FOOTPRINTING WITH IRON (II)-GENERATED HYDROXYL RADICAL

EDTA铁（II）与过氧化氢的反应产生羟基自由基，其已被证明是蛋白质-DNA和蛋白质- RNA复合物结构的强大且广泛使用的化学探针。 羟基自由基也是由电离辐射与水的相互作用产生的，因此是介导DNA辐射损伤的最接近的化学物质。该项目的长期目标是利用EDTA铁/过氧化氢系统或伽马辐照产生的羟基自由基的化学性质，制作功能复杂的蛋白质-DNA复合物的高分辨率化学“图像”。 待研究的系统包括活性转录RNA聚合酶、一组来自酵母的铜金属调节因子和Z-DNA结合RNA编辑酶ADAR 1。 该项目的一个附带目标是利用关于被羟基损伤的DNA的结构和特性的新知识，分离和鉴定最初识别DNA骨架辐射损伤的细胞蛋白质。 本课题的具体目标是：（1）以羟基自由基为探针，研究酵母铜金属调节因子Amt 1、Ace 1和Mac 1的DNA结合亚结构域与DNA的相互作用;（2）拟备一套三款的特别文件─损伤的DNA寡核苷酸，并使用它们来分离真核生物蛋白质，所述真核生物蛋白质识别由电离诱导的对DNA骨架的氧化损伤。辐射;（3）利用在过去资助期内进行的RNA聚合酶转录缺失核苷实验的结果，制备特异性缺口的模板DNA分子，以分析模板结构对转录的影响。 此外，还将制备具有弯曲和直腺嘌呤束的DNA模板，用于研究整体DNA结构对转录的影响;（4）对RNA编辑酶ADAR 1与左手Z-DNA的复合物进行羟基自由基足迹实验，最终目标是进行化学探针实验以确定功能RNA编辑系统的结构。 该项目的结果将提供关于细胞如何识别电离辐射对基因组造成的损害的新信息。 同样，对RNA聚合酶、酵母铜金属调节因子和ADAR 1的研究将为细胞用来解码基因组信息的一些复杂的蛋白质-DNA“机器”提供新的结构见解。

The reaction of iron(II) EDTA with hydrogen peroxide produces the hydroxyl radical, which has proven to be a powerful and widely- used chemical probe of the structure of protein-DNA and protein- RNA complexes. The hydroxyl radical also is produced by the interaction of ionizing radiation with water, and as such is the proximate chemical species that mediates radiation damage to DNA. The long-term goal of this project is to make use of the chemistry of the hydroxyl radical, produced either using the iron(II) EDTA/hydrogen peroxide system or by gamma irradiation, to make high-resolution chemical "images" of complicated functioning protein-DNA complexes. Systems to be studied include actively-transcribing RNA polymerase, a set of copper metalloregulatory factors from yeast, and the Z-DNA-binding, RNA -editing enzyme ADAR1. A collateral goal of the project is to use new knowledge on the structure and properties of DNA damaged by the hydroxyl radical to isolate and characterize cellular proteins that initially recognize radiation damage to the DNA backbone. The Specific Aims of the project are: (1) to use the hydroxyl radical as a chemical probe to dissect the interactions that the DNA-binding subdomains of the yeast copper metalloregulatory factors Amt1, Ace1, and Mac1 make with DNA; (2) to prepare a set of three specifically-lesioned DNA oligonucleotides and use them to isolate eukaryotic proteins which recognize oxidative damage to the DNA backbone that is induced by ionizing radiation; (3) to use the results of missing nucleoside experiments on transcribing RNA polymerase performed in the past grant period to prepare specifically-gapped template DNA molecules for analysis of the effect of template structure on transcription. As well, DNA templates with bent and straight adenine tracts will be prepared for studies on the effect of global DNA structure on transcription; (4) to perform hydroxyl radical footprinting experiments on the complex of the RNA editing enzyme ADAR1 with left-handed Z-DNA, with the eventual goal of performing chemical probe experiments to define the structure of a functioning RNA editing system. The results of this project will provide new information on how cells recognize damage to the genome cause by ionizing radiation. As well, the studies to be undertaken on RNA polymerase, the yeast copper metalloregulatory factors, and ADAR1 will give new structural insight into some of the complicated protein-DNA "machines" which the cell uses to decode the information in the genome.