Figure S5 Rad53 phosphorylation and the sensitivity to bleomycin of type I survivors is mainly dependent on Mec1. Rad53 phosphorylation was assayed by Western blot analysis. Proteins were prepared from strains in Figure 8B (top two panels) using trichloroacetic acid precipitation as described in Materials and Methods. Samples were separated on 7% SDS-polyacryl- amide gels and transferred to nitrocellulose membranes. The membranes were incubated with a 1:1000 dilution of anti-Rad53 antibody (gift of J. Diffley), followed by incubation with the secondary antibody. Membranes were developed using ECL chemiluminescence (GE) and exposed to autoradiographic film. It should be noted that Rad53 was partially phosphorylated in type I survivors before bleomycin treatment (5 mU/ml, 3 hours) and that was suppressed by MEC1 deletion. Found at: doi:10.1371/journal.pone.0008224.s005 (2.79 MB TIF) Figure S6 The distribution of Rad proteins in cells with short telomeres. Rad51 (red triangles) and Rad52 (blue ovals) are sequestered at chromosome ends in cells with short telomeres. References 1. Zakian VA (1995) Telomeres: beginning to understand the end. Science 270: 1601–1607. 2. Chan CS, Tye BK (1983) Organization of DNA sequences and replication origins at yeast telomeres. Cell 33: 563–573. 3. Marcand S, Gilson E, Shore D (1997) A protein-counting mechanism for telomere length regulation in yeast. Science 275: 986–990. 4. Wotton D, Shore D (1997) A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in Saccharomyces cerevisiae. Genes Dev 11: 748–760. 5. van Steensel B, Smogorzewska A, de Lange T (1998) TRF2 protects human telomeres from end-to-end fusions. Cell 92: 401–413. 6. Hayflick L (1974) The longevity of cultured human cells. J Am Geriatr Soc 22: 1–12. 7. Olovnikov A (2005) Lunasensor, infradian rhythms, telomeres, and the chronomere program of aging. Ann N Y Acad Sci 1057: 112–132. 8. Nauk DA (1971) Principle of marginotomy in template synthesis of polynucle- otides. SSSR 201: 1496–1499. 9. Olovnikov A (1971) Principle of marginotomy in template synthesis of polynucleotides. SSSR 201: 1496–1499. 10. Lundblad V, Blackburn EH (1993) An alternative pathway for yeast telomere maintenance rescues est1- senescence. Cell 73: 347–360. 11. Singer MS, Gottschling DE (1994) TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266: 404–409. 12. Teng SC, Zakian VA (1999) Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol Cell Biol 19: 8083–8093. 13. Krogh BO, Symington LS (2004) Recombination proteins in yeast. Annu Rev Genet 38: 233–271. 14. Bertuch AA, Lundblad V (2004) EXO1 contributes to telomere maintenance in both telomerase-proficient and telomerase-deficient Saccharomyces cerevisiae. Genetics 166: 1651–1659. 15. Cohen H, Sinclair DA (2001) Recombination-mediated lengthening of terminal telomeric repeats requires the Sgs1 DNA helicase. Proc Natl Acad Sci U S A 98: 3174–3179. 16. Johnson FB, Marciniak RA, McVey M, Stewart SA, Hahn WC, et al. (2001) The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase. Embo J 20: 905–913. 17. Teng SC, Chang J, McCowan B, Zakian VA (2000) Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif- inhibited recombinational process. Mol Cell 6: 947–952. 18. Tsai HJ, Huang WH, Li TK, Tsai YL, Wu KJ, et al. (2006) Involvement of topoisomerase III in telomere-telomere recombination. J Biol Chem 281: 13717–13723. 19. Chen Q IA, Greider CW (2001) Two survivor pathways that allow growth in the absence of telomerase are generated by distinct telomere recombination events. Mol Cell Biol 21: 1819–1827. 20. Enomoto S, Glowczewski L, Berman J (2002) MEC3, MEC1, and DDC2 are essential components of a telomere checkpoint pathway required for cell cycle arrest during senescence in Saccharomyces cerevisiae. Mol Biol Cell 13: 2626–2638. 21. IJpma AS, Greider CW (2003) Short Telomeres Induce a DNA Damage Response in Saccharomyces cerevisiae. Mol Biol Cell 14: 987–1001. 22. Karlseder J, Broccoli D, Dai Y, Hardy S, de Lange T (1999) p53- and ATM- dependent apoptosis induced by telomeres lacking TRF2. Science 283: 1321–1325. Redistribution of Rad Proteins Found at: doi:10.1371/journal.pone.0008224.s006 (2.50 MB TIF) Table S1 Strains used in this study Found at: doi:10.1371/journal.pone.0008224.s007 (0.09 DOC) Table S2 Oligonucleotide primers Found at: doi:10.1371/journal.pone.0008224.s008 (0.04 DOC) Supporting Information References S1 Found at: doi:10.1371/journal.pone.0008224.s009 (0.03 DOC) MB MB MB Author Contributions Conceived and designed the experiments: YHL. Performed the experi- ments: YHL CCC CWW. Analyzed the data: YHL CCC SCT. Wrote the paper: YHL SCT. 23. Karlseder J, Smogorzewska A, de Lange T (2002) Senescence induced by altered telomere state, not telomere loss. Science 295: 2446–2449. 24. Chin L, Artandi SE, Shen Q, Tam A, Lee SL, et al. (1999) p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis. Cell 97: 527–538. 25. Hemann MT, Rudolph KL, Strong MA, DePinho RA, Chin L, et al. (2001) Telomere dysfunction triggers developmentally regulated germ cell apoptosis. Mol Biol Cell 12: 2023–2030. 26. Goytisolo FA, Samper E, Martin-Caballero J, Finnon P, Herrera E, et al. (2000) Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals. J Exp Med 192: 1625–1636. 27. Wong KK, Maser RS, Bachoo RM, Menon J, Carrasco DR, et al. (2003) Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing. Nature 421: 643–648. 28. Rose MD, Winston F, Hieter P (1990) Methods in Yeast Genetics. NY, Cold Spring Harbor: Cold Spring Harbor Laboratory. 29. Yu X, Gabriel A (1999) Patching broken chromosomes with extranuclear cellular DNA. Mol Cell 4: 873–881. 30. Tseng S-F, Lin J-J, Teng S-C (2006) The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation. Nucl Acids Res 34: 6327–6336. 31. Tsai YL, Tseng SF, Chang SH, Lin CC, Teng SC (2002) Involvement of replicative polymerases, Tel1p, Mec1p, Cdc13p, and the Ku complex in telomere-telomere recombination. Mol Cell Biol 22: 5679–5687. 32. Yamamoto A, Guacci V, Koshland D (1996) Pds1p is required for faithful execution of anaphase in the yeast, Saccharomyces cerevisiae. J Cell Biol 133: 85–97. 33. Taggart AK, Teng SC, Zakian VA (2002) Est1p as a cell cycle-regulated activator of telomere-bound telomerase. Science 297: 1023–1026. 34. Bahler J, Wu JQ, Longtine MS, Shah NG, McKenzie A 3rd, et al. (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14: 943–951. 35. Feng Q, During L, de Mayolo AA, Lettier G, Lisby M, et al. (2007) Rad52 and Rad59 exhibit both overlapping and distinct functions. DNA Repair (Amst) 6: 27–37. 36. Lin CY, Chang HH, Wu KJ, Tseng SF, Lin CC, et al. (2005) Extrachromosomal telomeric circles contribute to Rad52-, Rad50-, and polymerase delta-mediated telomere-telomere recombination in Saccharomyces cerevisiae. Eukaryot Cell 4: 327–336. 37. Fisher TS, Taggart AK, Zakian VA (2004) Cell cycle-dependent regulation of yeast telomerase by Ku. Nat Struct Mol Biol 11: 1198–1205. 38. Sabourin M, Tuzon CT, Zakian VA (2007) Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae. Mol Cell 27: 550–561. 39. Huang CF, Chen CC, Tung L, Buu LM, Lee FJ (2002) The yeast ADP- ribosylation factor GAP, Gcs1p, is involved in maintenance of mitochondrial morphology. J Cell Sci 115: 275–282. 40. Lundblad V, Szostak JW (1989) A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57: 633–643. 41. Haber JE (2000) Lucky breaks: analysis of recombination in Saccharomyces. Mutat Res 451: 53–69. 42. Teng SC, Epstein C, Tsai YL, Cheng HW, Chen HL, et al. (2002) Induction of global stress response in Saccharomyces cerevisiae cells lacking telomerase. Biochem Biophys Res Commun 291: 714–721. 43. Nautiyal S, DeRisi JL, Blackburn EH (2002) The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 99: 9316–9321. PLoS ONE | www.plosone.org 11 December 2009 | Volume 4 | Issue 12 | e8224