DNA-Protein Dynamics in Base Excision DNA Repair

碱基切除 DNA 修复中的 DNA-蛋白质动力学

• 批准号: 8734457
• 负责人: Patrick J O'Brien
• 金额: $ 28.53万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-26 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734457
• 关键词: 3-methyladenine-DNA glycosylase; Address; Affect; Aging; Aging-Related Process; Alkylating Agents; Amaze; Amino Acids; Area; Base Excision Repairs; Binding; Biochemical; Biochemistry; Biological Assay; Biological Process; Carcinogens; Cell Death; Complex; DNA; DNA Damage; DNA Repair; DNA Structure; DNA glycosylase; DNA lesion; Data; Defect; Detection; Diffusion; Discrimination; Disease; Environmental Exposure; Enzyme Kinetics; Enzymes; Eukaryotic Cell; Excision; Exposure to; Failure; Fluorescence Spectroscopy; Funding; Genes; Genetic; Genetic Polymorphism; Genome; Genome Scan; Genomic DNA; Genomic Instability; Genomics; Goals; Human; In Vitro; Kinetics; Knowledge; Length; Lesion; Link; Microscopic; Mission; Molecular; Molecular Models; Motion; Mutagenesis; Mutation; National Institute of General Medical Sciences; Normal Cell; OGG1 gene; Outcome; Pathway interactions; Physiological; Positioning Attribute; Probability; Progress Reports; Protein Dynamics; Proteins; Public Health; Reaction; Relative (related person); Research; Resistance; Site-Directed Mutagenesis; Slide; Somatic Mutation; Source; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; Surveys; Testing; Thermodynamics; Work; age related; analog; assault; assay development; base; biophysical techniques; cancer risk; chemical reaction; genome sequencing; genome-wide; human DNA; human disease; in vivo; innovation; insight; molecular modeling; molecular recognition; mutant; normal aging; novel; public health relevance; rare variant; repair enzyme; repaired; research study

DESCRIPTION (provided by applicant): Spontaneous damage of DNA bases is a major source of genetic instability. Failure to correct this damage leads to somatic mutations that underlie many diseases associated with aging. The ability to safeguard against these spontaneous lesions relies largely on the base excision repair (BER) pathway whereby DNA glycosylases scan the genome to locate and excise base lesions. Many common forms of damage and proteins involved in the BER pathway have been identified and structurally characterized, but there is a fundamental gap in our understanding of how these enzymes accomplish this amazing task of genome-wide repair. Until this gap is filled, studies of DNA damage and mutagenesis will be largely observational and we will not be able to predict the effects of polymorphisms or exposure to novel carcinogens. Our long-term goal is to understand the molecular mechanisms and fundamental principles by which BER proteins locate and selectively act on a wide range of DNA lesions within genomic DNA. The central hypothesis, that protein-DNA dynamics are critical to the function of the BER pathway, particularly to those enzymes that recognize multiple forms of DNA damage, is strongly supported by data from our lab and others working in this area. Guided by progress and preliminary data from the past funding period, we propose to pursue four specific aims: 1) Determine the searching mechanism(s) of human DNA glycosylases in vitro; 2) Evaluate the hypothesis that facilitated diffusion contributes to efficient DNA repair in eukaryotic cells; 3) Quantify the catalytic specificity of AAG by employing competition kinetics with a wide variety of structurally diverse base lesions; 4) Determine the molecular mechanisms by which AAG achieves efficient recognition of a broad range of substrates. By combining the results from pre-steady state enzyme kinetics, fluorescence spectroscopy, and structure-activity relationships we are poised to dissect the protein-DNA dynamics important for recognition and repair of damaged bases. The approach is innovative, because we are developing novel biochemical assays and new molecular models to understand the kinetic and thermodynamic mechanisms of DNA repair. The proposed research is significant, because it has a high probability of expanding our understanding of BER mechanisms and to uncovering fundamental principles that are relevant for other DNA-templated biological processes. As BER is a critical component of the cellular defense against genomic instability, including endogenous damage, these studies will contribute both to our understanding of mutagenesis and the normal aging process.

描述(由申请人提供)： DNA碱基的自发损伤是遗传不稳定的一个主要来源。如果不能纠正这种损害，就会导致体细胞突变，这些突变是许多与衰老相关的疾病的基础。抵御这些自发损害的能力在很大程度上依赖于碱基切除修复(BER)途径，DNA糖基酶通过该途径扫描基因组来定位和切除碱基损害。已经确定了许多常见的损伤形式和参与BER途径的蛋白质，并对其结构进行了表征，但我们对这些酶如何完成这一令人惊叹的全基因组修复任务的理解存在根本差距。在填补这一空白之前，对DNA损伤和突变的研究将在很大程度上是观察性的，我们将无法预测多态或接触新致癌物的影响。我们的长期目标是了解BER蛋白在基因组DNA中定位和选择性作用于广泛DNA损伤的分子机制和基本原理。核心假设是，蛋白质-DNA动力学对BER途径的功能至关重要，特别是对于那些识别多种形式DNA损伤的酶，这一假设得到了我们实验室和其他在这一领域工作的人的数据的有力支持。在过去资助期间的进展和初步数据的指导下，我们建议追求四个具体目标：1)确定体外人DNA糖基酶的搜索机制(S)；2)评估促进扩散有助于真核细胞中有效的DNA修复的假设；3)通过与各种结构不同的碱基损伤的竞争动力学来量化AAG的催化专一性；4)确定AAG实现对广泛底物的有效识别的分子机制。通过结合稳态前酶动力学、荧光光谱和结构-活性关系的结果，我们准备剖析对识别和修复受损碱基重要的蛋白质-DNA动力学。这种方法是创新的，因为我们正在开发新的生化分析和新的分子模型来了解DNA修复的动力学和热力学机制。这项拟议的研究意义重大，因为它很有可能扩大我们对误码率机制的理解，并揭示与其他DNA模板生物过程相关的基本原理。由于BER是细胞抵御包括内源性损伤在内的基因组不稳定的关键组成部分，这些研究将有助于我们理解突变和正常的衰老过程。