TRANSCRIPTIONAL MUTAGENESIS BY IONIZING RADIATION

电离辐射转录诱变

• 批准号: 6376884
• 负责人: Paul William Doetsch
• 金额: $ 28.24万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-06 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6376884
• 关键词: DNA damage; DNA directed RNA polymerase; DNA repair; DNA replication; Escherichia coli; RNA biosynthesis; alkylating agents; gene expression; gene mutation; genetic transcription; guanine analog; ionizing radiation; mutant; nucleic acid sequence; oncoprotein p21; radiation genetics; site directed mutagenesis; tissue /cell culture; tumor suppressor genes; uracil

The biological endpoints of DNA damage are initiated primarily by its effects on the DNA and RNA synthesis machinery. RNA polymerase arrest at or near a DNA damage site is thought to be a key event in transcription-coupled repair. We have recently shown that in vitro, certain types of ionizing radiation-induced DNA damages can be efficiently bypassed by RNA polymerases and produce mutagenic insertions or deletions in the resulting transcript (transcription mutagenesis). We believe that such events have potentially profound implications for both DNA repair pathways as well as the cellular mechanisms that lead to the production of mutant proteins. Whether or not the failure of a given DNA damage to block transcription elongation has important biological consequences is presently unknown. One of the major goals of this proposal is to test the hypothesis that transcription mutagenesis can occur in living cells. For these studies we will utilize both in vitro and in vivo transcription systems. We will determine the effects of the ionizing radiation-induced base damage products uracil, dihydrouracil, and 8-oxoguanine and the alkylating agent-induced base damage product 06methylguanine. An important criterion for selecting these DNA base damage products is that they also cause mutagenic nucleotide insertions when encountered by DNA polymerases. Specifically we will: (1) place these defined base damage products into DNA templates and determine the effects of such lesions on the prokaryotic transcription elongation machinery in vitro; (2) determine the ability of these base damage products to generate mutant transcripts and proteins in vivo in stationary cultures of E. coli with different DNA repair backgrounds; (3) place these base damage products into DNA templates and determine the effects of such damages on the mammalian transcription elongation machinery in vitro; and (4) determine the ability of these base damage products to generate mutant transcripts and proteins in vivo in non- replicating mammalian cells. The in vitro studies will provide a direct indication of whether or not RNA polymerase bypass of specific types of ionizing radiation-induced DNA damages occurs and, if bypass occurs, whether or not such an event results in mutagenic base insertions in the resulting transcript. The in vivo studies will address whether or not such mutagenic insertions occurring at the level of transcription can alter protein expression in a non-dividing cell.

DNA 损伤的生物学终点主要是由其引发的 对 DNA 和 RNA 合成机制的影响。 RNA聚合酶阻滞 在 DNA 损伤位点或附近被认为是一个关键事件 转录耦合修复。 我们最近证明，在体外， 某些类型的电离辐射引起的 DNA 损伤可以 有效地被 RNA 聚合酶绕过并产生诱变插入 或所得转录本中的缺失（转录突变）。 我们相信此类事件对以下领域具有潜在的深远影响： DNA 修复途径以及导致 到突变蛋白的产生。 无论是否失败 鉴于 DNA 损伤阻止转录延伸具有重要意义 生物学后果目前未知。 主要目标之一 该提案的目的是检验转录的假设 突变可以发生在活细胞中。 对于这些研究，我们将 利用体外和体内转录系统。 我们将 确定电离辐射引起的碱基损伤的影响 产品尿嘧啶、二氢尿嘧啶和8-氧代鸟嘌呤及其烷基化 药剂诱导的碱基损伤产物 06 甲基鸟嘌呤。 一个重要的 选择这些 DNA 碱基损伤产物的标准是它们还 当遇到 DNA 时会引起突变核苷酸插入 聚合酶。具体来说，我们将：（1）放置这些定义的基础伤害 将产物转化为 DNA 模板并确定此类损伤的影响 体外原核转录延伸机制的研究； (2) 确定这些碱基损伤产物产生突变体的能力 大肠杆菌静态培养物中的体内转录物和蛋白质 不同的DNA修复背景； (3)放置这些基础伤害产品 进入 DNA 模板并确定这种损伤对 DNA 模板的影响 哺乳动物体外转录延伸机制； (4) 确定 这些碱基损伤产物产生突变转录本的能力 和非复制哺乳动物细胞中的体内蛋白质。 体外 研究将直接表明 RNA 是否 特定类型电离辐射诱导 DNA 的聚合酶旁路 发生损坏，如果发生旁路，无论是否发生此类事件 导致在所得转录物中插入诱变碱基。 这 体内研究将解决这种诱变插入是否 发生在转录水平可以改变蛋白质表达 一个不分裂的细胞。