Project Summary Every cell division requires the faithful duplication of genetic material from mother cell to daughter cell. Defects in the proper execution of the DNA replication program can directly result in the genome instability that is a hallmark of nearly all cancer cells. To ensure genome stability, the machinery responsible for DNA replication must be highly regulated. There must be enough flexibility built into the DNA replication program, however, to accommodate the dramatic changes in cell cycle and S phase progression that occur during the development of an organism. Understanding this regulation is imperative to determine how genome stability is maintained during development. We utilize the powerful developmental systems available in Drosophila to identify key regulators of the DNA replication program, with the ultimate goal of delineating the molecular underpinnings responsible for regulating genome duplication and stability during development. Our approach has been to systematically identifying factors associated with the DNA replication machinery in different developmental contexts. This has led us to three key research areas: 1) understanding replication-transcription conflicts and their impact on genome stability; 2) determining how the DNA replication machinery stably navigates through condensed chromatin, such as heterochromatin; 3) delineating how post-translational modifications, such as ubiquitylation, regulate the activity of the DNA replication machinery. By providing mechanistic insight into these processes, our goal is to provide fundamental insight into how genome stability is maintained during development.