Redistribution of Rad Proteins Figure 1. Type I survivors are hypersensitive to DNA-damaging agents. To measure the character of telomerase-minus cells, five-fold serial dilutions of isogenic wild type (WT), pre-senescent tlc1 mutant, type I or type II strains were spotted onto YPD plates containing indicated drugs with denoted concentrations. The plates were incubated at 30uC for three days before photograph. doi:10.1371/journal.pone.0008224.g001 restreaks (Figure 3A, top), even after Y’ amplification was generated at the 3rd restreak, as it was evident by Southern blot analysis (Figure 3B, top). Unlike type I survivors, although the tlc1 mutant also displayed sensitivity in the first two dilutions in liquid culture, increased resistance to bleomycin was observed immedi- ately after the development of type II survivors (Figure 3A, lower), in which telomere lengthening occurred rapidly by recombination of TG1–3 tracts (Figure 3B, lower). Because the major difference between two subtypes of survivors is the application of distinct recombination pathways to reconstruct their chromosomal ends, we speculated that it is the shorter TG1–3 tracts in early senescent tlc1 mutants and type I survivors that conferred their sensitivity to DSBs. Recruitments of Rad51 and Rad52 are reduced at an internal HO cut site but increased at telomeres in type I survivors Previous works have shown that many DNA repair proteins are often associated with telomeres. We speculated that the titration of DNA repair proteins to shortened chromosomal ends might decrease repair capacity at bona fide DSB sites. Since DSBs as the major cytotoxic lesions caused by bleomycin are preferably repaired by HR [41], we subsequently examined whether Rad51 and Rad52 were less efficiently recruited to an induced DSB in type I survivors. We investigated the recruitment of Rad51 and Rad52 to a single DSB by ChIP. The experimental system is shown in Figure 4A: a functional URA3 allele containing a copy of the actin intron as well as the Y–Z junction from the MATa locus, including the recognition sequence and cleavage site for the HO endonuclease, was transformed into a yeast strain that has been deleted of all MAT related sequences and contains an integrated galactose-inducible HO endonuclease gene. Upon growth in galactose media, the HO endonuclease created a unique DSB in the middle of the URA3 gene. Wild type, tlc1 and type I strains were grown in YEP media with raffinose as the sole carbon source until mid-log phase. Galactose was then added to induce expression of the HO endonuclease. At various times after galactose addition, aliquots were removed and genomic DNA was isolated to analyze for the formation of a DSB at the MAT locus using a PCR assay. The induction kinetics showed that all strains remained only approximately 20,27% of the original signal after two-hour induction (Figure S4). Figure 4B shows the quantitative results of ChIPs using a polyclonal antibody against Rad51 (left) or Rad52 (right). Immunoprecipitated DNA was analyzed by real-time PCR to detect MAT locus DNA from left side of the induced DSB (designated as ‘‘HOL’’). PCR primers PLoS ONE | www.plosone.org 4 December 2009 | Volume 4 | Issue 12 | e8224