SUMMARY- PROJECT 4 - Fork Repair: Mechanisms and consequences of stalled replication fork processing DNA replication is central to cancer etiology and treatment since errors during replication generate most cancer driver mutations, and many cancer treatments including PARP inhibitors and chemotherapeutic agents target DNA replication. We aim to discover the mechanisms of replication-coupled DNA repair and tolerance that are important for tumorigenesis and development of new therapeutics. The premise of this application is that ATP- dependent motor proteins act in pathways to control replication fork remodeling as a mechanism of replication stress tolerance and error avoidance. In order to overcome challenge, intermediates in these pathways are shunted towards alternative mechanisms, including translesion synthesis and repriming. The mechanism deployed may depend on the type of challenge, its persistence, and the inactivation of selected replication stress response and repair proteins that are common in cancer. While ensuring replication is completed, the use of error-prone mechanisms can generate the genetic alterations that drive tumor development and drug resistance. This genetic instability may also provide new opportunities for therapeutic intervention through synthetic lethal approaches. This project will answer fundamental questions about these replication stress response mechanisms using a combination of structural, biochemical, genetic, and cell biology approaches. Specifically, we will determine how ATP-dependent fork reversal enzymes control replication fork dynamics, identify the mechanisms that regulate replication stress tolerance pathways, and determine the consequences of damage tolerance and fork repair pathways for genome stability. These aims will be accomplished by the combined efforts of four investigators with expertise in cell biology, genetics, biochemistry, biophysics, and structural biology and a history of working in cooperation with each other and with the other project investigators and research cores.