DNA Degradation at Unprotected Telomeres in Yeast Is Regulated by the CDK1 (Cdc28/Clb) Cell-Cycle Kinase  Momchil D. Vodenicharov, Raymund J. Wellinger  Molecular Cell  Volume 24, Issue 1, Pages 127-137 (October 2006) DOI: 10.1016/j.molcel.2006.07.035 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Cell-Cycle-Regulated Generation of ssDNA in cdc13-1 Mutant Cells MVY10 (CDC13), MVY17 (cdc13-1), and MVY15 (cdc13-1 rad9) cells were arrested in either G1 or G2/M at 23°C. Synchronized cultures were split into four equal parts; two were released from arrest at 23°C or 37°C, respectively, while the other two were kept arrested and incubation was continued at either 23°C or 37°C. All cultures were incubated for 4 hr, and cell samples were processed for FACS analysis, genomic DNA extraction, or cell viability. (A) FACS analysis of relevant cultures following G1 arrest (left) or G2/M arrest (right). (B) At the end of 4 hr incubation, 10-fold serial dilutions of cells from each experimental condition were plated on YPD and regrown at the permissive temperature. Left, regrowth capacity after G1 arrest. Right, regrowth capacity after G2/M arrest. (C) Following G1 arrest (left) or G2/M arrest (right), genomic DNA derived from indicated strains and experimental conditions was analyzed for telomere degradation by nondenaturing in-gel hybridization. Native gels (top panels) were hybridized to a telomeric CA probe to detect exposed G-rich single-stranded DNA. Same gels were denatured and transferred onto nylon filter, and DNA loading was verified with a probe to chromosomal CEN4 sequences (bottom panels). M, size marker. ds+ssGT, controls for single-stranded G-rich and double-stranded telomeric repeats. Molecular Cell 2006 24, 127-137DOI: (10.1016/j.molcel.2006.07.035) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Forced Destruction of Telomere Cap Components in G1 Does Not Lead to Telomere Degradation (A) Rapid degradation of telomere capping factors fused to degron. At indicated time points after induction of degrons by addition of galactose and a shift to 37°C, whole-cell protein extracts from MVY81 (cdc13-td) and MVY86 (stn1-td) cells were analyzed by western blotting using antibodies against the DHFR moiety of the degron (upper panel), or HA antibodies recognizing the HA-tagged Ubr1p (middle panel). The Ponceau red-stained membrane after protein transfer (bottom panel) serves as a loading control. ∗, nonspecific protein crossreacting with anti-DHFR antibody. (B) Viability of MVY81 and MVY86 strains was tested at 37°C by plating 10-fold serial dilutions on YPD (degron is switched OFF) or YEP-galactose (YPG) (degron is switched ON) and compared to Y40038 (cdc105-td) strain, which served as a control for degron induction and depletion of an essential protein. As control, all strains were also grown on YPR plates at 23°C. (C) MVY81 and MVY86 were arrested in G1 in YPR at 23°C. One-half of the culture was then released from the block, and both synchronized and released cultures were split again in two; galactose was added to one culture for degron induction, while glucose was added to the other culture. All four cultures were shifted to 37°C for 6 hr, followed by isolation of total genomic DNA. Left, DNA was analyzed on nondenaturing gel for presence of telomeric damage as in Figure 1C. Right, the same gel was denatured and reprobed with a Y′-specific probe. (D) MVY81 and MVY86 strains were arrested in G2/M in YPR at 23°C. Synchronized cultures were split, glucose or galactose added, and incubation continued for 4 hr at 37°C. As a control, part of each G2/M-arrested culture was released into YEP-galactose at 37°C for the same period of time. Genomic DNA was extracted and analyzed for G-rich single-stranded telomeric DNA as in Figure 1C. (E) G1-arrested cells in YPR at 23°C were shifted to 37°C for 4 hr in either YPG (top panel) or YPD (bottom panel). The ability of cells to regrow after the treatments was tested by plating 10-fold dilutions on YPD at 23°C. (F) G2/M-arrested cells were treated as in (E). Molecular Cell 2006 24, 127-137DOI: (10.1016/j.molcel.2006.07.035) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Unprotected Telomeres Are Stable in Early S Phase (A and B) Cell-cycle trap experiments with MVY10 (CDC13), MVY15 (cdc13-1 rad9), and MVY17 (cdc13-1) strains to assess the timing of ssDNA formation after uncapping. G1-arrested cells were exposed to 37°C for 2 hr. They were then released from the G1 block into HU block for 2 hr at 37°C. Finally, the same cultures were released into nocodazole-containing media at 37°C. Cell-cycle position of the culture was monitored by FACS analysis (A), and the amount of ssDNA at telomeres in the particular cell-cycle stages was evaluated on native gel (B) as described in Figure 1C. (C and D) The kinetics of ssDNA formation at unprotected telomeres was followed by releasing the indicated strains from HU block into nocodazole-containing media at 37°C. Samples for FACS (C) and nondenaturing gel (D) analyses were taken at indicated time points after the release. Molecular Cell 2006 24, 127-137DOI: (10.1016/j.molcel.2006.07.035) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Inhibition of S-Cdk1 Suppresses ssDNA Formation at Unprotected Telomeres (A) Yeast strains MVY120 (CDC13 GAL-SIC1; WT), MVY121 (cdc13-1 GAL-SIC1), MVY122 (CDC13 GAL-SIC1-Δ3P), MVY123 (cdc13-1 GAL-SIC1-Δ3P), MVY124 (cdc13-1 rad9), and MVY125 (cdc13-1 rad9 GAL-SIC1-Δ3P) were arrested in G2/M in raffinose media with nocodazole at 23°C. Galactose was added to G2/M-arrested cultures, and GAL-SIC1 or GAL-SIC1-Δ3P expression was induced for 45 min at 23°C. All cultures were then shifted to 37°C for 2 hr before DNA extraction and analysis on native gel (top panel) as in Figure 1C. Two independent clones of each strain were tested. A portion of ethidium bromide-stained gel (bottom panel) shows approximately equal DNA loading. (B) MVY121 (cdc13-1 GAL-SIC1) cells, pregrown in raffinose media at 23°C, were arrested in G2/M and shifted to 37°C to induce telomere uncapping. Thirty minutes later, galactose was added to the culture to allow expression of GAL-SIC1 and samples for DNA isolation were withdrawn from the culture at indicated time intervals for native-gel analysis as in Figure 1C. (C) Same experiment as in (B) was carried out with the MVY123 (cdc13-1 GAL-SIC1-Δ3P) strain. Molecular Cell 2006 24, 127-137DOI: (10.1016/j.molcel.2006.07.035) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 S-Cdk1 Regulates the Formation of Cell-Cycle-Dependent G Tails at Native Telomeres (A and B) MVY120 (CDC13 GAL-SIC1; WT) and MVY122 (CDC13 GAL-SIC1-Δ3P) strains grown in YPR at 30°C were arrested in early S phase by HU. Galactose was added to HU-arrested cultures to induce GAL-SIC1 or GAL-SIC1-Δ3P expression for 30 min. The cells were then released from the block, and samples were taken at indicated time points for nondenaturing gel analysis (A) and FACS (B). Arrow points to the transient G tails observed in MVY120 strain. Molecular Cell 2006 24, 127-137DOI: (10.1016/j.molcel.2006.07.035) Copyright © 2006 Elsevier Inc. Terms and Conditions