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Two collections of chromosomally tagged yeast Saccharomyces cerevisiaestrains were previously designed to detect protein-protein interactions via the Cross-and-Capture system. Here, we used these strain collections in a different application to screen for proteins that are phosphorylated in response to DNA damage by electrophoretic shift analysis. Modification of a number of proteins that are known targets for checkpoint kinases was confirmed this way. Furthermore, we identified the mismatch repair protein Pms1 as a novel target for phosphorylation in the response to DNA damage and replication fork arrest. Genetic analysis revealed that this phosphorylation is dependent on checkpoint activation by ATM and ATR (yeast Mec1p and Tel1p) kinase. Hence, we demonstrated that the Cross-and-Capture system could be efficiently used to detect posttranslational modifications that modulate and control protein function in eukaryotic cells.