FOOTPRINTING WITH IRON(II)-GENERATED HYDROXYL RADICAL

为了表达其生物活性，DNA必须与 蛋白。加深对蛋白质与DNA相互作用的理解 将为疾病干预提供新的途径。的长期目标是 这项工作是为了获得关于复杂蛋白质-DNA的新信息 存在于细胞中的复合体。已经开发了新的方法来研究 DNA-蛋白质复合体的结构和能量学。这些方法 依赖于以过氧化氢为基团的铁(H)EDTA的化学 在溶液中产生羟基自由基的便捷方法。这个 羟基自由基通过提取氢原子来切割DNA骨架 从脱氧核糖中分离出来，在DNA中留下单链缺口。这 化学可以用来制作非常高分辨率的 与DNA结合的蛋白质。另一种基于羟基自由基化学的方法 是最近开发的缺失核苷实验，它提供了 关于蛋白质接触的重要能量的直接信息 用它的DNA结合部位。本申请的具体目的 是：(1)将缺失的核苷实验发展为定量 DNA中每个核苷酸相互作用自由能的量度 与其同源蛋白的结合部位。(2)使用羟基自由基 用于表征DNA的足迹和缺失的核苷实验 结合一系列突变的同源结构域蛋白，这些蛋白参与了 指明高等生物体的发育途径。(3)使用化学品 探测实验，包括失踪的核苷实验，以 确定参与的RNA聚合酶-DNA复合体的结构特征 在转录起始和延伸过程中。(4)开发新方法 在生活中制作DNA-蛋白质复合体的高分辨率足迹 细胞，利用伽马辐射在体内产生羟基自由基。这是一项新的 活体足迹方法将被应用于表征组织- 生长激素基因的特异性表达。

For its biological activity to be expressed, DNA must be associated with protein. Deeper understanding of the interactions of proteins with DNA will provide new avenues for disease intervention. The long term goal of this work is to gain new information on the complicated protein-DNA complexes that exist in the cell. New methods have been developed to study the structure and energetics of DNA-protein complexes. These methods depend on the chemistry of iron(H) EDTA with hydrogen peroxide as a convenient means of generating the hydroxyl radical in solution. The hydroxyl radical cleaves the DNA backbone by abstracting a hydrogen atom from a deoxyribose, leaving a single-stranded gap in the DNA. This chemistry can be used to make very high resolution "footprints" of proteins bound to DNA. Another method based on hydroxyl radical chemistry is the recently-developed Missing Nucleoside Experiment, which provides direct information on the energetically important contacted that a protein makes with its DNA binding site. The Specific Aims of this application are: (1) to develop the Missing Nucleoside Experiment into a quantitative measure of the free energy of interaction of each nucleotide in a DNA binding site with its cognate protein. (2) to use hydroxyl radical footprinting and the Missing Nucleoside Experiment to characterize the DNA binding of a series of mutated homeodomain proteins, which are involved in specifying developmental pathways in higher organisms. (3) to use chemical probe experiments, including the Missing Nucleoside Experiment, to determine the structural features of RNA polymerase-DNA complexes engaged in transcription initiation and elongation. (4) to develop a new method for making high resolution footprints of DNA-protein complexes in living cells, using gamma radiation to produce hydroxyl radical in vivo. This new in vivo footprinting methods will be applied to characterizing the tissue- specific expression of the growth hormone gene.