(Validated hits), the validated hits as well as essential proteins that are connected to two or more hits (Validated+essential connec- tors), the total number of validated MMS survival proteins identified by both screen and pathway validation (Validated+path- way hits), the Validated and pathway hits and the essential proteins that are connected to two or more hits (Validated+path- way hits+essential connectors), the total number of validated MMS survival proteins and all other components of the 13 MMS survival pathways (Validated+all in pathways), and finally the total number of validated MMS survival proteins, all other components of the 13 MMS survival pathways and the essential proteins that are connected to two or more hits or pathway proteins (Validated+all in pathways+essential connectors). Analyses were also provided to compare connectivity of real and randomly rewired PPI. Found at: doi:10.1371/journal.pgen.1000527.s017 (0.19 MB DOC) Methods for 8-oxo-guanine assay and notch reporter at: doi:10.1371/journal.pgen.1000527.s018 (0.03 MB Text S1 analysis. Found DOC) References 1. Altieri F, Grillo C, Maceroni M, Chichiarelli S (2008) DNA damage and repair: from molecular mechanisms to health implications. Antioxid Redox Signal 10: 891–937. 2. Shimada M, Nakanishi M (2006) DNA damage checkpoints and cancer. J Mol Histol 37: 253–260. 3. Begley TJ, Rosenbach AS, Ideker T, Samson LD (2004) Hot spots for modulating toxicity identified by genomic phenotyping and localization mapping. Mol Cell 16: 117–125. 4. Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER 3rd, Hurov KE, et al. (2007) ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316: 1160–1166. 5. Workman CT, Mak HC, McCuine S, Tagne JB, Agarwal M, et al. (2006) A systems approach to mapping DNA damage response pathways. Science 312: 1054–1059. 6. Kelley R, Ideker T (2005) Systematic interpretation of genetic interactions using protein networks. Nat Biotechnol 23: 561–566. 7. Ng A, Bursteinas B, Gao Q, Mollison E, Zvelebil M (2006) Resources for integrative systems biology: from data through databases to networks and dynamic system models. Brief Bioinform 7: 318–330. 8. Ramadan N, Flockhart I, Booker M, Perrimon N, Mathey-Prevot B (2007) Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. Nat Protoc 2: 2245–2264. 9. Drablos F, Feyzi E, Aas PA, Vaagbo CB, Kavli B, et al. (2004) Alkylation damage in DNA and RNA–repair mechanisms and medical significance. DNA Repair (Amst) 3: 1389–1407. 10. Rydberg B, Lindahl T (1982) Nonenzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-L-methionine is a potentially mutagenic reaction. Embo J 1: 211–216. 11. Hecht SS (1999) DNA adduct formation from tobacco-specific N-nitrosamines. Mutat Res 424: 127–142. 12. Liu L, Taverna P, Whitacre CM, Chatterjee S, Gerson SL (1999) Pharmacologic disruption of base excision repair sensitizes mismatch repair-deficient and - proficient colon cancer cells to methylating agents. Clin Cancer Res 5: 2908–2917. 13. Malet-Martino M, Gilard V, Martino R (1999) The analysis of cyclophospha- mide and its metabolites. Curr Pharm Des 5: 561–586. 14. Begley TJ, Rosenbach AS, Ideker T, Samson LD (2002) Damage recovery pathways in Saccharomyces cerevisiae revealed by genomic phenotyping and interactome mapping. Mol Cancer Res 1: 103–112. 15. Jelinsky SA, Samson LD (1999) Global response of Saccharomyces cerevisiae to an alkylating agent. Proc Natl Acad Sci U S A 96: 1486–1491. 16. Lee MW, Kim BJ, Choi HK, Ryu MJ, Kim SB, et al. (2007) Global protein expression profiling of budding yeast in response to DNA damage. Yeast 24: 145–154. 17. Murray JI, Whitfield ML, Trinklein ND, Myers RM, Brown PO, et al. (2004) Diverse and specific gene expression responses to stresses in cultured human cells. Mol Biol Cell 15: 2361–2374. 18. Wiles AM, Ravi D, Bhavani S, Bishop AJ (2008) An analysis of normalization methods for Drosophila RNAi genomic screens and development of a robust validation scheme. J Biomol Screen 13: 777–784. 19. Burgis NE, Samson LD (2007) The protein degradation response of Saccharomyces cerevisiae to classical DNA-damaging agents. Chem Res Toxicol 20: 1843–1853. Damage Survival Pathways Acknowledgments We thank Thomas Begley, University of Albany for sharing yeast MMS screen data. From UTHSCSA Center for Epidemiology and Biostatistics, we thank Gary Chisholm who performed initial statistical analyses and both John Cornell and Jonathon Gelfond for discussions and guidance. We also thank Fen Zhou and Montserrat Michelman of the Leder laboratory for cell line maintenance. We thank Kent Nybakken, Ram Dasgupta, and Herve´ Agaisse of Norbert Perrimon’s laboratory for cells, plasmids, and technical discussions and Lisa Ching for helping in the production of dsRNA. David Rodrigez of the GCCRI, UTHSCSA, provided support for the production of line art. Thank you to the members of the Bishop and Leder laboratories, as well as Dr. Don McEwen and particularly Dr. Vivienne Rebel for constructive criticism of the manuscript. Author Contributions Conceived and designed the experiments: DR PL AJRB. Performed the experiments: DR SB AJRB. Analyzed the data: DR AMW SB JR AJRB. Contributed reagents/materials/analysis tools: PL AJRB. Wrote the paper: DR AMW JR AJRB. Reviewed the manuscript: PL. 20. Shen C, Lancaster CS, Shi B, Guo H, Thimmaiah P, et al. (2007) TOR signaling is a determinant of cell survival in response to DNA damage. Mol Cell Biol 27: 7007–7017. 21. Rusyn I, Fry RC, Begley TJ, Klapacz J, Svensson JP, et al. (2007) Transcriptional networks in S. cerevisiae linked to an accumulation of base excision repair intermediates. PLoS ONE 2: e1252. doi:10.1371/journal.- pone.0001252. 22. Dickinson DA, Levonen AL, Moellering DR, Arnold EK, Zhang H, et al. (2004) Human glutamate cysteine ligase gene regulation through the electrophile response element. Free Radic Biol Med 37: 1152–1159. 23. Dann SG, Thomas G (2006) The amino acid sensitive TOR pathway from yeast to mammals. FEBS Lett 580: 2821–2829. 24. Ou YH, Chung PH, Sun TP, Shieh SY (2005) p53 C-terminal phosphorylation by CHK1 and CHK2 participates in the regulation of DNA-damage-induced C- terminal acetylation. Mol Biol Cell 16: 1684–1695. 25. Sinnberg T, Lasithiotakis K, Niessner H, Schittek B, Flaherty KT, et al. (2008) Inhibition of PI3K-AKT-mTOR Signaling Sensitizes Melanoma Cells to Cisplatin and Temozolomide. J Invest Dermatol. 26. Kao HY, Ordentlich P, Koyano-Nakagawa N, Tang Z, Downes M, et al. (1998) A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev 12: 2269–2277. 27. Kerrien S, Alam-Faruque Y, Aranda B, Bancarz I, Bridge A, et al. (2007) IntAct–open source resource for molecular interaction data. Nucleic Acids Res 35: D561–565. 28. Salwinski L, Miller CS, Smith AJ, Pettit FK, Bowie JU, et al. (2004) The Database of Interacting Proteins: 2004 update. Nucleic Acids Res 32: D449–451. 29. Alfarano C, Andrade CE, Anthony K, Bahroos N, Bajec M, et al. (2005) The Biomolecular Interaction Network Database and related tools 2005 update. Nucleic Acids Res 33: D418–424. 30. Watts DJ, Strogatz SH (1998) Collective dynamics of ‘small-world’ networks. Nature 393: 440–442. 31. Begley TJ, Samson LD (2004) Network responses to DNA damaging agents. DNA Repair (Amst) 3: 1123–1132. 32. Lynn S, Yew FH, Hwang JW, Tseng MJ, Jan KY (1994) Glutathione can rescue the inhibitory effects of nickel on DNA ligation and repair synthesis. Carcinogenesis 15: 2811–2816. 33. Mizumoto K, Glascott PA Jr, Farber JL (1993) Roles for oxidative stress and poly(ADP-ribosyl)ation in the killing of cultured hepatocytes by methyl methanesulfonate. Biochem Pharmacol 46: 1811–1818. 34. Harper JW, Elledge SJ (2007) The DNA damage response: ten years after. Mol Cell 28: 739–745. 35. Baudot A, Angelelli JB, Guenoche A, Jacq B, Brun C (2008) Defining a modular signalling network from the fly interactome. BMC Syst Biol 2: 45. 36. Barrios-Rodiles M, Brown KR, Ozdamar B, Bose R, Liu Z, et al. (2005) High- throughput mapping of a dynamic signaling network in mammalian cells. Science 307: 1621–1625. 37. Hwang D, Smith JJ, Leslie DM, Weston AD, Rust AG, et al. (2005) A data integration methodology for systems biology: experimental verification. Proc Natl Acad Sci U S A 102: 17302–17307. 38. Vidal M (2005) Interactome modeling. FEBS Lett 579: 1834–1838. 39. Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, et al. (2008) Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science 321: 1801–1806. PLoS Genetics | www.plosgenetics.org 17 June 2009 | Volume 5 | Issue 6 | e1000527