Protein-DNA Dynamics in Base Excision DNA Repair

碱基切除 DNA 修复中的蛋白质-DNA 动力学

• 批准号: 7667817
• 负责人: Patrick J O'Brien
• 金额: $ 28.88万
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7667817
• 关键词: 3-methyladenine; 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-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

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碱基的自发损害是引起癌症突变的主要来源。鉴于每天每个基因组产生的数千例病变，值得注意的是，癌症仍然是一个相对较少的事件，大多数病例在生命中较晚。随着预期寿命的增加和外源性DNA损伤药物的暴露，社会承担了诊断和治疗癌症的越来越多的成本。在细胞水平上，保护这些自发病变的能力在很大程度上取决于碱性切除修复（BER）途径，从而使DNA糖基酶扫描基因组以定位和切除碱病变。需要完全修复DNA的修复，需要阿源素（AP）特异性内核酸酶，AP-酯/DNA聚合酶和DNA连接酶的作用。我们的长期目标是了解BER蛋白如何定位和选择性地在基因组DNA中的广泛DNA病变上作用，以及蛋白质 - 蛋白质和蛋白质-DNA相互作用的动力学如何使多步，多酶修复途径的配位。最近的证据表明，核苷酸翻转是从DNA双链体中提取核苷酸并在活性位点袋中结合的过程，在区分受损和未损坏的碱基方面具有很大的选择性。我们建议通过直接观察DNA糖基酶对受损和未受损的核苷酸的翻转来检验这一假设（AIM 1）。通过检查每个DNA结合事件的许多核苷酸的检查，对罕见病变的基因组搜索促进，因此，我们将表征BER酶沿DNA移动并测量在扫描过程中损害部位有效地参与的效率的能力（AIM 2）。由于DNA修复中间体可能具有细胞毒性或诱变，因此完成的启动事件至关重要。我们建议研究BER中蛋白质蛋白相互作用的动力学，并确定它们在多种酶活性协调中的功能意义（AIM 3）。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，我们有一个独特的机会，可以剖析对损伤识别和修复至关重要的蛋白质-DNA动力学。由于BER是针对癌症的细胞防御的关键组成部分，并且由于这些途径对用于治疗癌症治疗的某些DNA损害剂具有拮抗作用，因此这些研究有可能为我们理解诱变和癌症治疗的进步做出贡献。