PROJECT ABSTRACT The overall goal of this project is to uncover novel mechanisms of genome maintenance during chromosomal replication. The progression of replication forks is frequently challenged by both environmental agents and intra-cellular causes. As such, cells must protect the fork structure in the face of replication stress in order to proliferate and to maintain genome stability. Defects in fork maintenance cause cancer, premature aging and other diseases. Paradoxically, obstruction of DNA replication is also a commonly used strategy for cancer treatment. Despite its critical importance in genome maintenance, tumorigenesis and cancer treatment, the molecular mechanisms of fork protection and maintenance remain poorly understood. In our effort to address this fundamental question, we have identified a novel calcium- and AMPK-dependent signaling pathway that protects fork structure upon replication stress by restraining the activity of the Exo1 nuclease to avoid deleterious processing of fork DNA. Disruption of this pathway causes fork degradation, chromosomal instability and compromised cell viability. Building on this initial finding, we describe experiments in this proposal to further decipher this new fork protection pathway by identifying key players and mechanisms that mediate its activation. In Aim 1, we will begin to define the molecular nature of the signal at stressed replication forks that directly induces the elevation of intracellular calcium essential for the activation of the overall pathway. Aim 2 seeks to identify key intermediate factors that mediate the induction of intracellular calcium upon replication stress by using a candidate gene approach and by carrying out an unbiased genome-wide CRISPR screen. In Aim 3, we will elucidate the key biochemical events that lead to the activation of AMPK for fork maintenance in the replication stress response. By identifying and characterizing the upstream signal, players and molecular processes, these studies will provide critical mechanistic insights into the novel calcium- and AMPK-dependent pathway in safeguarding the fork structure under stress. This project will not only expand our understanding of the replication stress response, but also lay a conceptual foundation for developing more effective cancer therapeutic strategies targeting DNA replication and AMPK. 1