Accurate DNA replication is an essential prerequisite of genome stability, and nature has evolved numerous mechanisms to ensure precise and complete genome duplication. Subversion of these protective mechanism results in genome instability, the hallmark of cancer and many human developmental syndromes. Paradoxically, recent evidence suggests that pharmacological interference with these mechanisms represents a promising therapeutic strategy, because cancer cells show a pronounced dependency on these pathways for their survival. One of the most important requirements for faithful DNA replication is the maintenance of replication fork stability under conditions of stress. A number of proteins involved in this process have been identified to date; however, our understanding about their post-translational regulation is rather vague. In particular, although their indispensable role in DNA damage signaling is firmly established, the role of ubiquitin and ubiquitin-like modifiers responding to DNA replication stress has so far not been addressed. We propose to combine high-content microscopy with reverse genetics to identify ubitiqutin-related factors and pathways in the response to DNA replication stress. To this end, we will set up a dual labeling protocol for the quantitative detection of DNA synthesis – an approach never exploited for high content studies. To realize the full potential of this approach, comprehensive expertise in high-content microscopy and RNAi based screens are essential – both of which are among the core competencies of the host institution. We will characterize novel regulators by a combination of basic and highly specialized, biochemical, molecular and cellular biology and physicochemical techniques. We are confident that the results of this study will reveal novel aspects of DNA replication and genome surveillance and their role in human disease, and provide new opportunities for cancer therapy.