TRANSCRIPTIONAL MUTAGENESIS BY IONIZING RADIATION

电离辐射转录诱变

基本信息

批准号：
6513303
负责人：
Paul William Doetsch
金额：
$ 29.08万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-07-06 至 2004-04-30
项目状态：
已结题

项目摘要

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由其启动对DNA和RNA合成机制的影响。 RNA聚合酶停滞在DNA损伤部位或附近，被认为是关键事件转录耦合维修。我们最近表明，体外，某些类型的电离辐射诱导的DNA损伤可能是有效地绕过RNA聚合酶并产生诱变插入或结果转录本中的缺失（转录诱变）。我们认为，此类事件对 DNA修复途径以及引导的细胞机制致突变蛋白的生产。是否失败给定DNA对块转录伸长的损害具有重要的生物学后果目前未知。主要目标之一该提议是为了检验转录的假设活细胞可能发生诱变。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诱导的碱基损伤的影响产品尿嘧啶，二氢酸和8-氧气以及烷基化代理诱导的基本损伤产物06methylGuanine。一个重要的选择这些DNA碱损伤产品的标准是 DNA遇到诱变的核苷酸插入聚合酶。具体而言，我们将：（1）放置这些定义的基本伤害产品进入DNA模板并确定此类病变的影响在体外的原核转录伸长机械上；（2）确定这些基本损伤产物产生突变体的能力大肠杆菌固定培养物中的成绩单和蛋白质与不同的DNA修复背景；（3）放置这些基本损坏产品进入DNA模板，并确定此类损害对体外哺乳动物转录伸长机械；（4）确定这些基本损伤产物产生突变转录本的能力在非复制哺乳动物细胞中体内的蛋白质。体外研究将直接表明RNA是否特定类型的电离辐射诱导的DNA的聚合酶旁路发生损害赔偿，如果发生旁路，是否发生此类事件导致诱变基础插入结果中的转录本。这体内研究将解决这种诱变插入是否在转录水平上发生可以改变一个非分明的单元。