and Cdc13-Dependent, but TG-Addition-Independent, Association of Est2 with the DSB
(A) Cleavage at the HO site and subsequent telomere repeat addition in wild-type and mutant strains expressing Myc-tagged Est2 was monitored by Southern blotting as in Figure 3A.
(B) Quantification by ChIP of binding of Myc-tagged Est2 to the DSB upon induction of cleavage at the HO site in the same genetic backgrounds as in (A).
Molecular Cell  , DOI: ( /j.molcel )