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。这些疾病病因学的分子机制可能涉及ROS引起的氧化DNA损伤。因此，了解RIS诱导DNA损伤的性质是非常重要的。这个提议的中心假设指出，氧化性DNA损伤的真实性质在几个重要方面与普遍接受的观点不同。这一论点的基础来自DNA寡聚体中ROS损伤的研究。寡聚体中DNA损伤的分析已经完成，而没有进一步降解受损的寡聚体。这些分析表明，许多主要的ROS诱导的病变是两个相邻的核苷都受损的病变。另一个发现是，如果氧可用于介导损伤过程，则嘧啶碱基分解产物很可能是主要的嘧啶碱基损伤。如果不存在氧作为介体，则6-羟基-5，6-二氢胸腺嘧啶修饰是主要损伤。最近的观察是，链断裂与附近鸟嘌呤损伤的氧化一起产生。我们的假设是，在寡聚体中观察到的这些和其他标志性病变也由细胞中的ROS产生。拟议的研究将在暴露于ROS的培养物中的细胞中检测这些标志性病变。大多数检测将采用32 p-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。将检查两种细胞系中被认为最能代表氧化性DNA损伤性质的病变。将使用三种不同的方法使细胞经受氧化应激。

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.