Dynamic Fluorescence Studies of DNA-Protein Complexes

描述(申请人提供)：这个项目的长期目标是阐明DNA聚合酶保真度的分子基础和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生理和细胞分裂过程中遗传信息的忠实传递是必不可少的。这个项目的重点是聚合酶-DNA复合体的动态构象变化，它与核苷酸掺入和32-52外核解核校对活动有关。我们将研究高保真的Klenow片段聚合酶，它提供了一个特征良好的模型系统。具体目标是：(1)建立单分子荧光方法监测DNA聚合酶的构象动力学。(2)确定DNA聚合酶的功能上的重要构象变化如何与正确的核苷酸底物的选择有关。(3)在单分子水平上实时检测DNA聚合酶活性。(4)了解在校对过程中，DNA引物/模板如何在分离的聚合酶和32-52个核酸外切酶之间移动。(5)确定聚合酶和核酸外切酶活性如何在物理上协调，以实现高保真的DNA复制。将建立新的单对FRET系统来监测核苷掺入过程中酶的构象变化，以及校对过程中DNA的移位。这些新工具将打开一扇独特的窗口，了解DNA聚合酶的内部工作原理，并将广泛适用于其他蛋白质-DNA复合体。

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.