Summary for Project 1 led by Research Project Leader Joonas Jamsen, PhD Environmental agents, endogenous cellular processes, and many cancer therapies induce accumulation of reactive oxygen species (ROS) that lead to oxidative DNA damage and chromosomal double-strand breaks (DSBs). DSBs are hazardous and if repaired erroneously or left unrepaired lead to cancer and disease. DSB repair pathways counter these breaks, yet little is currently known about the molecular details and structural transactions underlying the strategies and efficiency of mutation-prone or mutation-free repair that promote adverse human health outcomes. The long-term goal of our research program is to uncover fundamental mechanisms that enable DSB repair within multiprotein repair complexes. As part of the Center for Molecular Interactions in Cancer (CMIC), this COBRE Research Project will uncover strategies of DSB repair that are relevant to breast, ovarian, and other cancer types. We will test the central hypothesis that DNA polymerases (pols) λ and θ coordinate an alternative microhomology-mediated end joining (MMEJ) pathway, contributing to poly (ADP-ribose) polymerase inhibitor (PARPi) resistance in homologous recombination (HR)-deficient cancers. To test this hypothesis, Aim 1 will employ time-resolved X-ray crystallography and cryo-electron microscopy (cryo-EM) alongside biochemical and biophysical techniques to study the molecular features of pol λ and pol θ activity during MMEJ. Aim 2 will study cellular mechanisms of pol λ-mediated DSB repair and the impact on PARP inhibitor resistance. Aim 3 will develop small-molecule inhibitors to modulate DNA pols involved in DSB repair. This cutting-edge approach also will yield fundamental discoveries into DNA repair mechanisms by employing the state-of-the-art research environment and resources available in the COBRE-supported Structural Biology and Biomolecular Interactions Cores. Completion of the research project will enable me to develop new approaches for my research program. Innovative models of DSB repair will be pursued using advanced structural, biophysical and molecular biology techniques. Our work is significant because it will provide new insights into how pol-mediated reactions contribute to the repair of DNA damage known to impact cancer progression and resistance to therapy. Studying DNA polymerases involved in DSB repair will provide insight into how these reactions could be modulated to provide therapeutic approaches to impact cancer and disease.