Uracil Damage Recognition by Uracil DNA Glycosylase

尿嘧啶 DNA 糖基化酶对尿嘧啶损伤的识别

• 批准号: 8136286
• 负责人: JAMES T. STIVERS
• 金额: $ 37.67万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136286
• 关键词: Antineoplastic Agents; Base Excision Repairs; Base Pairing; Binding; Breathing; Cell Nucleus; Chemicals; Chromatin; DNA; DNA Damage; DNA Repair Enzymes; DNA-Protein Interaction; Diffusion; Enzymes; Escherichia coli; Event; Excision; Funding; Human; Humulus; Immune response; Intervention; Location; Measurement; Mediating; Methods; Molecular; Motion; Mutate; N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase; Natural Immunity; Nature; Nucleosomes; Pathway interactions; Process; Property; Public Health; Relaxation; Role; Site; Slide; Solutions; Stretching; Surface; System; Therapeutic Effect; Time; Uracil; Viral Pathogenesis; Virus; base; cancer therapy; carcinogenesis; fascinate; fluoropyrimidine; functional group; insight; molecular recognition; novel; public health relevance; repaired; scaffold; tool; uracil-DNA glycosylase

DESCRIPTION (provided by applicant): DNA repair enzymes detect damaged DNA bases in the dramatically different contexts of naked duplex DNA as well as nucleosomes and chromatin. These fascinating and essential molecular recognition processes require dynamic motions of the DNA base pairs to expose damaged bases to the enzyme, and also, facilitated diffusion of the enzyme along the DNA chain so that stretches of the DNA chain can be meticulously interrogated in a single bimolecular encounter event. A fundamental understanding of damage recognition in the contexts of naked DNA and nucleosomes requires a paradigm system that is especially amenable to study using a wide variety of biophysical tools. One such system is the uracil base excision repair pathway (UBER). This multienzyme pathway is initiated by the enzyme uracil DNA glycosylase (UNG), which efficiently locates and excises uracil bases from DNA. The biomedical relevance of UBER arises from the central role of this pathway in the adaptive immune response, in providing innate immunity against viruses, and in mediating the therapeutic effects of fluoropyrimidine anticancer drugs. In addition, aberrant UBER has been related to carcinogenesis. The characterization of the molecular nature of these interactions provides the basis for identifying new targets for biomedical intervention in the immune response, cancer therapy and viral pathogenesis. The focus of this proposal is to: (1) Elucidate the mechanism and molecular interactions that allow human UNG to execute intramolecular facilitated transfer between uracil sites in duplex DNA. Using NMR paramagnetic relaxation enhancement (PRE) methods, we will characterize transient and poorly populated binding modes of hUNG to nonspecific and specific DNA that are important for intramolecular site transfer. Based on unique structural insights, we will mutate specific residues on UNG, and make discrete perturbations of functional groups on the DNA, to uncover enzyme-DNA interactions important for intramolecular transfer. (2) Elucidate the role of nucleosomal DNA dynamics in uracil excision by hUNG. We will use newly conceived NMR and biophysical methods to explore the dynamic properties of DNA in mononucleosomes for the first time, and we will elucidate the mechanism by which UNG locates and repairs uracil sites imbedded in nucleosomal DNA. (3) Determine the mechanism and efficiency of intramolecular facilitated transfer by hUNG between uracil sites embedded in nucleosomes. The significance and mechanism of intramolecular site transfer in the context of mononucleosomes will be elucidated using novel biophysical and chemical approaches. In particular, we will discern whether the nucleosome surface provides a pathway for intramolecular transfer, whether the DNA scaffold is the primary conduit, or if site location occurs by diffusion from bulk solution. PUBLIC HEALTH RELEVANCE: The biomedical relevance of uracil base excision repair (UBER) arises from the central role of this pathway in the adaptive immune response, in providing innate immunity against viruses, and in mediating the therapeutic effects of fluoropyrimidine anticancer drugs. In addition, aberrant UBER has been related to carcinogenesis. The characterization of the molecular interactions in this pathway provides the basis for identifying new targets for biomedical intervention in the immune response, cancer therapy and viral pathogenesis.

描述（由申请人提供）：DNA修复酶在裸体双链DNA以及核小体和染色质的急剧不同的情况下检测到受损的DNA碱基。这些引人入胜且必需的分子识别过程需要DNA碱基对的动态运动，以将受损的碱暴露于酶中，并且还促进了酶沿着DNA链的扩散，以便在单个双肌肉encounter事件中精心审查DNA链的拉伸。在裸体DNA和核小体背景下对损害识别的基本理解需要一个范式系统，该系统尤其可以使用多种生物物理工具进行研究。这样的系统是乌拉质基碱切除修复途径（Uber）。该多酶途径是由尿嘧啶DNA糖基酶（UNG）启动的，该酶有效地定位并从DNA中摘除了尿嘧啶碱基。 Uber的生物医学相关性来自该途径在适应性免疫反应中的核心作用，在提供对病毒的先天免疫以及介导氟吡啶抗癌药物的治疗作用方面。此外，异常Uber与癌变有关。这些相互作用的分子性质的表征为鉴定在免疫反应，癌症治疗和病毒发病机理中生物医学干预的新靶标提供了基础。该提案的重点是：（1）阐明允许人UNG执行双方DNA中尿嘧啶位点之间的机制和分子相互作用。使用NMR顺磁性松弛增强（PRE）方法，我们将表征悬挂到非特异性和特异性DNA的瞬时和人口不足的结合模式，这对于分子内部位转移很重要。基于独特的结构见解，我们将在UNG上突变特定的残基，并使DNA上官能团的离散扰动，以发现对分子内转移至关重要的酶-DNA相互作用。 （2）阐明了悬挂的核小体DNA动力学在尿素切除中的作用。我们将首次使用新构想的NMR和生物物理方法来探索单核小体中DNA的动态特性，我们将阐明UNG在核小体DNA中嵌入和修复尿中心位点的机制。 （3）确定分子内促进在嵌入核小体中的乌拉西尔部位之间转移的机理和效率。在单核小体背景下，分子内部位转移的显着性和机制将使用新型的生物物理和化学方法阐明。特别是，我们将识别核小体表面是否提供了分子内转移的途径，DNA支架是主要导管，还是通过从大量溶液扩散而发生位置位置。 公共卫生相关性：尿嘧啶碱基切除修复（UBER）的生物医学相关性是由于该途径在适应性免疫反应中的核心作用，在提供对病毒的先天免疫以及介导氟吡咪定抗癌药物的治疗作用方面引起的。此外，异常Uber与癌变有关。在该途径中的分子相互作用的表征为鉴定在免疫反应，癌症治疗和病毒发病机理中生物医学干预的新靶标提供了基础。