Damage Survival Pathways Figure 6. Protein network of MMS survival proteins reveals an integrated, highly connected MMS survival network. (A) 150 of the validated MMS survival proteins (red nodes) are represented in the known Drosophila interactome, with protein:protein interactions represented by grey edges; 104 of these validated hits are ‘‘orphans,’’ not connected to other proteins. (B) Nodes and orphans from (A), including 11 proteins essential for viability (black nodes) that are connected to two or more validated hits, improves the connectivity of the DNA damage survival network. In this network, only 93 hits are orphans and a larger network of 36 proteins, containing 29 validated hits, is observed. (C) Nodes and orphans from (B), including additional pathway hits not found in screen (green nodes). In this network, 86 screen hits and an additional 54 pathway hits are orphans and a larger network of 63 proteins, containing 33 validated screen hits, 20 additional pathway hits and 10 essential proteins. (D) Nodes and orphans from (C), including all non-hit members of the 13 MMS survival pathways (open nodes). In this network, 60 screen hits and an additional 40 pathway hits are orphans and a larger network of 247 proteins, containing 79 validated screen hits (26 of which are within pathways), 35 additional pathway hits, 101 non-hit pathway proteins, and 32 essential proteins. (E) Network of proteins validated to be required after MMS treatment (red nodes), proteins essential for viability (black nodes), and 13 pathways (blue pathway nodes) validated to be involved in MMS survival. All proteins in each pathway, regardless of being a hit, are represented in its metanode, with node size representing protein number. After all pathway components are used to create the network, only 54 proteins are orphan, and 96 proteins are contained within the main interconnected network. Network connectivity coefficients are given at the lower right-hand corner for (A–D). doi:10.1371/journal.pgen.1000527.g006 both within and connecting to a pathway, even if the pathway components themselves are not critical to survival after MMS. To qualitatively visualize this result, each protein known to be in a pathway was grouped together and assigned to a ‘‘pathway node’’ (a single node within the interactome that retains the interactions of its constitutive proteins to proteins that are external to that pathway). This resulted in a highly connected interactome, or ‘‘MMS survival network’’ (Figure 6E), that now encompassed 179 of the 233 MMS survival proteins that are present in the PPI network (77%). Of the remaining 54 orphan proteins, 47 are only one protein removed from the MMS survival network, six are two proteins away, and only one is not connected at all. At each step in this analysis, we observed an improved network, either enlarged or better connected, not only when comparing the entire set of proteins in that network to a random set of proteins in the PPI, but also when randomizing the additional proteins added at each step (data not shown). Overall, our observations with the MMS survival network suggest that despite the general interconnectivity within protein interactomes, a pathway analysis is highly relevant because it may improve interactome connectivity and it simplifies a systems biology overview. Discussion Studies performed using yeast as a model organism to predict network response to MMS reported an astounding involvement of diverse biological pathways [31]. Considering that the genes that respond to environmental stress differ between mammalian and yeast cells [17], we presumed that damage response might be different or more complex in higher eukaryotes, especially considering the presence of paralogues and thus increased genetic redundancy. We therefore performed a genome-wide, RNAi based screen with Drosophila cells to investigate which genes are essential for survival following damage exposure with MMS. We were able to identify and validate 307 MMS survival genes, the majority of which had not previously been associated with alkylation damage survival. Of these genes, 146 were components of 13 different MMS survival pathways. With the five pathways examined in detail, we observed that four were functionally conserved in yeast and all five conserved in mouse with regard to their utilization following MMS treatment (Figure 5). In yeast, experimental validation of response to MMS by proteasome [19], the TOR pathway [20], and DNA repair pathways [21] was previously reported. Similarly, glutathione response to MMS was observed in mammalian cells [32], and our observation of an increase in GCLc expression provides an underlying mechanism for this phenomenon. Our demonstration of a dose-dependent increase in 8-oxoguanine after MMS exposure indicates that MMS also results in oxidative stress damage, as previous studies suggested [33]. Additionally, several recent studies have demonstrated a role for the proteasome in regulating several DNA repair pathways (reviewed in [34]), supporting our observation of increased proteasome activity in response to MMS. Thus, our screen and pathway identification have revealed a conserved set of MMS survival pathways. Our Drosophila based study has provided novel insights to the global cellular response to alkylation damage by identifying biological pathways whose functions are required for survival after this damage. The only other analogous genome-wide, loss-of- function screen for MMS survival genes was performed in yeast [14]. That study highlighted the general biological processes required for MMS survival based on gene ontology and integrated the identified proteins into a disorganized network [14]. Considering that pathways, whether signaling, metabolic, or enzymatic, have long been identifiable entities, it is logical to consider them as units within a network. Thus our experiments focused on identifying pathways required upon exposure to damage and validating the biological responsiveness of pathways following this damage exposure. Our results confirmed that these biological pathways are indeed functional in yeast, Drosophila, and mouse cells and therefore functional contribution of these biological pathways are pertinent to damage response in a network representation. In addition to the functional conservation of the survival pathway in response to alkylation damage, these same biological pathways appear to have roles in response to other types of damages. It is interesting to compare our results with an elegant study by Matsuoka et al. [4], which identified proteins that are phosphorylated following ionizing radiation in human cells. Their study identified proteins that are components of nine of our 13 MMS survival pathways, including four of the DNA repair pathways, DDR, mTOR, proteasome, basal transcription, and ribosome. These results suggest that different types of damage, not just alkylation damage, may utilize different components of a DNA damage survival network in a functionally conserved manner and reemphasize the functional conservation of pathways, if not the individual genes, between species. Our emphasis to reorganize the MMS survival network based on pathways is to facilitate the observation of biologically relevant interactions. Often protein:protein interaction networks may appear chaotic, but may be interrogated for simple sub- networks associated with protein(s) and pathways of interest [21]. However, when working with pleiotropic responses that encompass so many different biological processes, such as DDR, a chaotic network representation appears non-intuitive (Figure 6D). Thus, the integration of pathways within the conceptual framework of systems biology networking is logical. An additional advantage of including pathways is highlighted by our demonstration of MMS survival protein enrichment by detailed examination of pathways. Even with this detailed PLoS Genetics | www.plosgenetics.org 11 June 2009 | Volume 5 | Issue 6 | e1000527