Mammalian AP-endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are directly induced by environmental carcinogens or formed endogenously by free radicals during cellular metabolism, inflammatory diseases, carcinogenesis and aging. AP-sites are also induced by anti-tumoricidal agents or generated by excision of damaged bases by DNA glycosylases via the base excision repair (BER) pathway. Elevated levels of APE1 have been linked to resistance to chemotherapy, poor prognosis, and poor survival. Reducing the activity of the APE1 protein in cancer cells and tumors by small molecule inhibitors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. APE1-null mice died because of their inability to repair toxic endogenous AP-sites, showing the importance of APE1 in repair of toxic adducts. Exploring APE1 in isolation, as well as in cellulo condition, can help interpret and complement animal biology and translational research. APE1 has previously been cloned, expressed, and characterized. However, surprisingly, limited knowledge is available regarding the biophysical and biochemical properties of APE1 as well as its in cellulo mechanisms of AP-site repair, especially the repair of different types of AP-sites (regular, oxidized/reduced) in a comprehensive manner. Therefore, it is important to know the basic properties of APE1 before beginning animal and human studies for translational research. We hypothesize that in cellulo protein-protein and protein-DNA interactions play a crucial regulatory role in the repair of AP-sites of different kinds. We will test our hypothesis in the following two aims using proposed novel confocal microscopic techniques during the 2-year funding cycle:
Aim 1 : Compare the kinetics of interaction and repair of different AP-site DNA by APE1 in the nucleus of human cells.
This aim will be accomplished by assessing the localization of AP-site DNA constructs and their interactions with APE1 in the nucleus, especially in the context of chromatin. The DNA-protein interactions will be measured by co-localization and then by FRET-FLIM technique for direct binding. We will also measure repair kinetics of both types of AP-sites in intact human cells and then compare repair kinetics with interaction kinetics;
Aim 2 : Determine the effect of pre-treatment of human cells with DNA damaging agents on the interactions of APE1 and other BER proteins with different AP-site DNA in the nucleus of human cells by co-localization and FRET-FLIM (direct interaction) analysis. This portion of the study will elucidate in cellulo repair mechanisms by testing direct interaction of APE1 and other BER proteins with different AP-site DNA in intact human cells at basal level and under alkylating and oxidative stress conditions. APE1 inhibitors are currently in preclinical study for temozolomide-related cancer treatment. Thus, exploring the molecular basis of APE1 catalysis in vitro and in cellulo together may reveal better ways to explore more effective inhibitors for future use in cancer therapeutics and basic BER mechanism studies.

Public Health Relevance

Damage to cellular DNA causes mutations and development of cancer and neurodegeneration. The DNA in human cell undergoes several thousand to million damaging events per day, generated by both environmental pollutants including tobacco smoke and internal metabolic (endogenous) processes. Such DNA adducts are repaired by an endogenous preventive pathway, Base Excision Repair (BER). The goal of this project is to understand the mechanisms of regulation of BER pathway for highly toxic and mutagenic DNA adduct, abasic sites or baseless sites and devise strategies for modulating the expression of BER genes to improve the efficacy of chemopreventives and therapeutics.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Small Research Grants (R03)
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Cancer Etiology Study Section (CE)
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Shaughnessy, Daniel
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Georgetown University
Internal Medicine/Medicine
Schools of Medicine
United States
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