Structural Investigations Critical to Understanding DNA Damage Recognition and Repair in Cancer

University Of Vermont & St Agric College, Burlington VT

Investigators

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Abstract

SUMMARY The human genome is under constant threat of damage through both exogenous (ultraviolet rays, chemicals from cigarette smoke) and endogenous (oxygen for normal cellular respiration) sources. Safeguarding genomic integrity from damage is fundamental to human health and prevention of disease states including premature aging and cancer. While cellular oxidation-reduction systems exist for detoxifying a number of the metabolic byproducts, some fraction inevitably escapes to generate oxidized lesions in DNA. The damage can take the form of free radical damage to bases allowing mispairing during replication, sites of base loss and strand breaks. Cells endure tens of thousands of these sites of damage per day that, if left unrepaired, lead to cancer, cancer progression or render treatment regimens ineffective. Understanding the means by which these sites of damage are recognized and repaired error-free or bypassed in error-prone manners is essential to provide critical information regarding predictive outcomes and therapeutic strategies for cancer patients. This proposal utilizes biochemical, biophysical and structural biology techniques in support of two NCI-funded Programs aimed at elucidating the molecular details of key enzymes involved in DNA repair. Program 1 investigates structural and biochemical details of DNA polymerase β to determine mechanistic features governing the loss of replication fidelity observed for variants in the human population including those found in cancer. Pol β is responsible for filling DNA gaps generated by glycosylases of the base excision repair pathway upon excision of oxidative damages. Program 2 investigates the structural domains of the POLQ gene product DNA polymerase θ and its response to encountering unrepaired DNA during replication, in particular the processing of DNA double strand breaks in an error-prone manner during theta-mediated end joining (TMEJ). Upregulation of the POLQ gene strongly correlates with poor clinical outcome in breast cancer patients and Pol θ has emerged as a compelling drug target for combination therapy with radiosensitization. I serve diverse roles within this proposal including integration of multiple technical approaches to investigate protein interactions with damaged DNA from both Programs while characterizing functional or patient-derived variants of the enzymes. Additionally, I maintain the X-ray and structural biology support equipment while guiding trainees in their lab work under the NCI programs. Moreover, support from this proposal will allow generation of preliminary data for additional NCI proposals that synergistically complement the currently funded programs, in particular the renewal of the previously funded P01 program investigating DNA glycosylase structure and mechanism (Program 3). Collectively, this work is expected to generate important insights into the molecular mechanisms of DNA repair proteins. This proposal is expected to have a positive impact for NCI research because the detailed knowledge of enzyme molecular mechanisms coupled with studies of cancer variants will increase our understanding of cancer susceptibility and optimize treatments.