The goal of this proposal is to elucidate the eukaryotic DNA damage response through an integrated experimental/computational approach leading to in-silico models of signaling and regulatory networks. Comparative modeling of the networks induced by different damaging agents is likely to reveal rich new insights into cellular toxicity and, ultimately, cancer progression. Experimentally, we will focus on how the yeast DNA damage control network is reprogrammed by exposure to methyl methanesulfonate (MMS; years 1-2) or methyl-N'-nitro-N-nitrosoguanidine (MNNG; years 3-4), two different types of alkylating agent. The network will be characterized using high-throughput genomic technologies including chromatin immunoprecipitation in conjunction with promoter microarrays to identify protein-DNA interactions (chlP- chip); coimmunopreciptation followed by mass spectrometry to identify protein-protein interactions; and DNA microarrays to monitor genome-wide expression profiles resulting from systematic single and double gene knockouts. Computationally, we will integrate and model these data using tools for comparison of networks across multiple conditions (PathBLAST), statistical identification of expression-activated network regions (ActiveModules), and a specialized visualization platform and database we have developed for operating on network models (Cytoscape). A systems approach will be crucial for revealing the complex web of interactions between diverse cellular damage responses ranging from base-excision repair and homologous recombination to cell-cycle arrest, apoptosis, general stress response, protein metabolism, and those yet to be discovered. ? ?
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