SUMMARY Insuring genomic integrity is fundamental to human health, development as well as the prevention of numerous disease states including premature aging and cancer. The human genome is under constant threat of damage through both exogenous (UV Rays, chemicals from cigarette smoke) and endogenous (the oxygen required for normal cellular respiration) sources. While cellular oxidation-reduction systems exist for detoxifying a number of the metabolic byproducts, some fraction inevitably escapes to come in contact with the DNA and generate oxidized lesions that can lead to mutations during replication through free radical damage to bases, strand breaks and sites of base loss. It is estimated that each cell experiences in excess of 10,000 sites of damage per day that, if left unrepaired, lead to disease establishment and promote its progression. Understanding the means by which these sites of damage are recognized and repaired or bypassed is essential to providing critical information regarding predictive outcomes and therapeutic strategies for cancer patients. This proposal utilizes structural biology techniques including X-ray crystallography in support of thee NCI-funded programs aimed at elucidation of the molecular details of key enzymes involved in DNA repair. The focus of project 1 is the structural and biochemical investigations of DNA glycosylases that recognize and remove oxidative lesions within the DNA whereas program 2 investigates the mechanisms of replication fidelity of DNA polymerase β, the repair polymerase responsible for filling nucleotide gaps generated by a glycosylase. Program 3 investigates specialized DNA polymerases, particularly pol θ, and its response to encountering unrepaired DNA during replication. Pol θ processes DNA double strand breaks in an error-prone manner and upregulation of the POLQ gene strongly correlates with poor clinical outcome in breast cancer patients. Pol θ has thus emerged as a compelling drug target for combination therapy of radiosensitization. My role in this proposal will be to perform structural and biochemical investigations of complexes of polymerases and glycosylases bound to DNA damage. I will also be characterizing variants of these enzymes identified in cancer patients. Additionally, I maintain the X-ray equipment and manage the X-ray facility, am responsible for training of new users and guide students and postdocs in their structural work within these NCI programs. Collectively, this work is expected to generate important insights into the molecular mechanisms of DNA repair proteins. These results are expected to have a positive impact because the detailed knowledge of enzyme molecular mechanisms coupled with studies of cancer variants will increase our understanding of cancer susceptibility and optimize treatment protocols.