BIOLOGICAL CONSEQUENCES OF SITE-SPECIFIC DAMAGE TO DNA

DNA 特定位点损伤的生物学后果

• 批准号: 3180834
• 负责人: Louis J Romano
• 金额: $ 9.19万
• 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 1989-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3180834
• 关键词: Escherichia coli; acetylaminofluorene; benzopyrenes; covalent bond; enzyme mechanism; guanine; high performance liquid chromatography; nucleic acid hybridization; protein sequence; radiotracer

It is well accepted that most mutagens exert their biological effect by covalently binding to and altering the genomic DNA. However, there are a multitude of difficulties associated with specifying the chemical nature of the mutgenic DNA alteration, both because of the variety of DNA reaction products induced by many mutagens and because of the rarity of most mutational response. The goal of this proposal is to overcome these intrinsic problems by preparing DNA molecules containing chemically defined, biologically relevant damage at a specific location, to subject these templates to in vivo and in vitro replication, and to characterize the products both genetically and chemically. Using DNA from the well-defined M13 mp8/mp9 system, we will first prepare vectors capable of hybridizing to and ligating with site-specifically modified oligonucleotides. Next, we will extend our current studies on preparing oligonucleotides containing site-specific aminofluorenes, to include other adducts and other methods of preparation. Two approachs will be employed: chemical synthesis of oligonucleotides by the phosphotriester method using a specificlly modified nucleotide as one of the precursors, and nucleotide-specific reactions of various mutagens with preformed oligonucleotides. The types of DNA damage we will initially focus on include O6-ethylguanine, aminofluorene, and acetylaminofluorene benzo[a]pyrene. Site-specifically damaged M13DNA molecules will be transfected into appropriate strains of E. coli, representative mutants selected, and the relevant DNA sequences determined. Using this method we will be able to precisely correlate the chemical nature of a DNA lesion with a particular mutational event. The DNA molecules containing site-specific adducts will also serve as templates for DNA synthesis by several available DNA polymerases and in our well-characterized in vitro T7 DNA replication system. The availability of these templates will allow us to determine the precise effect these adducts have on both DNA synthesis by purified DNA polymerases alone and on the DNA replication process by a biologically relevant in vitro system.

人们普遍认为，大多数诱变剂通过以下方式发挥其生物效应： 共价结合并改变基因组DNA。 然而，有一个 与指定化学性质相关的许多困难 DNA 突变的改变，都是由于 DNA 反应的多样性 由许多诱变剂诱导的产品，并且由于大多数诱变剂的稀有性 突变反应。 本提案的目标是克服这些问题 制备含有化学物质的 DNA 分子的内在问题 在特定位置定义的、与生物相关的损害 这些模板可用于体内和体外复制，并表征 基因和化学产品。 使用来自 定义明确的 M13 mp8/mp9 系统，我们首先准备能够 与位点特异性修饰杂交并连接 寡核苷酸。 接下来，我们将扩展目前的研究准备 含有位点特异性氨基芴的寡核苷酸，包括其他 加合物和其他制备方法。 将采用两种方法： 通过磷酸三酯法化学合成寡核苷酸 作为前体之一的经过特定修饰的核苷酸，以及 各种诱变剂与预先形成的核苷酸特异性反应 寡核苷酸。 我们首先关注的 DNA 损伤类型 包括O6-乙基鸟嘌呤、氨基芴和乙酰氨基芴 苯并[a]芘。 位点特异性受损的 M13DNA 分子将被 转染到合适的大肠杆菌菌株中，代表性突变体 选择并确定相关的DNA序列。 使用这种方法我们 将能够精确关联 DNA 损伤的化学性质 与特定的突变事件。 DNA分子含有 位点特异性加合物也将作为 DNA 合成的模板 几种可用的 DNA 聚合酶和我们充分表征的体外 T7 DNA复制系统。 这些模板的可用性将使我们能够 确定这些加合物对 DNA 合成的精确影响 单独纯化 DNA 聚合酶并通过 DNA 复制过程 生物学相关的体外系统。