SUMMARY DNA repair maintains genome stability to prevent cancer and provides resistance to cancer treatments; yet, DNA repair defects lead to cancer-causing mutations and are an Achilles heel for cancer-targeted treatments. Structural Cell Biology of DNA Repair Machines 5 (SBDR-5) coordinates leaders in DNA repair (DR) to work together synergistically for comprehensive mechanistic and structural knowledge of DR processes. Four interacting Projects (P1-4) target DR pathways implicated in cancer etiology, treatment, and resistance: P1 investigates repair of base lesions from chemotherapy including a paradigm-shifting model for RNA damage responses to alkylating agents. P2 defines inter-locking pathways for repair of dsDNA breaks (DSBs), a result of radiation therapy as well as replication encounters with DNA lesions. P3 examines PARylation-mediated phase condensates and their impacts on DR for PARP inhibitor therapies. P4 determines mechanisms in replication fork stress responses, key consequences of many cancer treatments. Our highest priorities are to solve actionable structures in each Project and apply our teamwork to biochemically and biologically test molecular insights and hypotheses based on our structures. Two experimental Cores will enable efficient preparation of DR assemblies and determination of their structures. Removing bottlenecks within individual laboratories and promoting collaborative efforts, SBDR Projects and Cores together enable a comprehensive cross-pathway knowledge of dynamic multi-functional DR machines – knowledge best achieved through multi-disciplinary approaches and concerted efforts by multiple groups. Our four Program aims are: 1) Determine actionable and biologically-validated structures of DR complexes, interfaces, and conformations. 2) Dissect multi-functionality of DR machineries tested by structure-based design of separation-of-function mutations and chemical inhibitors to inform on predicted therapeutic sensitivity and development of resistance. 3) Define DR pathway interactions and crosstalk with replication dynamics to delineate DR pathway outcomes. 4) Discover synthetic lethality from mutations or chemical agents that target specific DR activities and which become essential only in conjunction with another DR defect to promote successful therapeutic interventions. SBDR will provide lasting structure-based knowledge for interpretation of mutations from The Cancer Genome Atlas and of mechanisms that inform system level knockout and knockdown correlations. Through concerted efforts, SBDR-5 will establish biologically-validated structures and synthetic lethality relationships with durable biological and medical utility. We will share constructs, mutants, inhibitors, data, and technologies to enhance synergy, reproducibility, and efficiency. Our target structures and mechanisms are both challenging and timely: the expected results will have indelible impacts on ongoing NIH research in other labs, on assessment ...