Author Summary Cellular damage is known to elicit a pleiotropic response, but the relative importance of the constituent compo- nents in cell survival is poorly understood. To provide an unbiased identification of the proteins utilized in damage survival, we performed an RNAi survival screen in fly cells with methyl methanesulfonate (MMS). The genes identi- fied are involved in 13 biologically diverse pathways. Comparison with analogous yeast data demonstrated a lack of conservation of the individual MMS survival genes but a conservation of pathways. We went on to demonstrate the MMS responsiveness for five representa- tive pathways in both fly and mouse cells. We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue compari- sons fail. Incorporation of pathway data in interactome analysis also improved connectivity and, more importantly, revealed a biologically intuitive, highly inter-connected ‘‘MMS survival network.’’ This pathway conservation and inter-connectivity implies extensive interaction between pathways; for diseases such as cancer, such crosstalk may dictate disparate cellular responses not necessarily expect- ed and confound treatments that are not tailored to the individual molecular context. individual proteins significantly altered cell viability following methyl methanesulfonate (MMS) exposure. MMS is a prototypical alkylating agent that attacks nucleophilic groups, such as those found in nucleic acid [9]. The resultant base methylation destabilizes the glycosidic bond, facilitating the production of the most biologically relevant cellular lesion of MMS, an abasic site in DNA. Other common sources of alkylation damage include endogenous S-adenoysyl methionine [10], the tobacco carcinogen N-nitorsoamine [11] and chemotherapeutics such as temozolo- mide [12], carmustine (BCNU) [9], and cyclophosphamide [13]. Considering the physiological relevance of alkylation damage, several recent studies using yeast as model organism investigated the global effects of alkylation damage induced by MMS. Viability [14], gene transcription [15] and protein expression [16] were measured in these studies, all of which provided insights into the diverse nature of biological responses to alkylation damage. However when transcriptional responses to various environmen- tal stresses were compared between mammalian and yeast cells [17], Murray et al. reported clear distinctions and suggested that this might be the result of different selective pressures between multi-cellular organisms and single-cell organisms. In the attempt to understand diverse responses required for damage survival in a system that is evolutionarily closer to mammals than yeast, we performed a genome-wide, RNAi-based screen using cells derived from Drosophila melanogaster. Our experiments were based on ‘‘loss of function’’ and an assessment for cellular viability following exposure to MMS. Here we present results from this MMS survival screen, the pathways that were identified, and a comparative analysis of pathways to understand conservation of these pathways in yeast and mouse cells. Additionally, we present a novel approach of assembling a protein interaction network based on defined biological path- ways, which facilitates network consolidation. By including biological pathways in our genomic data and protein network analysis, we were able to determine the commonality across species, which was obscured by direct orthologue comparison, and provide a simplified representation of the global survival responses that we identified. Damage Survival Pathways Results Genome-wide RNAi screen and validation An RNAi screen was designed to identify those genes that modulate cellular survival following exposure to a level of MMS- induced damage that resulted in only a limited amount of cell death. A linear decrease in cell viability was observed from 0.002% (w/v) of MMS to 0.008% (w/v) of MMS (Figure S1). A dose of 0.004% (w/v; 40 mg/mL) MMS was chosen, which resulted in a statistically significant decrease to approximately 65% viability, while allowing an additional, measurable decrease in cell survival. RNAi screens were performed using the Drosophila RNAi Screening Center (DRSC) version 1 library (about 23,000 D. melanogaster open reading frames) [8]. Kc167 Drosophila cells were exposed to three days of dsRNA to allow protein knock-down, followed by three days of growth in either the absence or presence of MMS exposure (Figure 1A). To identify those genes required for cell viability following MMS treatment, viability results from the MMS treated RNAi screen were compared with that of the untreated (control) screen as previously described [18]. 1,398 different open reading frames were identified that affected MMS survival in a continuous distribution of cell survival values (see [18] and http://gccri. uthscsa.edu/ABPublished_Data.asp for original data), of which 996 had a unique assigned FlyBase gene number (FBgn; denotes known genes) (see Figure 1B and Table 1). Of these 996 genes, the top 537 were selected for validation analysis by a previously described, stringent validation method [18]. Whereas in [18] we validated normalization methods, here we independently validated individual genes using dsRNA targeting a different region of each gene. 202 protein knock-downs validated with a significant MMS viability effect, while 55 more had a notable trend effect without meeting our stringent statistical criteria (Table S1 and Table S2). An examination of gene ontology (GO) on the 202 validated MMS survival genes revealed a significant enrichment for genes involved in DNA metabolism, gene transcription, and cellular metabolism (Table 2). The overall variety of GO categories observed was quite broad, consistent with the findings reported for an analogous yeast screen [3] (data not shown). Surprisingly though, no significant enrichment between the gene orthologues for the two organisms was observed (G-test p-values of 0.29 and 0.057 or Fisher Exact test p-values of 0.37 and 0.08, yeast and fly, respectively). To further test this, we examined the MMS sensitivity following knock-down of 183 fly genes that were orthologues of to 118 yeast MMS survival genes [3], but only found conservation of MMS survival phenotype with 20 (Table S3). This apparent lack of gene enrichment between species has also been reported when comparing transcriptional profiles between mammalian and yeast cells in response to a variety of stresses [17]. Overall, these results suggest that there may be conservation of the biological processes that respond to MMS rather than the individual genes. Identification of pathways responsive to MMS Assuming that biological function is a more informative measure of damage response than a requirement of individual genes, we therefore endeavoured to identify those MMS survival proteins within known signaling, metabolic, and enzymatic pathways. Using both a priori knowledge and KegArray, we identified 13 pathways that together included 41 MMS survival proteins (Figures 1, 2, 3 and Figures S2, S3, S4, S5), among them, five DNA repair pathways (Base Excision Repair; Nucleotide Excision Repair; Mismatch Repair; Homologous Recombination Repair; and RECQ). Many of these pathways have a statistically significant number of MMS survival proteins (Table S5), and this PLoS Genetics | www.plosgenetics.org 2 June 2009 | Volume 5 | Issue 6 | e1000527