! 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 the parental grant 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. 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. Specific to this research supplement, we have established a three-color single-molecule TIRFM system to characterize the multiprotein BER complex. Using this approach, Dr. Fausto Varela will determine how OGG1 and APE1 coordinate on the DNA during the repair of 8-oxoG. Simultaneously, he will learn new technical expertise, publish impactful research, and acquire the key preliminary data for a competitive postdoctoral fellowship. Furthermore, the results gleaned from the proposed experiments with OGG1 will directly complement the parental grant and ongoing experiments in the lab looking at substrate channeling between APE1 and pol ?, which are two downstream enzymes in BER.
DNA damage arising from oxidative stress promotes multiple human diseases, and the primary cellular defense against this oxidative DNA damage is the multi-protein base excision repair (BER) pathway. The overarching goal of this proposal is to address a pressing set of questions towards an atomic level understanding of key BER enzymes including the structural and dynamic interactions within the entire BER multi-protein/DNA complex. We will employ elegant biophysical approaches to elucidate inherent BER complexities, while providing both a foundation for interpreting the biological response and the subsequent development of rational therapeutic treatments towards enhancing human health.
