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How Damaged DNA Forms, and its Subsequent Chemistry: Fundamental Studies and Applications

受损 DNA 是如何形成的及其后续化学：基础研究和应用

基本信息

批准号：
10413873
负责人：
MARC M GREENBERG
金额：
$ 65.68万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2025-05-31
项目状态：
未结题

项目摘要

Our research group addresses fundamental questions concerning how nucleic acids are damaged and what the biochemical consequences of damage are. We also capitalize on the fundamental discoveries made in these investigations to create enzyme inhibitors, radiosensitizing agents, and tools that are useful in biotechnology. To bring these research projects to fruition, we utilize organic chemistry, biochemistry, as well as molecular and cell biology. Over more than two decades, this research approach has enabled us to uncover novel pathways of DNA damage, adjudicate mechanistic controversies, and reveal biochemical effects of damaged DNA that illustrate that nucleic acid damage itself is not always the end of the story. We request support to continue all 3 aspects of this research program. We will utilize our ability to independently generate reactive intermediates to elucidate questions concerning oxidative damage in free and nucleosomal DNA. For instance, we will examine the reactivity of nitrogen radicals, which we demonstrated are capable of initiating tandem lesion formation via hydrogen atom abstraction, unlike most carbon radicals. Tandem lesions are a deleterious form of DNA damage that are a hallmark of g-radiolysis. Some of the nitrogen radicals are also chameleon-like in that their pKa's are sufficiently high that reasonable quantities of the respective radical cations are present at neutral pH. Radical cations are important species produced from the direct effect of ionizing radiation and initiate hole transfer in DNA. We will study hole transfer in nucleosomal DNA by independently generating radical cations in nucleosome core particles (NCPs) at defined sites. This will enable us to determine the effects of NCP structure on hole migration, a topic that is of increasing interest due to the realization that hole transfer is important in signaling between proteins and DNA. Efforts on understanding the effects of DNA damage will focus on chemistry in NCPs and the consequences of DNA damage-induced histone modification. We will build upon our discoveries that alkylated DNA forms DNA-protein cross-links (DPCs) with histones and that histone catalyzed chemistry of oxidized abasic sites results in modification of lysine residues. These studies will range from experiments in test tubes to cells to determine the prevalence of histone modifications formed in cells and to identify their biochemical ("downstream") effects. We will also determine whether DPC formation occurs in NCPs when DNA is alkylated in the minor groove. Finally, we will utilize halogenated purines to potentiate the effects of DNA alkylation by stabilizing the DPCs formed. This research will contribute to our fundamental understanding of DNA damage and its connection to the etiology and treatment of disease.

我们的研究小组致力于解决有关核酸如何被破坏以及核酸被破坏的基本问题。损伤的生化后果是。我们还利用这些领域的基本发现研究创造酶抑制剂、放射增敏剂和可用于生物技术的工具。到为了使这些研究项目取得成果，我们利用有机化学、生物化学以及分子和细胞生物学。二十多年来，这种研究方法使我们能够发现新的途径 DNA 损伤、裁决机制争议并揭示受损 DNA 的生化效应说明核酸损伤本身并不总是故事的结局。我们请求支持以继续所有 3 个任务该研究计划的各个方面。我们将利用我们独立生成反应中间体的能力阐明有关游离 DNA 和核小体 DNA 氧化损伤的问题。例如，我们将检查氮自由基的反应性，我们证明氮自由基能够通过以下方式引发串联损伤形成：与大多数碳自由基不同，氢原子抽象。串联损伤是 DNA 损伤的一种有害形式这是 g 放射分解的标志。一些氮自由基也像变色龙一样，因为它们的 pKa 是足够高，使得在中性pH下存在合理数量的相应自由基阳离子。激进的阳离子是电离辐射直接作用产生的重要物种，并引发空穴传输脱氧核糖核酸。我们将通过在核小体中独立产生自由基阳离子来研究核小体 DNA 中的空穴转移位于指定位置的核心颗粒（NCP）。这将使我们能够确定 NCP 结构对孔的影响迁移，由于认识到空穴转移在信号传导中的重要作用，这个话题越来越受到人们的关注介于蛋白质和DNA之间。了解 DNA 损伤影响的努力将集中在 NCP 中的化学上以及 DNA 损伤引起的组蛋白修饰的后果。我们将基于我们的发现烷基化 DNA 与组蛋白形成 DNA-蛋白质交联 (DPC)，并且组蛋白催化化学反应氧化的无碱基位点导致赖氨酸残基的修饰。这些研究范围包括测试实验管到细胞以确定细胞中形成的组蛋白修饰的普遍性并鉴定其生化（“下游”）效应。我们还将确定当 DNA 烷基化时 NCP 中是否会形成 DPC 在小凹槽中。最后，我们将利用卤化嘌呤来增强 DNA 烷基化的效果稳定形成的 DPC。这项研究将有助于我们对 DNA 损伤的基本理解及其与疾病的病因学和治疗的联系。