Dynamic Fluorescence Studies of DNA-Protein Complexes

DNA-蛋白质复合物的动态荧光研究

• 批准号: 6914855
• 负责人: David P MILLAR
• 金额: $ 30.79万
• 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 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6914855
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primers; DNA replication; active sites; chemical binding; chemical kinetics; confocal scanning microscopy; conformation; electrospray ionization mass spectrometry; enzyme mechanism; enzyme structure; enzyme substrate; exonuclease; fluorescence resonance energy transfer; fluorescence spectrometry; fluorescent dye /probe; nucleic acid sequence; nucleotides; protein sequence; protein structure function

DESCRIPTION (provided by applicant): The goal of this project is to understand the mechanisms by which DNA polymerases achieve high fidelity DNA replication, using the Kienow fragment of E. coli (pol I family) and RB69 bacteriophage DNA polymerase (p01alphaI family) as model systems. Single-molecule fluorescence methods will be developed to monitor large scale motions of protein domains and of the DNA substrate that occur during nucleotide selection and exonucleolytic proofreading. A detailed description of the conformational dynamics of DNA polymerases will lead to a deeper understanding of how these enzymes regulate and coordinate their different activities to achieve high fidelity DNA replication. We will investigate how the different domain arrangements in Kienow fragment and RB69 polymerase determine the pathway by which DNA is transferred between polymerase and exonuclease active sites during proofreading. Mutations will be introduced into the tip and base of the thumb subdomain to determine whether this domain helps to guide the DNA substrate between the two sites. The role of duplex melting in active-site switching will be investigated for both polymerases. Conformational dynamics of the fingers subdomain will be observed in the presence and absence of nucleotide substrates to elucidate the role of the fingers in nucleotide selection. The rate of template base flipping will be measured to determine whether these transitions are triggered by closure of the fingers. We will also test the hypothesis that finger domain movements control the rate of exonucleolytic proofreading by modulating the site-switching kinetics.

描述（由申请人提供）：该项目的目标是了解DNA聚合酶实现高保真DNA复制的机制，使用大肠杆菌Kienow片段（pol I家族）和RB69噬菌体DNA聚合酶（p01alpha - I家族）作为模型系统。单分子荧光方法将用于监测在核苷酸选择和核外溶校对过程中发生的蛋白质结构域和DNA底物的大规模运动。对DNA聚合酶的构象动力学的详细描述将使我们更深入地了解这些酶如何调节和协调它们不同的活动以实现高保真DNA复制。