BIOLOGICAL CONSEQUENCES OF SITE-SPECIFIC DAMAGE TO DNA

DNA 特定位点损伤的生物学后果

• 批准号: 3180829
• 负责人: Louis J Romano
• 金额: $ 10.22万
• 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 1992-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3180829
• 关键词: DNA repair; 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.

该提案的目的是继续开发 制备寡脱氧核糖核苷酸的通用方法 含有独特且特定的化学明确损伤 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，并 尝试将这些结构变化与诱导的 突变。 该提案集中于三个重要且良好的方面： 研究了致癌DNA加合物——氨基芴、乙酰基 氨基芴和苯并芘 - 由于化学物质 这些特殊结构的稳定性。 然而我们的长期 目标是开发将任何 DNA 损伤置于 特定的 DNA 序列，包括需要化学反应的序列 现在不知道准备病变或不稳定的地方 目前可用的寡核苷酸合成的损害 技术是令人望而却步的。 目前正在使用两种方法 制备位点特异性修饰的寡核苷酸： 这些致癌物衍生物的核苷酸特异性反应 与预先形成的寡核苷酸，以及全化学合成 使用含有加合物的核苷酸前体制备寡核苷酸。 我们将使用这些修饰的寡核苷酸和各种 光谱和酶技术来确定这些效果 加合物具有DNA双链体的三维结构。 利用我们在过去的项目中开发的技术 期间，我们将几个修饰的寡核苷酸定位到 有缺口的异源双链体形成位点特异性修饰， 具有生物活性的DNA分子。 经过广泛 表征这些产品后，将它们引入大肠杆菌中 细胞和通过 DNA 测序确定的诱导突变 后代。