Figure 2. A CEN-based plasmid containing TLC1 complemented the sensitive phenotype of type I survivors. (A) Wild-type, pre- senescent tlc1 mutant, type I or type II strains carrying pRS316 or pRS316-TLC1 plasmid were five-fold serially diluted and spotted onto YEPD plates containing bleomycin in various concentrations. (B) Southern blot analysis of the XhoI-digested genomic DNA isolated from strains described in panel A probed to detect telomeric sequences. doi:10.1371/journal.pone.0008224.g002 specific to the ASP3 gene (which is on a different chromosome from MAT) were included as an internal control. Compared to wild type cells, type I survivors showed only 8% and 16% in bindings of Rad51 and Rad52, respectively, to the HO-induced DSB, while the pre-senescent tlc1 mutant displayed similar results as wild type cells. To confirm that the reduced bindings of Rad51 and Rad52 in type I survivors was not due to lower protein expression levels, expression levels of both proteins were compared. As shown in Figure 4C, type I survivors expressed a higher level of Rad51 than that of wild type, and approximately the same level of Rad52 as that of wild type. This is consistent with the finding that Rad51 expression is up-regulated during senescence in genome-wide studies of expression response to telomere shortening [42,43]. Therefore, it was not the lower protein expression that caused the decreased bindings of repair proteins to the HO cut site. To exclude the possibility that the reduced recruitments of HR proteins in type I survivors is caused by the different phases which WT and type I cells may be in, we conducted the ChIP experiment with both strains arrested in G2/M phase, and similar results were obtained (data not shown). From the data above, we propose that the sensitive phenotype to DNA-damaging agents in cells with short telomeres may be resulted from their impaired ability to recruit sufficient DNA repair proteins to damaged sites. Redistribution of Rad Proteins To determine whether the gathering of repair proteins at short telomeres perturbed their recruitment to DSBs, the amount of Rad51 and Rad52 bound at the left telomere of chromosome VII (VII-L) was examined by ChIP in strains containing the modified chromosomes (Figure 5A). Since telomeres were constantly subject to recombinant events upon loss of telomerase and thus were likely to relocate or lose the sequence designed for quantification, the maintenance of the URA3 marker in the telomere of type I survivors were confirmed by Southern blot before ChIP analysis (data not shown). ChIP samples were quantitated by real-time PCR. As shown in Figure 5B (left), there was a 3.2-fold enrichment of telomere sequences in the anit-Rad51 immunoprecipitate of type I cells, whereas both wild-type and pre-senescent tlc1 mutant strains showed a 1.9-fold enrichment. Similar results were found with Rad52 (Figure 5B, right). While the short VII-L telomeric DNA was enriched 4.3-fold in the anti-Rad52 immunoprecipitate of type I survivors, there was only 1.5 and 1.6-fold enrichment in wild-type and pre-senescent tlc1 mutant strains, respectively. As expected, the two-hour treatment with bleomycin (1.5 mU/ml) before ChIP samples were collected did not decrease the level of Rad52 bound to the telomere in type I survivors (data not shown). However, to exclude the possibility that the higher association of Rad proteins to the modified telomere in type I cells was attributed to some artifacts formed during the ChIP procedure, we further investigated whether these sequestered repair proteins can be released from telomeres to bona fide DSBs upon subjection to exogenous DNA damage. Towards this aim, before ChIP samples were collected, a portion of yeast culture was treated with high concentration of bleomcyin (7.5 mU/ml) for two hours. As a result, the bindings of both Rad proteins at VII-L reduced to the wild-type level in type I cells after treated with bleomycin (Figure 5B). In contrast, both wild-type and pre-senescent tlc1 mutant strains displayed indistinguishable bindings of Rad proteins at VII-L before and after bleomycin treatment. All together, these results demonstrate that sequestration of Rad51 and Rad52 at short telomeres reduces the efficiency of their recruitments to an induced DSB site and that the mere onset of telomerase deprivation in pre-senescent tlc1 mutant does not lead to the same level of biased distribution of Rad proteins as shown in type I survivors. To confirm that type I survivors sequester the key repair protein Rad52 to short telomeres at the cellular level, we investigated the redistribution of Rad52 in wild-type and type I cells by immunofluorescence using YFP-marked Rad52 and Myc-tagged Rap1. Since spontaneous Rad52 foci form mainly at chromosome breaks during normal DNA replication, to easily obtain signal, cells were arrested in G1 and then released into S-phase [44]. The percentage of cells presenting HR foci was determined micro- scopically. Consistent with the fact that short telomeres require the recombination protein Rad52 to be rescued, we found that type I survivors showed higher levels of Rad52 foci compared to WT cells (Figure 6A). To determine whether the higher level of spontaneous Rad52 foci formed in type I cells reflects HR proteins being sequestered at short telomeres, colocalization of Rad52 with the telomeric DNA-binding protein Rap1 (tagged with Myc) was investigated. While wild-type strains formed only 9.3% of the Rad52 foci colocalized with a Rap1 focus, type I cells displayed more than 20% colocalization prior to bleomycin treatment (Figure 6B, black bars) and this level of colocalization slightly decreased when subjected to DSBs (Figure 6B, gray bars). These results confirm our ChIP data (Figure 5) that HR proteins were ‘‘hyper-recruited’’ to the telomeres in Type I survivors and that high concentration of bleomycin released HR proteins from their associated telomeres, even though this redistribution may not be PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8224