Damage Survival Pathways Figure 2. Base excision repair, DNA damage response, and proteasome pathways utilized for MMS survival. CG numbers are given for each Drosophila pathway component, as well as the protein names or complex names for their human orthologues. Pathway entry points are noted with Roman numerals at the top, and end points are at the bottom. A key for the following symbols is provided. Symbols encircled with thick lines represent proteins that act together or in a complex, while symbols encircled with thin lines represent paralogues or proteins that may substitute for one another. Proteins found to affect MMS survival are noted as down (death) or up triangles (resistance). Statistically significant proteins are indicated with black triangles, while trend hits are indicated with grey triangles. Essential genes are noted with a thick bar and any with downwards or upwards pointing boxes were also validated as conferring death or resistance, respectively, to MMS upon knock-down. Shaded squares are proteins not found to be hits after validation, and open squares were not tested in our validation. Yeast orthologues previously found to be required for MMS survival [3] are noted with a dot under the symbol. (A) Base Excision Repair. (B) DNA Damage Response. (C) Proteasome. doi:10.1371/journal.pgen.1000527.g002 exposure in Drosophila. In general we observed comparable results in MEFs to Drosophila (compare Figures 4 and 5). MMS increased the amount of AP sites in MEFs (Figure 5A); in the absence of XRCC1, however, we observed a significant increase in AP sites following MMS exposure (Figure 5A; p#3.7E-4). For DDR in MEFs, we assessed the phosphorylation status of Chk1 (a kinase that, once phosphorylated, phosphorylates p53) [24], total p53 protein, and the phosphorylation status of p53 (Figure 5B), not to examine p53 expression levels, but to obtain a result equivalent to that of Drosophila result: that DDR is activated by MMS exposure. Also, similar to the observation in Drosophila cells, MEFs had increased proteasome activity following MMS treatment (Figure 5D; p#2.0E-4); knock-down of either proteasome component, PSMC1 or PSMD1 (orthologues to Rpn2 and Pros26.4, respectively), resulted in decreased proteasome activity following MMS exposure (PSMC1: p#8.1E-5; PSMD1: p#4.1E- 4). These results are comparable with Drosophila: it is clear that proteasome activity is responsive to MMS in MEFs. It should also be noted that, though we were able to demonstrate the same dose- dependent decrease in S6K phosphorylation following MMS exposure in MEFs as seen in Drosophila cells (Figure 5E) and that knock-down of TSC1 disrupted this MMS-induced effect, the disruption was not as evident as observed with Drosophila, probably due to inefficient knock-down of the TSC1 protein in MEFs (data not shown). Having observed the functionality of these pathways in response to MMS and the ability of the five siRNA to modulate the MMS response of their respective pathway, we also examined the effect of each knock-down on MMS survival in MEFs. Three of the protein knock-downs, GCLC, PSMC1 and PSMD1, affected MMS survival in MEFs (data not shown). Overall, it would appear that Drosophila could be used to accurately predict MMS survival pathways in mouse. Utilization of the MMS survival pathways in response to Temozolomide Given that MMS causes alkylation damage, it would be reasonable to suppose the identified biological pathways are generally involved in the response to other alkylating agents. Temozolomide is an alkylating agent used clinically in the treatment of cancer [12,25]. It has already been demonstrated that BER and TOR are necessary for Temozolomide survival [12,25]. Temozolomide causes DNA damage by increasing the number of AP sites, and similar to our MMS results, inhibition of BER increases sensitivity to temozolomide [12]. It has also been reported that rapamycin inhibition of the TOR pathway increased cellular sensitivity to temozolomide [25]. To further demonstrate the utilization of MMS survival pathways in response to temozolomide, we examined the DDR pathway, glutathione levels, and proteasome activity in MEFs and observed the same responses as observed following MMS exposure (Figure S8). Further, using HEK293 cells, we observed that both MMS and temozolomide exposure repressed Notch reporter activity (Figure S9 and Text S1). Absence of functional Notch protein has been shown to repress the activity of a downstream transcriptional activator (RBP-Jk) [26], therefore this result demonstrates a functional Notch pathway response in alkylation damage expo- sure. Taken together, these data suggest a functional conservation of the MMS survival pathways responses with other similarly acting agents. Network integration of MMS survival pathways Of the 307 identified MMS survival genes (202 validated screen hits plus 105 from pathway analyses), we were able to assign 146 to 13 pathways (Figures 1, 2, 3 and Figures S2, S3, S4, S5). Because 161 MMS survival protein remain unassigned, we re-examined the inter-relationship between the identified MMS survival proteins using the currently available Drosophila protein:protein interactome (PPI) map [27–29]. To measure the connectivity among the MMS survival genes, we took the largest connected components of the PPI network and discarded the other, smaller components. The largest connected component contained 7364 of the 7504 proteins in the original PPI network. Taking the 202 original validated MMS screen hits as an unbiased starting point, we determined the number of proteins that were directly connected to one another, compared to a random set of the same number of proteins from the PPI, and observed a significant enrichment (Figure 6A and Table S7; p#2.1E-10). Similar results were obtained for other assessments of network connectivity (Table S7). Because the MMS screen hits had more connections on average than a set of random genes, which might have biased the above analysis, we also compared the connectivity of the MMS screen hits in the real PPI network with the connectivity of the same set of proteins in a randomly rewired PPI network. Degrees were preserved in the random rewiring process. As shown in Table S7, the MMS screen hits have statistically significantly more direct interactions than would be expected in a randomly rewired network (p = 0.01). On the other hand, the randomized network had smaller average distance and higher global efficiency than the real network, which could be attributed to the well-known property of real-world networks: they usually have slightly longer average distances (and correspondingly, lower global coefficients) than their degree- preserving randomly rewired counterparts [30]. Because we were unable to assess the effect of essential proteins (proteins whose knock-down resulted in cell death regardless of treatment) on MMS survival, we repeated the connectivity analysis while including those essential proteins that were connected to two or more MMS survival proteins, since these are the ones most likely to have a functional role in MMS survival. This increased the size and significantly improved the connectivity within the resultant network (Figure 6B and Table S7; p#1.3E-26). After pathway analysis, we identified an additional 105 MMS pathway hits, which were validated. In order to expand the network to include the proteins in these relevant pathways, these hits were then incorporated and a larger, and equally well-connected network was observed (Figure 6C and Table S7; p#4.6E-26). Considering the apparent importance of pathways over the individual genes, we then included all components of the 13 identified MMS survival pathways and observed that the PLoS Genetics | www.plosgenetics.org 7 June 2009 | Volume 5 | Issue 6 | e1000527