ABSTRACT Oxidative stress is a prevalent and dangerous cellular condition resulting in deleterious modifications to the structure of DNA. These modifications promote mutagenesis and consequently the development of numerous human maladies, including cancer. The base excision repair (BER) pathway is the cells primary defense against oxidative DNA damage and is a vital guardian of genome stability. While the roles of individual enzymes during a classical BER cycle are largely established, it remains enigmatic how these enzymes function together in a multi-protein/DNA complex to facilitate the channeling of toxic DNA repair intermediates between each protein. Importantly, BER not only occurs on naked duplex DNA, but also within chromatin that is composed of nucleosomes. These nucleosomes present a barrier to BER enzymes accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome is poorly understood. The major goals of this proposal are to understand the molecular mechanisms of each BER factor both individually and within larger multi- protein/DNA complexes using naked duplex DNA and chromatin; and to decipher the molecular mechanism by which telomerase replicates the telomere. Elegant biophysical approaches are required to elucidate these BER complexities and to provide both a foundation for interpreting the biological response and the development of therapeutic treatments. We are in a unique position to advance this scientific front based on my strong track record in DNA damage and repair, assembled team of collaborators, and multidisciplinary approach. To meet this goal, we utilize a comprehensive approach of time- lapse X-ray crystallography, molecular dynamic simulations, enzyme kinetics, single-molecule total internal reflection microscopy, and cryo-EM. Using these methodologies, we will determine 1) how do new fundamental mechanistic steps alter the DNA polymerase and telomerase mechanism; 2) how do individual BER enzymes assemble into a multi- protein/DNA complex to facilitate the channeling of toxic DNA intermediates; 3) how are multi-protein/DNA BER complexes structurally organized; 4) how is DNA damage identified and repaired within nucleosomes; and 5) how are multi-protein/DNA BER complexes formed on nucleosomes containing DNA damage. This set of questions will go from an atomic level mechanistic understanding of key BER components to the structural and dynamic interactions within the entire BER multi-protein complex. By doing this, we will lay the foundation to address an inherent challenge in establishing cellular models and developing new therapeutic treatments that target DNA repair. With this information in hand, we will be closer to our long-term goal of providing a basis for rational drug design towards the development of more effective chemotherapeutics and synergistic drug combinations that target proteins involved in the DNA damage respo...