THE TRUE NATURE OF OXIDATIVE DNA DAMAGE

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Reactive oxygen species (ROS) and oxidative stress have been implicated in cancer, aging and in several neurodegenerative diseases. The molecular mechanism underlying these disease etiologies likely involves oxidative DNA damaged engendered by ROS. Consequently, it is important to know the nature if RIS-induced DNA damage. The central hypothesis of this proposal states that the true nature of oxidative DNA damage differs in several important aspects from generally accepted views. The basis for this contention comes from studies of ROS damage in DNA oligomers. The analyses of DNA damage in oligomers has been accomplished without further degradation of the damaged oligomers. These analyses indicate that many of the main ROS-induced lesions are lesions in which two adjacent nucleosides are both damaged. Another finding is that pyrimidine base breakdown products are by all odds the main pyrimidine base damage if oxygen is available to mediate the damage process. If oxygen is not present as mediator, the 6-hydroxyl-5,6-dihydrothymine modification is a principal lesion. A recent observation is that strand breaks are produced in conjunction with oxidation of a nearby guanine lesion. Our hypothesis is that these and other hallmark lesions observed in oligomers are produced by ROS in cells as well. The proposed research will assay for these hallmark lesions in cells exposed in culture to ROS. Most assays will employ 32p-post-labeling for detection and will be made unequivocal through the use of carriers synthesized in this laboratory. Two cell lines will be examined for the lesions deemed most representative of the nature of oxidative DNA damage. Three different methods for subjecting cells to oxidative stress will be utilized.