Table 1. Summary of MMS survival genes identified by genome-wide RNAi screen. Normalized RNAi screen results BS_QN cellHTS2 total FBgn Death 1183 594 1326 935 Resistance 40 40 72 61 Total 1223 634 1398 996 Numbers of death and resistance hits found by background subtraction followed by quantile normalization (BSQN) and cellHTS2 are given. The total number of unique hits and the total number of hits whose amplicons map to genes with FlyBase gene number (FBgn) are also given. doi:10.1371/journal.pgen.1000527.t001 genes they found to be responsive to MMS. As previously noted, we did not observe a significant overlap between the two screens when comparing MMS survival gene orthologues, however, with this pathway comparison, we mapped orthologues of yeast MMS survival genes to 10 of the 13 Drosophila MMS survival pathways (Figures 2 and 3; Figures S2, S3, S4, S5; Table S6). The three Drosophila MMS survival pathways without yeast MMS survival genes either are not conserved in yeast or have almost all of the pathway components are essential for viability in yeast, thus making them refractory to MMS viability analysis (Table S5). The gene enrichment with each pathway between species is highly supportive of a conservation of processes involved in MMS survival. A notable absentee in the Drosophila screen, which was observed in yeast, is 3-methyladenine-DNA-glycosylase (MAG1/ AlkA), the principal protection against MMS-induced DNA damage in yeast, but no direct orthologues exist in animals. One of the two glycosylase found in Drosophila that act at the same step in BER, namely Thd1, was found to be required for survival after MMS treatment. Furthermore, several of the pathways observed in both species, such as proteasome [19], the TOR pathway [20], and DNA repair pathways [21], have been shown to be functionally responsive to MMS in yeast. Altogether, these results suggest a conservation of pathway function, if not individual genes, in response to MMS between species. To demonstrate that the identified Drosophila pathways are functioning in the expected manner in response to MMS, five were selected for further examination – base excision repair (BER), DDR, glutathione metabolism, proteolysis by proteasomal degra- dation (proteasome), and the TOR pathway. Two of these pathways, BER and DDR, are expected to play a role in MMS survival [9], while the others were selected for their apparent breadth of function and their non-canonical role in damage survival. For each pathway, an appropriate functional assay was chosen, and when possible, an appropriate upstream MMS survival protein within that pathway was knocked-down (Figure S6A) to demonstrate modulation of the MMS induced response. Functional conservation of MMS survival pathways in Drosophila and mouse First, we tested the two expected MMS survival pathways BER and DDR. MMS-induced DNA damage results in the production of apurinic/apyrimidic (AP) sites, a DNA damage typically repaired by BER [9]. We therefore quantified the amount of AP sites per microgram of genomic DNA following MMS treatment and observed a statistically significant increase compared to control (Figure 4A; p#7.6E-8). Knock-down of the BER Damage Survival Pathways component XRCC1 resulted in an increased amount of AP sites in and of itself (p#3E-3), but following MMS treatment, the amount of AP sites in the absence of XRCC1 was increased further (Figure 4A; compared with untreated XRCC1 knock- down, p#3.3E-8; compared to MMS treated luciferase (Luc) control, p#3.7E-3); MMS therefore produces the expected form of DNA damage to which BER appropriately responds. p53, a central component of the DDR pathway, is regulated at expression, protein stabilization, and posttranslational modifica- tion levels. As expected, p53 gene expression increased in response to MMS exposure (Figure 4B; p#1.6E-3). We then examined three MMS survival pathways that are not part of the canonical DDR and DNA repair process: glutathione metabolism, proteasome, and the TOR pathway. An increased activity of the glutathione synthesis pathway following MMS exposure was demonstrated by measuring total glutathione content per milligram of protein lysate (Figure 4C; p#3.4E-3), as well as by examining the expression of the rate-limiting enzyme for glutathione synthesis, GCLc [22] (p#8.8E-4). Therefore, as expected, knock-down of this same protein, GCLc, significantly reduced the total amount of glutathione present compared with control (p#3.4E-3), but its knock-down also prevented the cells from increasing the level of glutathione in response to MMS exposure (Figure 4C). Since glutathione synthesis is considered to be an oxidative stress response, we demonstrated that MMS resulted in a dose-dependent increase in the level of 8-oxoguanine, a DNA lesion normally associated with oxidative damage (Figure S7 and Text S1). Thus it appears that MMS results in not only alkylation damage but also damage from oxidative stress. For the analysis of the proteasome degradation pathway, we measured proteasome activity and found that it increased following MMS treatment (Figure 4D; p#4.2E-2). Protein knock-down of the proteasome components Rpn2 or Pros26.4 significantly reduced proteasome activity compared to cells without knock-down that were either unexposed (Rpn2: p#1.7E-2; Pros26.4: p#3.7E-3) or exposed to MMS (Rpn2: p#1.4E-4; Pros26.4: p#7.0E-5). Following Rpn2 knock-down, cells were unable to mount a detectable increase in proteasome activity following MMS exposure, although we were unable to demonstrate this for Pros26.4 knock-down (p#1.7E-3). Overall, it appears that MMS exposure results in increased proteasome activity. Finally, to investigate TOR pathway activity, S6K phosphor- ylation status was monitored. TOR is a kinase that phosphorylates S6K to promote growth through ribosome biogenesis and is negatively regulated by the tumor suppressor protein Tsc1 [23]. MMS exposure resulted in a dose-dependent decrease in S6K phosphorylation, suggesting an inhibition of TOR activity (Figure 4E). This MMS-induced decrease in S6K phosphorylation was dependent on Tsc1 (Figure 4E). MMS exposure therefore elicited a down-regulation of the growth promoting TOR pathway, suggesting that this pathway is also a coordinated component of DDR similar to observations in yeast [20]. This supports a previously published observation by Matsuoka et al. [4], who showed that some components of the mammalian TOR pathway are phosphorylated following ionizing radiation expo- sure. In conclusion, the results of these functional assays validate the identification of the ‘‘MMS survival pathways’’ by RNAi screening and that the functionality of these pathways, or lack thereof, affects MMS survival. Having thus identified 13 Drosophila MMS survival pathways, we went on to investigate their functional conservation in mammals. Using primary mouse embryonic fibroblasts (MEFs), we examined the same five pathways that were modulated following MMS PLoS Genetics | www.plosgenetics.org 4 June 2009 | Volume 5 | Issue 6 | e1000527