DNA damage. Studies in telomerase-minus mice have suggested that telomere length correlates with organismal sensitivity to radiation [26,27]. However it remains an open question of how the dysfunctional telomeres contribute to DSB sensitivity. Here we observed that yeast cells with short dysfunctional telomeres displayed MEC1-dependent hypersensitivity to DSBs and a robust redistribution of HR proteins from the endogenous DSBs to the telomeres, providing a molecular aspect of the relationship between aging and impaired DNA repairing capacity. Materials and Methods See the Supplemental Data for additional experimental details. Strains and plasmids All the yeast operations were performed by standard methods [28]. Yeast strains used in the study are listed in Table S1. All strains constructed in this study were isogenic to YPH501 (MATa/ MATa ura3-52/ura3-52 lys2-801 amber/lys2-801 amber ade2-101 ochre/ade2-101 ochre trp1D63/trp1D63 his3D200/his3D200 leu2-D1/ leu2-D1), BY4743 (MATa/a his3D1/his3D1 leu2D0/leu2D0 LYS2/ lys2D0 met15D0/MET15 ura3D0/ura3D0), AGY673 [29], or BY4743 (Invitrogen). YPH499 tlc1::LEU2 complemented with pRS316-TLC1 was described previously [12]. For spotting assays, the dnl4::KanMX4 and rad59::HIS3 mutants were constructed by transforming dnl4::KanMX4 and rad59::HIS3 PCR fragments, respectively, into the YPH499 strain, and were crossed with YPH500 tlc1::LEU2 to obtain heterozygous diploids. The rad50 mutant and mec1 sml1and tel1 mutants were described as previously [17,30,31] and were crossed with YPH500 tlc1::LEU2 to obtain heterozygous diploids. The G418 resistant rad9, rad24, chk1, pds1 and dun1 mutants (Invitrogen) were crossed with the BY4741 tlc1::HIS3 mutant (constructed by PCR-based replacement of the TLC1 sequence) to obtain heterozygous diploids. The pds1::LEU2 ts mutant was created in BY4742 by using pAY55 plasmid (a kind gift from Dr. D. Koshland) as previously described [32] and was crossed with tlc1::HIS3 mutant to obtain heterozygous diploids. The mrc1::HIS3 was constructed by PCR-based replacement of the MRC1 ORF in BY4742 and the resulting strain was crossed with BY4741 tlc1::LEU2 to obtain heterozygous diploids. All heterozy- gous diploids were sporulated for haploid single, double, or triple mutants. For HOcs ChIP analysis, tlc1 mutant was constructed by transforming AGY67 [29] with the tlc1:: TRP1 PCR fragment amplified from STY161 (YPH499 tlc1::TRP1) genomic DNA using TLC1-59 and TLC1-39 as primer pairs. For telomere ChIP analysis, YPH499 UT strain containing modified chromosome VII-L was described previously [33], and both wild type and tlc1::LEU2 spore colonies used in this study were derived from tetrad dissection of YPH501 UT tlc1::LEU2/TLC1 diploids. For immunofluorescence assay, the Myc-tagged RAP1 strain was constructed by double crossing over the chromosomal RAP1 of YPH499 with a 13Myc PCR fragment from pFA6a-13Myc- KanMX6 [34]. This strain was transformed with pWJ1213 [35], a plasmid carrying Rad52-YFP and the HIS3 marker (a kind gift from Dr. R. Rothstein) and its chromosomal RAD52 was deleted by PCR-based replacement using a RAD52::URA3 PCR fragment. The resulted strain was crossed with YPH500 tlc1::LEU2 to obtain heterozygous diploids and sporulated for YPH499 wild type and tlc1::LEU2 haploid cells containing KanMX6, HIS3 and URA3 markers. All type I survivors were confirmed by the telomere restriction fragment (TRF) assay. All pre-senescent tlc1 mutants used in this study were derived directly from spore colonies (about 25 Redistribution of Rad Proteins generations). Genotype was confirmed by Southern blot, PCR and/or sequencing analysis. Primer sequences for PCR, real-time PCR amplification and sequencing are listed in Table S2. DNA damage sensitivity assays For spotting assays, log-phase cultures were adjusted to an OD600 of 0.1 or 1 for type I survivors, and serial five-fold dilutions were plated on YPD followed by exposure to different tempera- tures, ionizing radiation (IR), UV light, or YPD containing various concentrations of glucose, bleomycin (BLM), methyl methanesul- fonate (MMS), H2O2, or hydroxyurea (HU). Yeast culture condition, sample preparation, enzyme digestion, gel electrophoresis, and Southern blot analysis Each spore colony was inoculated into YEPD and grown at 30uC. Several independent isolates for each genotype were analyzed. Spore cells were serially diluted into or restreaked onto YEPD medium. Liquid cultures were generated by inoculating spore colonies from the tetrad plate into 10 ml of liquid YEPD medium. Cultures were diluted repeatedly 1:10,000 into fresh medium at 48 or 72 hours. A solid-plate study was performed by repeatedly streaking spore colonies from the tetrad plates on YEPD plates. Genomic DNA preparation and Southern blot analysis were performed as previously described [31,36]. To examine the DNA from individual colonies, each colony was expanded in 2 ml of liquid medium to obtain DNA for Southern blot analysis. The DNA was digested with XhoI in order to observe the type I pattern or with a mixture of HaeIII, HinfI, HinpII, and MspI four-base cutters to observe the type II pattern. A 500-bp C1–3A fragment was randomly labeled with the Random Prime Labeling system (Invitrogen) and used in Southern hybridization. Data shown under ‘‘Results’’ are representative of two or more experiments from independent spore colonies. Protein extraction, trichloroacetic acid (TCA) precipitation and Western blot analysis Yeast cells were grown overnight at 30uC in 5 ml YEPD medium and refreshed from OD600 = 0.2 to OD600 = 0.5 in 10 ml of YEPD. Cells were washed with 1 ml of sterile water and resuspended in 1 ml of cold 0.25 M NaOH/1% 2-mercaptoeth- anol, followed by incubation on ice for 10 min. Samples were added with 160 ml of 50% TCA and incubated on ice for additional 10 min. Cells were pelleted at full spin for 10 min and then washed with 1 ml of acetone. After air dry, 100 ml of 2x SDS sample buffer was added to resuspend protein extracts. Western blot analysis was performed as described [18] using an anti-Rad51 antibody (Santa Cruz sc-33626), an anti-Rad52 antibody (Santa Cruz sc-50445), or an anti-Pgk1 antibody (Molecular Probes A- 6457) for detection. Chromatin immunoprecipitation (ChIP) Yeast cells were grown overnight in 5 ml of YEPD at 30uC, washed twice with ddH2O, and refreshed to an OD600 = 0.2 in 100 ml YEP medium with 2% raffinose until mid-log phase (OD600,0.5). One gram of galactose was then added into 50 ml of culture, while the other half was remained in YEP medium containing raffinose. After two hours of incubations at 30uC, cells were spin down and cross-linked with 1% formaldehyde and ChIP was conducted as described [37] using polyclonal anti-Rad51 and anti-Rad52 antibodies or normal rabbit IgG as a control. DNA was extracted and dissolved in 100 ml of TE. PCR primers specific to the ASP3 gene (which is on a different chromosome from the MAT) were included as an internal control. ChIP samples were PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8224