whereas we did not observe this in our pre-senescent tlc1mutant. The discrepancy can be explained by the divergent strain backgrounds that generate different rates of senescence. Addition- ally, our ChIP results are consistent with the observation that pre- senescent tlc1 mutant is not as sensitive as senescent cells or type I survivors to bleomycin (Figure 3A). The idea of competition between DNA damage sites has also been reported elsewhere: the inhibitory effect of NHEJ on the initiation of DSB resection in G1 phase is able to be suppressed by four or more DSBs, indicating that some components of NHEJ may become limiting in the presence of multiple breaks [51]. Because shortened telomeres mimic DSBs, we speculate that DSB repair proteins, either for HR or NHEJ, might become limiting as well in the presence of shortened telomeres that resemble DSB sites. One surprising finding was that the MEC1 removal leads to a dramatic increase in the bleomycin resistance in type I survivors (Figures 8B), indicating that type I sensitivity to bleomycin is dependent on MEC1. Type I survivors persistently displayed phosphorylated Rad53 even in the absence of bleomycin and the bleomycin-induced Rad53 mobility shift was almost abolished in conjunction with the abrogation of MEC1 gene (Figure S5), suggesting that type I sensitivity to DSBs might be attributed to the persistent Mec1-related checkpoint activation. These findings were quite striking, because checkpoint activation is normally expected to coincide with improved damage resistance. However, here we observed that diminished checkpoint activation by deleting MEC1 dramatically promoted bleomycin tolerance of type I survivors. Nonetheless, similar results have been reported previously [52], as the abolished Rad53 phosphorylation in mec1 mutants is suppressed by deletion of MDT1. This improved checkpoint activation paradoxically worsened bleomycin tolerance compared to mec1 single mutants. Moreover, Alabert et al. have reported similar conflicting observations that Mrc1-dependent checkpoint activation prevents homolgous recombination at DNA DSBs [44]. They proposed that ssDNA exposed at stalled forks is the key signal to recruit HR machinery and suppress potentially dangerous HR at DSBs, since ssDNA is already present at the stalled fork and DSB repair depends on the formation of the 39 ssDNA overhangs. These findings prompt us to hypothesize that short telomeres distinguish themselves from DSBs through exposed 39 overhangs to trigger Mec1-dependent checkpoint activation and thus prevent HR at DSBs that still need resection. In fact, the idea that Mec1 is required to recognize signals emanating from short telomeres was supported by a recent ChIP result showing that Mec1 interacts physically with critically short telomeres, thereby sensing and transducing the signal for G2/M cell-cycle arrest [53]. In this study, even though type I survivor formation seems to be one major alternative pathway to repair the damage caused by dysfunctional telomeres, these survivors, nevertheless, resemble the senescent cells that suffer from a prolonged arrest. This prolonged arrest indicates an attempt to repair ‘‘chromosomal end damage’’ in type I cells. Combined with the ChIP and IF data, it is tempting to speculate that in the absence of Mec1, short chromosomal ends are not properly recognized as DNA damage sites, thereby becoming less competitive for DNA repair proteins that are normally recruited to bona-fide DSBs (Figure S6). Further investigations are needed to clarify this possibility. Altogether, this study suggests that the translocation of Rad51, Rad52, and likely other repair factors to telomeres contributes to the Mec1- dependent hypersensitivity to DSBs in telomerase deficient cells. Studies of the role of telomerase in the maintenance of genome integrity in mammalian cells have provided insight into the molecular aspects in aging and cancer, and in particular, epithelial Redistribution of Rad Proteins carcinogenesis [54]. A growing number of evidence also supports that various pathways of DNA repair become less efficient in aged cells [55]. The work presented here suggests another mechanism by which aged organisms may suffer from increased rates of unrepaired DNA damage and its concomitant risk of cancer. Careful considerations of the ways in which effective therapeutics are designed may help for the treatment and prevention in the aged. Acknowledgments We thank Dr. Tsai-Kun Li for his discussion and critical comments. We also thank Drs. A. Gabriel, V. Zakian, D. Koshland, J. Diffley, A. Sugino, and R. Rothstein for their plasmids, strains and antibodies. Finally, we would like to thank our anonymous reviewers that helped clarify several vital points in the manuscript. Supporting Information Figure S1 Patterns of Y’ telomeres are indistinguishable between sensitive and resistant type I survivors. Equal amounts of the XhoI- digested genomic DNA isolated from wild-type and type I survivors of tlc1 mutants (6th streak) were loaded for Southern blot analysis, using a Y’ fragment as a probe [1]. BLMR indicates strains that are resistant to bleomycin at 2 mU/ml and BLMS indicates sensitive strains. Arrows denote the positions of Y’-long and Y’-short elements. Marker sizes are indicated at the left (Kb). Found at: doi:10.1371/journal.pone.0008224.s001 (4.25 MB TIF) Figure S2 Survival rates of early type I survivors on bleomycin plates. Survival rates were measured as the number of viable patches on YEPD containing 2 mU/ml of bleomycin over that on YEPD without bleomycin. Type I survivors from nine independent spore colonies were examined. A total of 447 survivors were scored for the 6th streak, 52 for the 9th and 52 for the 12th streaks, respectively. Found at: doi:10.1371/journal.pone.0008224.s002 (1.06 MB TIF) Figure S3 Measurement of mutation rates. (A) Assay for mutation rate measurement. Strains harbor a can1 mutation are resistant to canavanine. (B) To measure the rate of forward mutation to canavanine resistance (Canr), at least six yeast cultures were started from single colonies and grown to stationary phase in 10 ml liquid YEPD medium. Cells were plated with appropriate dilutions onto complete medium containing L-canavanine (60 mg/ml) and lacking arginine for Canr mutant count, and onto complete medium lacking arginine for viable count. The pol2- 4 (STY1609) mutant, previously reported to express a mutator phenotype [2], was used as a positive control. YIpBI (kindly provided by Dr. A. Sugino) was used to create the pol2-4 mutant in YPH499 background by the method previously described [3]. Mutation rates were determined by the method of the median [4]. Found at: doi:10.1371/journal.pone.0008224.s003 (0.98 MB TIF) Figure S4 Kinetics of DSB induction at the HO cut site. Galactose (2%, w/v) was added to cells in mid-log phase in order to induce HO endonuclease expression. Genomic DNA was purified at various time points, and PCR was performed using primers RAG513 and RAG 515 that flank the HO cut site (HOcs) from 114 bp CEN distal of the HOcs to 946 bp CEN proximal. The DSB was detected as a loss of PCR product. Primers specific to the ASP3 gene were included in the PCR as a control. DSB bands were quantitated by the ImageQuant software, normalized to the ASP3 bands, and indicated as the percentage of starting signal (‘‘% remaining product’’) at the bottom of the panel. Found at: doi:10.1371/journal.pone.0008224.s004 (2.53 MB TIF) PLoS ONE | www.plosone.org 10 December 2009 | Volume 4 | Issue 12 | e8224