Dynamic Fluorescence Studies of DNA-Protein Complexes

DNA-蛋白质复合物的动态荧光研究

• 批准号: 7995966
• 负责人: David P MILLAR
• 金额: $ 36.29万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7995966
• 关键词: Active Sites; Biological Models; Carcinogens; Cell division; Cell physiology; Complex; DNA; DNA Damage; DNA Polymerase I; DNA Primers; DNA Repair; DNA biosynthesis; DNA-Directed DNA Polymerase; Detection; Disease; Enzymes; Equilibrium; Event; Exonuclease; Exposure to; Family; Fingers; Fluorescence; Fluorescence Resonance Energy Transfer; Genetic; Goals; Hereditary Disease; Human Genetics; Individual; Investigation; Lesion; Link; Malignant Neoplasms; Methods; Molecular; Molecular Conformation; Monitor; Nucleotides; Pathway interactions; Play; Polymerase; Proteins; Role; Scheme; Site; Sunlight; System; Techniques; Testing; Time; Travel; Work; base; conformational conversion; design; molecular site; novel; protein complex; single molecule; small molecule; tool; transmission process

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the molecular basis for DNA polymerase fidelity and the role of protein conformational dynamics in polymerase function. Accurate nucleotide selection and proofreading by DNA polymerases are essential for proper cellular physiology and the faithful transmission of genetic information during cell division. This project will focus on dynamic conformational changes of the polymerase-DNA complex that are linked to the nucleotide incorporation and 32-52 exonucleolytic proofreading activities. We will study the high fidelity Klenow fragment polymerase, which provides a well-characterized model system. The specific aims are: (1) Develop single-molecule fluorescence methods to monitor the conformational dynamics of DNA polymerases. (2) Establish how functionally important conformational changes of DNA polymerase are linked to selection of a correct incoming nucleotide substrate. (3) Detect DNA polymerase activity at the single-molecule level in real-time. (4) Discover how a DNA primer/template travels between the separate polymerase and 32-52 exonuclease sites during proofreading. (5) Establish how the polymerase and exonuclease activities are physically coordinated to achieve high fidelity DNA replication. Novel single-pair FRET systems will be established to monitor enzyme conformational changes during nucleotide incorporation and translocation of DNA during proofreading. These new tools will open a unique window into the inner workings of DNA polymerase enzymes and will be broadly applicable to other protein-DNA complexes.

描述（由申请人提供）：该项目的长期目标是阐明 DNA 聚合酶保真度的分子基础以及蛋白质构象动力学在聚合酶功能中的作用。准确的核苷酸选择和 DNA 聚合酶的校对对于细胞分裂过程中正确的细胞生理学和遗传信息的忠实传递至关重要。该项目将重点关注与核苷酸掺入和 32-52 核酸外切校对活动相关的聚合酶-DNA 复合物的动态构象变化。我们将研究高保真 Klenow 片段聚合酶，它提供了一个良好表征的模型系统。具体目标是：（1）开发单分子荧光方法来监测DNA聚合酶的构象动力学。 (2) 确定 DNA 聚合酶的功能上重要的构象变化与选择正确的引入核苷酸底物之间的关系。 (3)单分子水平实时检测DNA聚合酶活性。 (4) 发现校对过程中 DNA 引物/模板如何在单独的聚合酶和 32-52 核酸外切酶位点之间移动。 (5) 确定聚合酶和核酸外切酶活性如何物理协调以实现高保真 DNA 复制。将建立新型单对 FRET 系统来监测校对过程中核苷酸掺入和 DNA 易位过程中酶构象的变化。这些新工具将为了解 DNA 聚合酶的内部运作打开一扇独特的窗口，并将广泛适用于其他蛋白质-DNA 复合物。