Redistribution of Rad Proteins Figure 8. Type I sensitivity to bleomycin is in a largely checkpoint-dependent manner. (A) Signaling of MEC1/TEL1-regulated checkpoint pathway [56]. (B) Exponentially growing cells were five-fold serially diluted and spotted onto YPD plates containing bleomycin with indicated concentrations. Except for the plates shown in the last panel, which were incubated at 25uC, all of the strains were incubated at 30uC for three days. TI represents type I survivors derived from each mutant spore, and all the others are pre-senescent spore colonies. doi:10.1371/journal.pone.0008224.g008 because these cells display decreasing vitality and eventually die within a narrow timeframe. In this study, we observed that type I survivors are exclusively hypersensitive to DSBs (Figure 1). We have proved that this phenotype was not caused by some permanent genetic distortions since it could be complemented by a plasmid-borne TLC1 and elevated mutation rates were not detected in type I cells (Figure 2 and S3). We further discovered that the sensitive response to DSBs is not only restricted to type I survivors, but also applies to tlc1 senescent cells before any survivor formation (Figure 3A). Once TG1–3 tracts are lengthened through the recombination pathway, such as in type II survivors, cells can be recovered from the DSB-sensitive phenotype (Figure 3A). Southern blot analysis (Figure 3B) demonstrated that the overall TG1–3 tracts of senescent tlc1 mutants and type I survivors are relatively shorter than those of wild type or type II survivor strains. This indicates a correlation between short telomeres and ineffective DSB repair. Our finding of the reduced binding of HR proteins to a single cut site along with the observation of increased association of HR proteins with telomeres in type I survivors (Figures 4, 5, and 6) provides a potential mechanistic basis for the impaired DSB response in organisms with dysfunctional telomeres. Similar observations have been proposed in two recent studies [49,50], both of which reported a robust Rad52 localization with the telomere in the est2 mutant. Meyer and Bailis [49] have also demonstrated a correlation between this sequestration of Rad52 to telomeres in the est2 mutant and reduced localization to an induced DSB formation. One apparent difference with our results is that the pre-senescent est2 mutant strain in the Meyer et al. experiments showed increased binding of Rad52 to the telomere, PLoS ONE | www.plosone.org 9 December 2009 | Volume 4 | Issue 12 | e8224