THE TRUE NATURE OF OXIDATIVE DNA DAMAGE

活性氧（ROS）和氧化应激与癌症、衰老和几种神经退行性疾病有关。这些疾病病因的分子机制可能与活性氧引起的DNA氧化损伤有关。因此，了解ris诱导的DNA损伤的性质是很重要的。该提案的中心假设指出，氧化性DNA损伤的真实性质在几个重要方面不同于普遍接受的观点。这一论点的基础来自对DNA低聚物中活性氧损伤的研究。低聚物DNA损伤的分析已经完成，而没有进一步降解受损的低聚物。这些分析表明，许多主要的ros诱导病变是两个相邻核苷均受损的病变。另一个发现是，如果有氧气介导损伤过程，那么嘧啶基分解产物很可能是主要的嘧啶基损伤。如果氧不作为介质存在，6-羟基-5,6-二氢胸腺嘧啶修饰是主要损害。最近的一项观察发现，链断裂是与附近鸟嘌呤损伤的氧化一起产生的。我们的假设是，在低聚物中观察到的这些和其他标志性病变也是由细胞中的ROS产生的。拟议的研究将在暴露于ROS培养的细胞中检测这些标志性病变。大多数检测将采用32p后标记进行检测，并通过使用本实验室合成的载体进行明确。将对两种细胞系进行检查，以寻找最能代表氧化性DNA损伤性质的病变。将使用三种不同的方法使细胞受到氧化应激。

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.