quantitated by real-time PCR, calculated as previously described [38], and were normalized to the value of each non-induced counterpart [29]. Data are expressed as ‘‘relative IP’’ using wild type as 100% enrichment. To collect samples for telomeric ChIPs, yeast cells were grown overnight in 5 ml of synthetic complete medium that lacked uracil at 30uC and refreshed to an OD600 = 0.2 in 100 ml of YEPD medium until mid-log phase (OD600,0.5). Bleomycin was then added into 50 ml of culture to a final concentration of 7.5 mU/ml for 2 hr at 30uC, while the other half was remained in YEPD medium without treatment. Data for Rad proteins binding at telomeric regions were normalized to input DNA and expressed as the amount of VIIL (telomeric sequence on the left arm of chromosome VII) over that of DDI2 gene (internal control). Samples were amplified in triplicate to obtain an average value and ChIPs were repeated at least three times for each strain. Immunofluorescence All experiments were performed in 23uC to allow the YFP chromophore to form efficiently. Cells were fixed with 4% formaldehyde and processed for fluorescence microscopy as descried previously [39]. Rap1 foci were detected by using anti- Myc monoclonal antibody (9E10, Roche). To stain genomic DNA, 0.3 mg/ml DAPI was added to the mounting solution. All images were captured at 63-fold magnification using a Plan-Apochromat 663, 1.4 objective lens. Cells were mounted and analyzed under a confocal microscope (Leica TCS SP5, Wetzlar). The illumination source was a 100-W mercury arc lamp. Images were acquired and pseudo-colored using Leica LAS AF software. For each field of cells, 22 images were obtained at 0.2-mm intervals along the z axis to allow inspection of all focal planes of each cell. At least 300 cells were counted to determine the percentage of cells with Rad52-YFP foci. For the percentage of Rad52-YFP foci colocalized with Rap1-Myc13, we calculated the percentage of YFP coinciding with anti-Myc signals, rather than the converse. Three persons independently scored at least 200 cells for 50% or more overlap of yellow and red traced areas, each of which was defined as a colocalization. Results Type I survivors are hypersensitive to DNA DSBs To characterize the property of telomerase-minus cells, we first challenged wild-type, pre-senescent telomerase-minus cells, senes- cent type I or type II survivors with different stresses. Interestingly, type I survivors were exquisitely hypersensitive to strand break- inducing agents, including BLM, MMS, IR, and UV light, but not to different temperatures, altered glucose concentrations, oxidative free radical-forming agent H2O2 or replication stress-inducing drug HU (Figure 1 and data not shown). The reason why type I cells were more resistant to HU than type II survivors is unclear. In order to confirm that growth inhibition induced by bleomycin was not restricted to a single source of type I survivors, we further tested the sensitivity of 448 type I survivors derived from nine independent tlc1 spore colonies to bleomycin. After three days of incubation at 30uC, survival rates of these type I survivors on YEPD plates containing bleomycin (2 mU/ml) were scored (data not shown). 55% (248 of 448) of tested type I survivors were able to grow on plates containing bleomycin, while the rest 45% (200 of 448) were not. To determine if the Y’-short and Y’-long elements that form the major part of type I telomeres contribute to the generation of two distinct populations with different bleomycin sensitivity, genomic DNA of 24 sensitive and 24 resistant type I survivors was digested with XhoI and subjected to Southern blot Redistribution of Rad Proteins analysis with a Y’ probe. However, no apparent differences in Y’ patterns between the two populations were observed (Figure S1), suggesting that Y’-long or Y’-short preference did not affect the bleomycin sensitivity of type I survivors. All type I survivors are eventually sensitive to bleomycin following sufficient passages The results above drove us to quest whether the bleomycin sensitivity of type I cells was due to epigenetic events which rendered two interchangeable phenotypes, or constant events caused by irreversible genetic alterations. To distinguish these, we passaged both sensitive and resistant populations on solid plates for up to 12 streaks (240,300 generations) and tested their sensitivity to bleomycin. At the 12th streak, virtually all survivors (51/52) ceased to proliferate on plates containing bleomycin (Figure S2). This result implies that most of the type I survivors given sufficient outgrowth are eventually sensitive to bleomycin. Viewed in this finding, the sensitivity of type I can be regarded as a constant phenotype rather than a random event. It was notable that Southern blot analysis of Y’ elements also showed no apparent differences in the distribution of the two types of Y’ between the only resistant survivor and other sensitive ones at the 12th streak (data not shown). Sensitive phenotype of type I survivors is attributed to the inactivation of telomerase It has been reported previously that progressive telomere shortening leads to chromosome instability [40]. To test this possibility that critical genetic aberrations might arise from telomere attrition and thus contribute to the bleomycin-sensitive phenotype, the RNA component of telomerase from a CEN-based plasmid containing TLC1 (pRS316-TLC1) was delivered into type I cells as well as control strains. As shown in Figure 2A, type I survivors became resistant to bleomycin after complemented with a plasmid-borne TLC1, suggesting that the sensitive phenotype of type I survivors was attributed to the inactivation of telomerase. A Southern blot analysis on the telomere pattern was also performed to monitor the change of telomere length in these strains (Figure 2B). Further measures on the rate of forward mutation to canavanine resistance (Canr) also showed no striking difference in the spontaneous mutation rate in type I survivors compared to control cells (Figure S3). These results indicate that rather than permanent genetic mutations, the loss of telomerase is more likely the major cause of sensitive response of type I survivors to DNA- damaging agents. Cells undergone progressive telomere attritions are susceptible to DSB attack In order to paint a detailed picture regarding exactly when the tlc1 mutant becomes DSB-sensitive and what causes this distinct response between type I and type II survivors to DSBs, we tracked the change in the response to bleomycin from a tlc1 spore colony till a survivor. Type I and type II survivors were obtained by repeatedly restreaking the tlc1 spore colony on solid plates and continuous dilutions in liquid culture, respectively. Yeast colonies from each restreak (corresponds to 20–25 generations) or each dilution (corresponds to 13 generations) were analyzed by spot assays on YEPD plates with or without bleomycin (Figure 3A), and the timing of survivor formation was monitored by Southern blot analysis (Figure 3B). When the tlc1 mutant was maintained on solid plates, cells displayed sensitivity to bleomycin from the first streak, and they showed much lower viability on bleomycin plates for the following PLoS ONE | www.plosone.org 3 December 2009 | Volume 4 | Issue 12 | e8224