Skip to main content
. 2010 Mar 1;24(5):502–515. doi: 10.1101/gad.1869110

Figure 3.

Figure 3.

A TGn-HO set of strains to study telomere healing at HO-induced DSBs. (A) Schematic representation of the modified Chr VII–L to generate the TGn-HO set of strains. The ADH4 locus was replaced by the ADE2 or URA3 gene followed by telomere seed sequences of different sizes (no telomeric seed, 5, 11, 17, and 81 bp of TG1–3 repeats), followed by the HO endonuclease cleavage site. The location of the probe used for Southern blotting (star on a bar) is also shown. (B) Quantitation of telomere addition frequency, as determnined by αAA resistance. The strains tested were PIF1 (WT; black bars) and pif1-m2 (gray bars) derivatives of strains TG0-HO, TG5-HO, TG11-HO, TG17-HO, and TG81-HO. The data are presented as the mean ± SEM (N ≥ 3). (C) The pif1-m2 mutation stimulates telomere addition in the TG11-HO strain. Cultures of wild-type and pif1-m2 derivatives of TG11-HO rad52Δ were arrested with nocodazole. HO expression was induced using galactose, and samples were taken at the indicated time points. In addition, Lys+ and Lys− derivatives of TG11-HO rad52Δ pif1-m2 were collected from the genetic assay based on αAA selection. A Southern blot analysis of EcoRV-cut genomic DNA probed with a section of the URA3 gene is shown. The band labeled PRE represents the EcoRV-digested fragment from Chr VII–L. After cleavage with HO, this fragment is converted into a new band (CUT). The URA3 probe also detects the ura3-52 locus (INT) and serves as a loading control. New telomere elongation forms a smear above the CUT band. (D–F) Quantitation of telomere addition frequency, as determnined by αAA resistance. The strains tested were wild-type (WT), rrd1Δ, and est2Δ derivatives of pif1-m2 TG0-HO (D), pif1-m2 TG5-HO (E), or TG11-HO (F). The data are presented as the mean ± SEM (N ≥ 3). (G) Quantitation of telomere addition frequency of wild-type, rrd1Δ, yku80Δ, rrd1Δ yku80Δ, and est2Δ derivatives of pif1-m2 TG0-HO. The data are presented as the mean ± SEM (N ≥ 3). (H) Quantitation of telomere addition frequency of wild-type, rrd1Δ, yku80Δ, and rrd1Δ yku80Δ derivatives of TG0-HO. The data are presented as the mean ± SEM (N ≥ 3).