Dynamic Fluorescence Studies of DNA-Protein Complexes

描述（由申请人提供）：本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。通过DNA聚合酶进行精确的核苷酸选择和校正对于适当的细胞生理学和细胞分裂期间遗传信息的忠实传递至关重要。本项目将重点研究聚合酶-DNA复合物的动态构象变化，这些变化与核苷酸掺入和32-52核酸外切校正活动有关。我们将研究高保真Klenow片段聚合酶，它提供了一个良好的特征模型系统。具体目标是：（1）发展单分子荧光法监测DNA聚合酶的构象动力学。(2)确定DNA聚合酶功能上重要的构象变化如何与正确的核苷酸底物的选择相联系。(3)实时检测单分子水平的DNA聚合酶活性。(4)发现DNA引物/模板如何在校对过程中在单独的聚合酶和32-52个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。新的单对FRET系统将建立监测酶的构象变化，在核苷酸掺入和易位的DNA校对过程中。这些新工具将为DNA聚合酶的内部工作打开一扇独特的窗户，并将广泛适用于其他蛋白质-DNA复合物。

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.