BIOLOGICAL CONSEQUENCES OF SITE-SPECIFIC DAMAGE TO DNA

DNA 特定位点损伤的生物学后果

• 批准号: 3180836
• 负责人: Louis J Romano
• 金额: $ 13.89万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-05-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3180836
• 关键词: DNA repair; DNA replication; Escherichia coli; acetylaminofluorene; adduct; benzopyrenes; chemical carcinogen; chemical structure function; circular dichroism; conformation; molecular cloning; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; oligonucleotides; restriction mapping; site directed mutagenesis

The objective of this proposal is to continue the development of a general method for the preparation of oligodeoxyribonucleotides containing chemically well-defined damage at unique and specific locations, to use these molecules to determine how specific adducts affect the three-dimensional structure of a DNA duplex, and to attempt to relate these structural changes to the induced mutations. This proposal is focussed on three important, and well- studied carcinogenic DNA adducts-aminofluorene, acetyl aminofluorene, and benzol(a)pyrene-because of the chemical stability of these particular structures. However our long-term objective is to develop the techniques to place any DNA lesion into a specific DNA sequence, including those where the chemistry needed to prepare the lesion is not now known or where the instability of the lesion to currently available oligonucleotide synthesis technology is prohibitive. Two approaches are currently being used to prepare the site-specifically modified oligonucleotides: nucleotide-specific reactions of derivatives of these carcinogens with pre-formed oligonucleotides, and the total chemical synthesis of oligonucleotides using adduct-containing nucleotide precursors. We will use these modified oligonucleotides and a variety of spectral and enzymatic techniques to determine the effect these adducts have on three-dimensional structure of a DNA duplex. Utilizing the techniques we have developed during the past project period, we will position several modified oligonucleotides into gapped heteroduplexes to form site-specifically modified, biologically active DNA molecules. After extensively characterizing these products, they will be introduced into E. coli cells and the induced mutations determined by DNA sequencing of the progeny.

该提议的目的是继续发展 一种制备寡脱氧核糖核苷酸的一般方法 在独特和特定的 位置，使用这些分子来确定特定加合物如何 影响DNA双链体的三维结构，然后 尝试将这些结构性变化与诱导的 突变。 该提议的重点是三个重要，良好的 研究的致癌DNA加合物 - 氨基氟，乙酰基 氨基氟和苯二（A）苯甲酸苯甲酸苯甲酸苯甲酸酯，因为该化学物质 这些特定结构的稳定性。 但是我们的长期 目的是开发将任何DNA病变置于 特定的DNA序列，包括需要化学的DNA序列 现在不知道病变或不稳定的地方 当前可用的寡核苷酸合成病变 技术是过敏的。 目前正在使用两种方法 准备特定于特定修饰的寡核苷酸： 这些致癌物的衍生物的核苷酸特异性反应 与预成型的寡核苷酸和总化学合成 使用含有加合物的核苷酸前体的寡核苷酸。 我们将使用这些修饰的寡核苷酸和各种 光谱和酶促技术来确定这些作用 加合物具有DNA双链体的三维结构。 利用我们在过去项目中开发的技术 时期，我们将将几个修改的寡核苷酸定位在 间隙杂波形成特定于位点的修改， 生物活性DNA分子。 广泛之后 这些产品表征，它们将被引入大肠杆菌 细胞和通过DNA测序确定的诱导突变 后代。