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. In addition, it is poorly understood how deviations in the classical BER pathway affect the DNA repair process and genome stability. These deviancies range from mismatched-, damaged-, and ribo-nucleotides inserted by a DNA polymerase, to the coordinated repair of “dirty” or damaged DNA ends that block BER. These scenarios become particularly biologically relevant during times where there is an increase in genome instability (i.e., in cancer cells and/or during therapeutic treatments). The overarching goal of this proposal is to understand the molecular mechanisms of each BER component individually and to place these activities within the larger BER co-complex with damaged DNA repair intermediates. Elegant biophysical approaches are required to elucidate these BER complexities and to provide both a foundation for interpreting the biological response and the subsequent 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, neutron crystallography, small angle neutron scattering, molecular dynamic simulations, enzyme kinetics, and single-molecule total internal reflection microscopy. Using these methodologies, we will determine 1) How the location of DNA damage alters the DNA polymerase mechanism during repair; 2) How does the poorly characterized APE1 exonuclease reaction process damaged RNA and DNA repair blocks; 3) What are the mechanistic roles of protons during DNA damage and repair; 4) How are DNA repair complexes formed and structurally organized? 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 D...