TRANSCRIPTIONAL MUTAGENESIS BY IONIZING RADIATION

DNA损伤的生物学终点主要由其 影响DNA和RNA合成机制。 RNA聚合酶阻滞 在DNA损伤部位或附近被认为是 转录偶联修复 我们最近发现，在体外， 某些类型的电离辐射诱导的DNA损伤可以 被RNA聚合酶有效绕过并产生诱变插入 或在所得转录物中缺失（转录诱变）。 我们认为，这些事件可能对以下方面产生深远影响： DNA修复途径和细胞机制 突变蛋白质的生产。 无论一个人的失败 鉴于DNA损伤对阻断转录延伸具有重要的 生物学后果目前尚不清楚。 主要目标之一 这个提议的目的是检验转录 诱变可发生在活细胞中。 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基区损伤的影响 产物尿嘧啶、二氢尿嘧啶和8-氧代鸟嘌呤， 药剂诱导的碱基损伤产物06甲基鸟嘌呤。 一个重要 选择这些DNA碱基损伤产物的标准是它们还 当被DNA遇到时， 聚合酶具体来说，我们将：（1）将这些定义的基础损伤 DNA模板，并确定这种病变的影响 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 转录本和蛋白质在体内的静止培养的E.杆菌 不同的DNA修复背景;（3）将这些碱基损伤产物 DNA模板，并确定这种损害对 体外哺乳动物转录延伸机制;以及（4）确定 这些碱基损伤产物产生突变转录物能力 和非复制哺乳动物细胞中的体内蛋白质。 体外 研究将提供一个直接的指示， 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，无论此类事件是否 导致在所得转录物中插入诱变碱基。 的 体内研究将解决是否这样的诱变插入 在转录水平上发生的改变可以改变蛋白质的表达， 不分裂的细胞

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.