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

• 批准号: 10161792
• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10161792
• 关键词: Address; Biochemical; Biochemistry; Biotechnology; Breathing; Carbon; Cations; Cells; Cellular biology; Chemistry; DNA; DNA Alkylation; DNA Damage; Disease; Enzyme Inhibitor Drugs; Etiology; Fruit; Health; Histones; Human; Hydrogen; Investigation; Ionizing radiation; Lesion; Lysine; Malignant Neoplasms; Minor Groove; Modification; Molecular; Molecular Biology; Nitrogen; Nucleic Acids; Nucleosome Core Particle; Organic Chemistry; Oxides; Pathway interactions; Prevalence; Proteins; Purines; Radiation-Sensitizing Agents; Research; Research Project Grants; Signal Transduction; Site; Structure; Testing; Tube; adjudicate; crosslink; design; experimental study; histone modification; interest; migration; novel; oxidative damage; programs; tool

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.

我们的研究小组解决了关于核酸是如何被破坏的以及什么是 损害的生化后果是。我们还利用了在这些领域所取得的基本发现 研究开发在生物技术中有用的酶抑制剂、辐射增敏剂和工具。至 将这些研究项目付诸实施，我们利用有机化学、生物化学以及分子和细胞 生物学。在二十多年的时间里，这种研究方法使我们能够发现 DNA损伤，裁决机制争议，并揭示损伤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 这项研究计划的各个方面。我们将利用我们独立生成活性中间体的能力来 阐明有关游离和核小体DNA氧化损伤的问题。例如，我们将检查 我们证明了氮自由基的反应性能够通过以下途径启动串联损伤的形成 氢原子抽象化，与大多数碳基不同。串联损伤是一种有害的DNA损伤形式 这是G-射线松解术的标志。一些氮自由基也像变色龙一样，因为它们的pKA是 足够高，以至于在中性pH下存在合理数量的相应自由基阳离子。根部 阳离子是由电离辐射的直接作用和引发空穴转移而产生的重要物种 DNA我们将通过在核小体中独立地产生自由基阳离子来研究核小体DNA中的空穴转移 指定地点的核心粒子(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递中的重要性，一个越来越感兴趣的话题 在蛋白质和DNA之间。了解DNA损伤的影响的努力将集中在NCP中的化学 以及DNA损伤导致的组蛋白修饰的后果。我们将以我们的发现为基础， 烷基化DNA与组蛋白形成DNA-蛋白质交联(DPC)，组蛋白催化化学 氧化的碱基导致赖氨酸残基的修饰。这些研究将从测试中的实验开始。 试管到细胞，以确定在细胞中形成的组蛋白修饰的流行率并鉴定它们的生化 (“下游”)效果。我们还将确定当DNA烷基化时，NCP中是否会形成DPC 在小凹槽里。最后，我们将利用卤化嘌呤来增强DNA烷基化的效果，方法是 稳定形成的民主党全国委员会。这项研究将有助于我们从根本上理解DNA损伤 以及它与疾病的病因和治疗的联系。