PROJECT SUMMARY Numerous environmental and cellular factors can induce DNA lesions or create other types of challenges that can slow DNA replication, leading to replication stress. Failure to alleviate this stress and restart stalled replication forks can cause genome instability, which can drive cancer initiation and progression and affect the cellular response to chemotherapy. Hence, deciphering the cellular response to replication stress––the long- term goal of this application––is imperative for understanding fundamental aspects of tumorigenesis. One crucial aspect of the replication stress response involves replication fork reversal, a process that leads to the formation of a four-way junction structure by the remodeling of both nascent and parental DNA strands. There are a family of ATP-dependent translocases that promote fork reversal in vitro and in cells, but why so many enzymes are involved in this process is not understood. We showed that one of these translocases, HLTF, prevents a stress-resistant mode of replication by promoting fork reversal, and we elucidated the basic mechanism by which cells continue replication upon HLTF loss. The object of this application is to further elucidate the mechanisms by which HLTF mediates the replication stress response, characterizing its unique role in this process. HLTF is a multi-functional protein and recent studies using a panel of HLTF mutants that are each deficient for one of its activities have revealed unexpected roles for these activities in HLTF’s known functions, raising a number of new and exciting questions. This application will address questions about the molecular mechanisms by which HLTF promotes fork slowing and fork reversal in cells and determine the impact of its loss on replisome stability, genome stability, cell proliferation. Aim 1 will investigate which domains of HLTF are needed for fork slowing, fork reversal, and resistance to replication stress, and HLTF’s association with the replication fork. HLTF’s dynamic interaction with the fork will also be probed. Aim 2 will investigate a newly discovered function for HLTF in stabilizing the replication fork. These experiments will employ a combination of molecular, cellular, genetic and proteomic approaches as well as single-molecule imaging and tracking methods in Xenopus extracts to solve fundamental questions about how cells respond to replication stress.