TI - Discussion . AB - The impact of telomere shortening on senescent budding yeast upon loss of telomerase function is rather difficult to estimate 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 , 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 . Additionally , 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 3' ssDNA overhangs . These findings prompt us to hypothesize that short telomeres distinguish themselves from DSBs through exposed 3' 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 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 .