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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生物化学的后果。我们还利用了这些领域的基本发现，研究创造酶抑制剂、放射增敏剂和生物技术中有用的工具。到为了实现这些研究项目，我们利用有机化学，生物化学以及分子和细胞生物学在过去的二十多年里，这种研究方法使我们能够发现新的途径， DNA损伤，裁定机制争议，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结局。我们请求支持继续所有3个这个研究项目的各个方面。我们将利用我们独立产生活性中间体的能力，阐明有关游离和核小体DNA氧化损伤的问题。例如，我们将检查氮自由基的反应性，我们证明了能够启动串联损伤形成，氢原子抽象，不像大多数碳原子团。串联损伤是DNA损伤的一种有害形式这是G-辐解的标志。一些氮自由基也是变色龙样的，因为它们的pKa是足够高，使得在中性pH下存在合理量的相应自由基阳离子。阳离子是由电离辐射的直接效应产生的重要物质， DNA.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中的空穴转移核心颗粒（NCP）在定义的网站。这将使我们能够确定NCP结构对空穴的影响迁移，由于认识到空穴传输在信令中的重要性，蛋白质和DNA之间的联系理解DNA损伤影响的努力将集中在NCP的化学上以及DNA损伤诱导的组蛋白修饰的后果。我们将以我们的发现为基础，烷基化的DNA与组蛋白形成DNA-蛋白质交联（DPC），并且组蛋白催化的氧化的脱碱基位点导致赖氨酸残基的修饰。这些研究将包括测试中的实验以确定细胞中形成的组蛋白修饰的普遍性，并鉴定其生物化学性质。（“下游”）影响。我们还将确定当DNA被烷基化时，DPC的形成是否发生在NCP中在小沟里最后，我们将利用卤代嘌呤来增强DNA烷基化的作用，稳定形成的DPC。这项研究将有助于我们对DNA损伤的基本理解以及它与疾病的病因学和治疗的联系。