FOOTPRINTING WITH IRON (II)-GENERATED HYDROXYL RADICAL

铁（II） EDTA与过氧化氢反应产生羟基自由基，羟基自由基已被证明是一种强大且广泛应用的蛋白质- dna和蛋白质- RNA复合物结构的化学探针。氢氧自由基也由电离辐射与水的相互作用产生，因此是介导辐射对DNA损伤的近似化学物质。该项目的长期目标是利用利用铁（II） EDTA/过氧化氢系统或伽马辐射产生的羟基自由基的化学作用，制作复杂功能蛋白质- 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，以及z - dna结合RNA编辑酶ADAR1。该项目的一个附带目标是利用羟基自由基损伤DNA的结构和性质的新知识，分离和表征最初识别辐射损伤DNA骨干的细胞蛋白质。该项目的具体目标是：(1)利用羟基自由基作为化学探针，剖析酵母铜金属调节因子Amt1、Ace1和Mac1的DNA结合亚域与DNA的相互作用；(2)制备一组三种特异性损伤DNA寡核苷酸，并利用它们分离识别电离辐射诱导的DNA主干氧化损伤的真核蛋白；(3)利用过去资助期缺失核苷对转录RNA聚合酶的实验结果，制备特异性缺口的模板DNA分子，分析模板结构对转录的影响。同时，将制备具有弯曲和直的腺嘌呤束的DNA模板，用于研究DNA整体结构对转录的影响；(4)在RNA编辑酶ADAR1与左旋Z-DNA复合物上进行羟基自由基足迹实验，最终目的是进行化学探针实验，以确定功能性RNA编辑系统的结构。该项目的结果将为细胞如何识别电离辐射引起的基因组损伤提供新的信息。此外，对RNA聚合酶、酵母铜金属调节因子和ADAR1的研究将为一些复杂的蛋白质- dna“机器”提供新的结构见解，这些“机器”是细胞用来解码基因组信息的。

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.