Damage Survival Pathways Figure 1. The experimental design of the Drosophila RNAi MMS survival screen and summary of the workflow and results. (A) A schematic representation of the experimental design used to screen for genes involved in survival of an MMS exposure using RNAi in Drosophila. (B) A schematic representation of the workflow from the initial RNAi screen, the normalization and validation of results, and the follow-up work conducted, with a summary of the number of dsRNA amplicons and genes (FBgn; FlyBase gene number) involved at each step in the process. Also provided are the final numbers of MMS survival proteins and MMS survival pathways (BER: base excision repair; NER: nucleotide excision repair; MMR: mismatch repair; HRR: homologous recombination repair; RECQ: RecQ helicases; DDR: DNA damage response; Proteasome; GSH: glutathione synthesis; TOR: TOR pathway; Transcription: basal transcription; Ribosome; ATPase; Notch: Notch signaling pathway). doi:10.1371/journal.pgen.1000527.g001 is without accounting for pathway ‘‘branching’’ or ‘‘subdivision.’’ Considering the likely role of these pathways in MMS survival, we used an RNAi assay method, which is both more sensitive and stringent than the original screen [18], to determine whether other members of the 13 identified pathway also affected MMS survival (Table S1). By individually interrogating the 346 pathway members of the 13 pathways that were not identified in the original screen, an additional 105 MMS survival genes were discovered. This observation significantly enriched the number of MMS survival genes in each of the 13 pathways (Figures 1, 2, 3 and Figures S2, S3, S4, S5) and provides additional support to the hypothesis that each of the identified pathways are involved in MMS survival. Though this result highlights the possibility of false negative screen data, compared to our previous false negative rate of 23.6% from randomly selected data, we observe a significant enrichment for false negatives within pertinent pathways ((x2 p = 3.9E-5). In total we identified 146 MMS survival genes in 13 ‘‘MMS survival pathways,’’ encompassing 25% to 86% of all non- essential genes within each pathway (Table S4). Using the genes we mapped to the MMS survival pathways, we then compared pathways identified from our Drosophila MMS screen with the analogous screen performed in yeast [14] using PLoS Genetics | www.plosgenetics.org 3 June 2009 | Volume 5 | Issue 6 | e1000527