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Protein-DNA Dynamics in Base Excision DNA Repair

碱基切除 DNA 修复中的蛋白质-DNA 动力学

基本信息

批准号：
8097478
负责人：
Patrick J O'Brien
金额：
$ 28.01万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-26 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8097478
关键词：
3-methyladenine-DNA glycosylase Active Sites Affect Affinity Alkylation Base Excision Repairs Base Pairing Binding Biochemical Biological Assay Cancer Etiology Chemotherapy-Oncologic Procedure Complex DNA DNA Binding DNA Damage DNA Ligases DNA Repair DNA biosynthesis DNA glycosylase DNA lesion DNA-(apurinic or apyrimidinic site) lyase DNA-3-methyladenine glycosidase II DNA-Directed DNA Polymerase DNA-Protein Interaction Diagnosis Diffusion Embryo Enzyme Kinetics Enzymes Equilibrium Escherichia coli Event Excision Exposure to Fluorescence Spectroscopy Genetic Transcription Genome Genome Scan Genomics Goals Health Human Hydrogen Bonding Hydrolysis In Vitro Individual Kinetics Lesion Life Life Expectancy Lyase Malignant Neoplasms Measures Methods Mus Mutagenesis Mutation Normal Cell Nucleotides Oligonucleotides Pathway interactions Predisposition Process Protein Dynamics Proteins Rare Lesion Reaction Relative (related person)Repair Complex Research Personnel Role Rotation Scaffolding Protein Scanning Series Site Societies Source Structure Structure-Activity Relationship Substrate Specificity Testing Thermodynamics Time Tyrosine Ursidae Family Virus Inhibitors analog base cancer therapy chemical reaction cost cytotoxic enzyme structure genome-wide human APEX1 protein novel nucleotide analog oxidation programs protein protein interaction reconstitution repair enzyme repaired research study

项目摘要

DESCRIPTION (provided by applicant): Spontaneous damage of DNA bases is a major source of cancer-causing mutations. Given the thousands of lesions generated per genome every day, it is remarkable that cancer remains a relatively infrequent event with the majority of cases arising relatively late in life. With increasing life expectancy and exposure to exogenous DNA damaging agents, society bears the ever increasing cost of diagnosing and treating cancer. At the cellular level 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. The action of an apurinic (AP)-specific endonuclease, AP-lyase/DNA polymerase, and DNA ligase are required to complete repair of the DNA. Our long-term goals are to understand how BER proteins locate and selectively act on a wide range of DNA lesions within genomic DNA and how the dynamics of protein-protein and protein-DNA interactions enable coordination of multi-step, multi-enzyme repair pathways. Recent evidence suggests that nucleotide flipping, the process by which a nucleotide is extracted from the DNA duplex and bound in an active site pocket, provides much of the selectivity in distinguishing damaged and undamaged bases. We propose to test this hypothesis by directly observing flipping of damaged and undamaged nucleotides by DNA glycosylases (Aim 1). The genomic search for rare lesions is facilitated by the examination of many nucleotides with each DNA binding event, therefore we will characterize the ability of BER enzymes to move along DNA and measure the efficiency with which sites of damage are productively engaged during a scanning encounter (Aim 2). As DNA repair intermediates are potentially cytotoxic or mutagenic, it is critical that initiated BER events be completed. We propose to investigate the dynamics of protein-protein interactions in BER and determine their functional significance in the coordination of multiple enzymatic activities (Aim 3). By combining the results from pre-steady state enzyme kinetics, fluorescence spectroscopy, and structure-activity relationships we have a unique opportunity to dissect the protein-DNA dynamics important for damage recognition and repair. As BER is a critical component of the cellular defense against cancer, and because these pathways are antagonistic toward some DNA damaging agents used in the treatment of cancer, these studies have the potential to contribute both to our understanding of mutagenesis and to advances in cancer therapy.

描述（由申请人提供）：DNA 碱基的自发损伤是致癌突变的主要来源。鉴于每个基因组每天都会产生数千个病变，值得注意的是，癌症仍然是一种相对罕见的事件，大多数病例发生在生命的较晚时期。随着预期寿命的延长和接触外源 DNA 损伤剂的增加，社会承担着不断增加的诊断和治疗癌症的费用。在细胞水平上，防止这些自发损伤的能力很大程度上依赖于碱基切除修复（BER）途径，DNA糖基化酶通过该途径扫描基因组以定位和切除碱基损伤。完成 DNA 修复需要无嘌呤 (AP) 特异性核酸内切酶、AP 裂解酶/DNA 聚合酶和 DNA 连接酶的作用。我们的长期目标是了解 BER 蛋白如何定位并选择性地作用于基因组 DNA 内的各种 DNA 损伤，以及蛋白质-蛋白质和蛋白质-DNA 相互作用的动态如何实现多步骤、多酶修复途径的协调。最近的证据表明，核苷酸翻转（从 DNA 双链体中提取核苷酸并结合在活性位点口袋中的过程）在区分受损和未受损碱基方面提供了很大的选择性。我们建议通过直接观察 DNA 糖基化酶对受损和未受损核苷酸的翻转来检验这一假设（目标 1）。通过检查每个 DNA 结合事件的许多核苷酸，可以促进对罕见损伤的基因组搜索，因此我们将表征 BER 酶沿着 DNA 移动的能力，并测量在扫描过程中有效参与损伤位点的效率（目标 2）。由于 DNA 修复中间体具有潜在的细胞毒性或致突变性，因此完成启动的 BER 事件至关重要。我们建议研究 BER 中蛋白质-蛋白质相互作用的动态，并确定它们在协调多种酶活性中的功能意义（目标 3）。通过结合前稳态酶动力学、荧光光谱和结构-活性关系的结果，我们有一个独特的机会来剖析对于损伤识别和修复至关重要的蛋白质-DNA 动力学。由于 BER 是细胞防御癌症的关键组成部分，并且这些途径与癌症治疗中使用的一些 DNA 损伤剂具有拮抗作用，因此这些研究有可能有助于我们对突变的理解和癌症治疗的进步。