Structure and Mechanism of Base-Excision DNA Repair

碱基切除DNA修复的结构和机制

• 批准号: 6712756
• 负责人: GREGORY Lawrence VERDINE
• 金额: $ 38.68万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6712756
• 关键词: DNA damage; DNA repair; N glycosidase; X ray crystallography; Xenopus oocyte; active sites; carcinogenesis; enzyme mechanism; fluorescent dye /probe; mutagens; protein structure function; stereochemistry; time resolved data

DESCRIPTION (provided by applicant): Spontaneous damage to the four bases of DNA is a major cause of the mutations that give rise to cancer. Most of these genetic lesions are corrected by a pathway known as base-excision DNA repair (BER). The key components of BER are DNA glycosylases, professional lesion-hunting enzymes that scan the genome in search of particular kinds of base damage, then catalyze excision of the damaged base from the DNA backbone. The long-term goals of our studies are to understand how these enzymes locate damaged bases amidst the vast excess of normal DNA, and the precise reaction pathways that they utilize. A comprehensive, fundamental understanding of DNA damage recognition and removal represents the solution to a major aspect of the tumorigenesis puzzle. In the proposed studies, we will focus on the cellular resistance to the genotoxic effects of oxidative stress. Specifically, we will study base-excision repair of the highly mutagenic lesion 8-oxoguanine (oxoG) by two DNA glycosylases, human Ogg1 protein and bacterial MutM. Here we outline a broad-based, interdisciplinary approach that employs chemical synthesis of substrate analogs, semisynthetic site-specific modification of the proteins, high-resolution structural analysis, single-molecule fluorescence spectroscopy, and in vitro biochemistry to elucidate significant unresolved issues in the structure and function of these proteins. In particular, we aim to develop a structural picture of the multistep lesion-processing reaction cascade catalyzed by Ogg1 and MutM, by stalling the reaction at various points along the way to capture static structures, and by using time-resolved X-ray to observe the base-excision reaction in real time. We propose furthermore to determine whether the base-excision process as studied in vitro is a reasonably faithful representation of repair in vivo, by using Xenopus oocyte extracts as a model for the latter. Finally, we propose to extend our understanding of structure/function in BER to include the MutY protein, which functions in eukaryotes and prokaryotes to correct the mutagenic damage resulting from misreplication of 8-oxoguanine.

描述（由申请人提供）：对DNA的四个基础的自发损害是导致癌症突变的主要原因。这些遗传病变中的大多数通过称为碱基脱位DNA修复（BER）的途径校正。 BER的关键成分是DNA糖基酶，专业的狩猎酶，它们扫描基因组以寻找特定种类的碱基损伤，然后从DNA骨架中催化损坏的碱基的切除。我们研究的长期目标是了解这些酶如何在大量过量的正常DNA以及它们使用的确切反应途径中找到受损的碱。对DNA损伤识别和去除的全面，基本的理解代表了肿瘤发生难题的主要方面的解决方案。 在拟议的研究中，我们将重点关注对氧化应激的遗传毒性作用的细胞抗性。具体而言，我们将通过两个DNA糖基化酶，人OGG1蛋白和细菌MUTM研究高度诱变病变8-氧气的基础脱离修复。在这里，我们概述了一种基于基础的跨学科方法，该方法采用了底物类似物的化学合成，蛋白质的半合成站点特异性修饰，高分辨率结构分析，单分子荧光镜头和体外生物化学，以阐明这些蛋白质和这些蛋白质的结构和功能中的显着单分析。特别是，我们旨在通过将反应在捕获静态结构的途中陷入静态的各个点，并使用时间分辨的X射线来实时观察基础效果反应，从而开发由OGG1和MUTM催化的多步骤病变反应级联反应的结构图。我们建议通过使用Xenopus oocyte提取物作为后者的模型来确定体外研究的基础拆卸过程是否是体内修复的合理忠实表示。最后，我们建议将对BER中的结构/功能的理解扩展到包括Muty蛋白的理解，该蛋白质在真核生物和原核生物中起作用，以纠正由于8-氧气的修复而造成的诱变损害。