Signaling pathways activated by stress or cellular cues modify existing proteins post-translationally by phosphorylation, acetylation and ubiquitination, in order to rapidly alter physiology. We will examine regulation through post-translational modifications (PTMs) in response to several forms of stress, paying special attention to alterations in phosphorylation and ubiquitination in response to DNA damage. Our examination of the ubiquitin pathway has two primary areas of focus: ubiquitin linkage analysis and substrate identification. Ubiquitin chains are formed on substrates using any of ubiquitin's seven lysines. We take a genetic approach in yeast to explore the significance of these chains by identifying mutants that have synthetic genetic interactions with ubiquitin lysine mutants unable to form particular chain types. To identify ubiquitin ligase substrates, we developed a method called Ligase Trapping, in which we fuse a poly-ubiquitin binding domain onto a ubiquitin ligase, which increases the affinity of the ligase with its ubiquitinated substrate, allowing substrate identification via mass spectroscopy. We have carried this out in both yeast and human cells, and will follow-up on several interesting hits. We are particularly interested in ubiquitin-mediated protein turnover in response to DNA damage. DNA damage-regulated protein turnover typically occurs after a substrate is phosphorylated by one of several checkpoint kinases. Checkpoint kinases, such as ATR, CHK1 and CHK2 are activated upon DNA damage and regulate a large number of pathways. We will continue our effort to identify substrates of the DNA damage checkpoint, focusing on targets involved in either cell cycle regulation or metabolism. As with our examination of ubiquitin ligase substrates, we will generate alleles that cannot be modified and examine their effects on cellular physiology. Finally, we have developed a method by which phosphatases, de-ubiquitinases, HDACs, or other enzymes can be localized individually to each protein in yeast. We will use this technology to identify modifications that are essential for viability either in unperturbed cells, or in respose to stresses such as DNA damage. Together with our substrate identification studies, this will allow us to generate a global, functional picture of protein modification.