RADIATION DAMAGE IN DNA

这是CA46838《辐射分析》的第二次修订应用 DNA中的损伤。已在 完整的DNA寡聚体。高分辨率高效液相色谱已用于分离受损的 低聚物带有不同的病变。这些病变的识别 是通过使用核磁共振波谱学来完成的，并补充了 通过快原子轰击(FAB)质谱仪。其他损害 会被鉴定出来，特别是在受辐射的四聚体和六聚体中。 初步研究显示，高效液相色谱分析与 辐照单链齐聚物的产物轮廓及其产物 来自相同齐聚物的双链辐照的轮廓。这个 双链形式明显干扰了 特定的损伤。继续研究的一个目标是 确认这些损伤。 辐射修饰的DNA低聚物是一种重要的资源。这个 以特定损伤为底物的DNA寡聚体的行为 可以研究核酸内切酶、激酶和聚合酶。例如，它 已经在这个实验室中证明了许多辐射引起的 病变抑制核酸酶Pl。这一现象导致了一种 检测DNA中耐Pl损伤的敏感方法。这个 已经对所谓的甲酰胺损伤进行了检测。化验结果 将进一步改进和扩展到分析从 暴露的细胞。该检测还将扩展到其他病变，其中许多 这与氧化应激和辐射损伤有关。 另一个目标是将每一个特定的病变与 该病变诱发突变的倾向。一种体外试验是 建议评估损伤对DNA功能的影响 聚合酶。同样，提议的实验的基本资源是 带有特定损伤的低聚物，用作底物 测试聚合酶功能。

This is the second revised application of CA46838 "Analysis of Radiation Damage in DNA". Twenty-five different lesions have been identified in intact DNA oligomers. High resolution HPLC has served to isolate damaged oligomers bearing different lesions. The identification of these lesions has been accomplished through the use of NMR spectroscopy, complemented by fast atom bombardment (FAB) mass spectrometry. Additional lesions would be identified, especially in irradiated tetramers and hexamers. Preliminary studies have revealed stark differences between the HPLC product profile from irradiated single-stranded oligomer and the product profile from the same oligomer irradiated in duplex form. The double-stranded form manifestly interferes with the formation of particular lesions. An objective of the continuing research would be to identify these lesions. Radiation-modified DNA oligomers constitute an important resource. The behavior of a DNA oligomer bearing a specific lesion as substrate to endonucleases, kinases and polymerases can be studied. For example, it has been demonstrated in this laboratory that many radiation-induced lesions inhibit nuclease Pl. This phenomenon led to the design of a sensitive assay for the detection of Pl-resistant lesions in DNA. The assay has been implemented for the so-called formamido lesion. The assay would be further refined and extended to the analysis of DNA taken from exposed cells. The assay would also be extended to other lesions many of which are associated with oxidative stress as well as radiation damage. Another objective is to associate with each specific lesion a measure of the propensity of that lesion to induce mutation. An in vitro assay is proposed to evaluate the effect of the lesion on the -function of DNA polymerase. Again, the basic resource for the proposed experiments are oligomers bearing a specific lesion which are to be used as substrate for testing polymerase function.