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碱基的自发损害是遗传不稳定的主要来源。无法纠正这种损害会导致躯体突变，这些突变是许多与衰老相关的疾病的基础。保护这些自发病变的能力主要依赖于碱性切除修复（BER）途径，从而使DNA糖基酶扫描基因组以定位和消除碱病变。已经确定并在结构上表征了许多常见的损害和蛋白质形式，但是我们对这些酶如何完成基因组修复的这一惊人任务的理解存在根本的差距。在填补了这一差距之前，对DNA损伤和诱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性或暴露于新型癌症的影响。我们的长期目标是了解BER蛋白在基因组DNA中定位并有选择地作用于BER蛋白的分子机制和基本原理。中心假设是，蛋白质-DNA动力学对于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，我们的实验室和其他在该领域工作的酶得到了强有力的支持。在过去资金期的进步和初步数据的指导下，我们建议追求四个具体目标：1）确定体外人类DNA糖基酶的搜索机制； 2）评估促进扩散的假设有助于真核细胞中有效的DNA修复； 3）通过使用具有多种结构多样的碱性病变的竞争动力学来量化AAG的催化特异性； 4）确定AAG通过有效识别广泛底物的分子机制。通过结合稳态的状态酶动力学，荧光光谱和结构活性关系的结果，我们有望剖析蛋白质-DNA动力学对于识别和修复受损碱基的识别和修复很重要的蛋白质DNA动力学。这种方法具有创新性，因为我们正在开发新型的生化测定和新的分子模型，以了解DNA修复的动力学和热力学机制。拟议的研究很重要，因为它具有很高的可能性，可以扩展我们对BER机制的理解，并发现与其他DNA模拟生物学过程相关的基本原理。由于BER是针对基因组不稳定性的细胞防御的关键组成部分，包括内源性损害，这些研究将有助于我们对诱变和正常衰老过程的理解。