Genome instability refers to changes in chromosome sequence, structure, or number that affect normal cell function. Such instability is characteristic of cancer, as well as certain developmental and neurological defects, and aging. Data from multiple organisms suggests that DNA replication stress is a key contributor to genome instability. Mechanisms that stabilize replication forks, prevent abnormal divisions, and promote DNA repair are a primary barrier to disease; therefore, understanding their function and the consequences of their disruption has direct relevance to human health. This proposal employs an established model cell biology system, the fission yeast S. pombe, to characterize how living cells respond to replication stress. S. pombe is a well-established genetic model for chromosome biology that shares many features with human cells. Significantly, nearly all the genes under study have orthologues in humans that have been associated with disease.. A key aspect of the approach is to use live cell imaging to characterize the response to replication stress and characterize its long term consequences. The overarching goal is to understand the dynamics of replication stress and its resolution in normal and mutant cells. This includes determining how the cell deploys molecular mechanisms to allow damage resolution and ensure chromosome segregations. We address the cellular and genetic consequences of division under stress; investigate how replication occurs late in G2 or mitosis to facilitate resolution; and examine the three-dimensional organization of repair structures. We have previously shown that the pericentromere is a fragile site, and we have expanded that to examine the ribosomal DNA and the role of phase separation in contributing to gene integrity, as well as identification of other fragile regions. A novel component is the analysis of replication stress during meiosis as a contributor to chromosome rearrangements associated with birth defects and infertility. By combining this cell biological approach with superb yeast gene-discovery tools, and identifying the molecular events that lead to abnormal divisions and further stress, this project tackles a critical gap in current understanding. What are the pathways that contribute to different responses to stress and their associated pathologies and how do they affect the biology of living cells? Together, these studies provide a holistic picture of how conserved proteins interact to maintain genome stability in a eukaryotic cell, identifying markers and risk factors for human disease.