Redistribution of Rad Proteins Figure 5. Sequestration of repair proteins at telomeres in type I survivors. (A) The left arm of chromosome VII-L in YPH499 UT strain was modified by integration of fragments containing the URA3 and telomeric sequence at the ADH4 locus. Telomeres are represented by hatched ovals; arrows indicate primers used for real-time PCR. (B) ChIPs were performed and quantitated by real-time PCR as described in Materials and Methods. Error bars represent standard deviations for at least three independent experiments. Before the addition of bleomycin (black bars), bindings of Rad51 and Rad52 to the VII-L telomere in type I cells differed from those in wild-type in a statistically significant manner (P values = 5.3661028 and 0.010, respectively), and their bindings to the VII-L telomere was statistically indistinguishable in pre-senescent tlc1 mutant and wild-type strains. In type I cells, bindings of Rad51 and Rad52 to the VII-L telomere before bleomycin treatment (black bars) differed from those after treatment (gray bars) in a statistically significant manner (P values = 0.0004 and 0.014, respectively). This discrepancy was not observed in both pre-senescent tlc1 mutant and wild-type strains. doi:10.1371/journal.pone.0008224.g005 sufficient to repair DNA damage caused by excessive dose of bleomycin. The sensitivity of type I cells to bleomycin behaves in a checkpoint-dependent manner Previous studies have indicated that the short telomeres cause cell cycle arrest [20,21,45,46,47], suggesting that compromised telomeres are recognized as damaged DNAs and are capable of triggering the DNA repair machinery during an arrest. Several proteins in the HR pathways and factors involved in the DNA damage machinery have been reported to associate with the ALT pathway or to participate in the process when compromised telomeres are detected as DSBs [31,46,48]. Therefore, we are interested in whether these genes impinge on the bleomycin sensitivity of type I survivors. We first asked whether factors controlling the fate of repair in telomerase-minus cells contribute to this sensitivity. Both RAD50 and RAD59, which belong to the RAD52 epistasis group, are required for telomere maintenance in telomerase-minus cells and are not essential for type I pathway [17,19]. Elimination of RAD50 or RAD59 in type I cells was subjected to spotting assays as described in Figure 1. As shown in Figure 7, deletion of either RAD50 or RAD59 slightly increased the sensitivity of type I cells to bleomycin, whereas abrogation of a major nonhomologous end joining (NHEJ) component DNL4 resulted in no further pheno- typic alterations. These data further underline the role of HR proteins in the sensitivity to DNA damage of type I cells. Given that we have additionally observed a chronic cell cycle delay and persistent Rad53 phosphorylation in type I survivors (Figure S5), spotting assays were performed to investigate if checkpoint activation (Figure 8A) correlates with type I sensitivity to bleomycin. Strikingly, ablation of MEC1 greatly enhanced type I survival rate in response to bleomycin, whereas deletion of TEL1 only mildly promoted its survival (Figure 8B). To ensure that these results were accompanied with modified checkpoint competence, the phosphorylated level of Rad53 in various strains was determined by Western blot analysis. Indeed, the absence of MEC1 completely abolished persistent checkpoint activation in type I survivors (Figure S5). Moreover, MEC1 deletion largely hampered Rad53 phosphorylation induced by bleomycin treat- ment in type I survivors (Figure S5). Since similar results were not observed in tel1 deletion mutants, the persistent checkpoint activation, the associated cell cycle arrest and the bleomycin sensitivity might be mainly dependent on MEC1. These observa- tions prompted us to further study if the ablation of MEC1 downstream genes (Figure 8A) surmounts the sensitivity phenotype of type I cells. As shown in Figure 8B, deletion of RAD9, RAD24, MRC1, CHK1, DUN1 or PDS1 (partially deleted as a temperature sensitive mutant, see Materials and Methods) promoted the proliferation of type I cells on plates containing bleomycin. Notably, we were unable to determine whether RAD53-deleted cells display the same effect since the spore colonies were too sick to survive on YEPD plates during the second restreak and no type I survivors could be assayed. Altogether, these results indicate that a Mec1-related checkpoint signal pathway regulates the hyper- sensitivity to DSBs in cells with short telomeres. Discussion The impact of telomere shortening on senescent budding yeast upon loss of telomerase function is rather difficult to estimate PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8224