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

Figure 5.

Figure 5.

Tethering Cdc13 to a DSB overrides the Rrd1/Pph3-Mec1 regulatory network. (A) Schematic representation of the modified Chr VII–L in the GBD fusion assay. The restriction sites (EcoRV) and the probe location (star on a bar) used for Southern blotting are depicted. Telomere healing events stimulated by the GBD fusion can be detected via the loss of LYS2 and the retention of ADE2. (B) Quantitation of telomere addition frequency of wild-type (WT), yku80Δ, rrd1Δ, and rrd1Δ aur1∷RRD1 derivatives of the GAL4UAS rad52Δ strain in the presence of either an empty GBD-expressing plasmid (−) or the GBD-Est1 fusion (Est1). The data are presented as the mean ± SEM (N ≥ 3). (C) Wild-type and rrd1Δ cells were grown in galactose to induce HO expression, and samples were taken at the indicated time points. A Southern blot of EcoRV-cut genomic DNA was probed with a portion of the ADE2 gene. The band labeled PRE represents the EcoRV restriction-digested fragment from Chr VII–L. After HO cleavage, this fragment is converted into a new band (CUT). New telomere elongation appears as a smear above the CUT band. (D) Quantitation of telomere addition frequency of wild-type and mec1Δ derivatives of a GAL4UAS sml1Δ rad52Δ strain (black bars), and wild-type and rrd1Δ derivatives of a GAL4UAS rad52Δ strain (gray bars). All strains expressed the GBD-Est1 fusion. The experiments were carried out in nocodazole-arrested cells. The data are presented as the mean ± SEM (N ≥ 3). (E) Quantitation of telomere addition frequency of wild-type, yku80Δ, and rrd1Δ derivatives of a GAL4UAS rad52Δ strain expressing the GBD-Cdc13RD fusion. As a control, we also determined the telomere addition frequency of a GAL4UAS rad52Δ strain expressing the nonfunctional GBD-Cdc13RD-est fusion. The data are presented as the mean ± SEM (N ≥ 3). (F) Quantitation of telomere addition frequency of wild-type and mec1Δ derivatives of a GAL4UAS sml1Δ rad52Δ strain (black bars), and wild-type and rrd1Δ derivatives of a GAL4UAS rad52Δ strain (gray bars). All strains expressed the GBD-Cdc13RD fusion. The experiments were carried out in nocodazole-arrested cells. The data are presented as the mean ± SEM (N ≥ 3).