Dynamic Fluorescence Studies of DNA-Protein Complexes

描述（由申请人提供）：本项目的目的是了解DNA聚合酶实现高保真DNA复制的机制，使用大肠杆菌Kienow片段。coli（pol I家族）和RB69噬菌体DNA聚合酶（p01 α I家族）作为模型系统。将开发单分子荧光方法来监测在核苷酸选择和核酸外切校对期间发生的蛋白质结构域和DNA底物的大规模运动。DNA聚合酶的构象动力学的详细描述将导致这些酶如何调节和协调其不同的活动，以实现高保真的DNA复制更深入的理解。 我们将研究Kienow片段和RB69聚合酶中的不同结构域排列如何决定DNA在校对过程中在聚合酶和核酸外切酶活性位点之间转移的途径。将突变引入拇指亚结构域的尖端和基部，以确定该结构域是否有助于引导两个位点之间的DNA底物。双链体熔化的作用，在活性位点切换将研究这两种聚合酶。 将在存在和不存在核苷酸底物的情况下观察指状物亚结构域的构象动力学，以阐明指状物在核苷酸选择中的作用。将测量模板碱基翻转的速率，以确定这些转变是否由手指闭合触发。我们还将测试的假设，指域运动控制外切核酸的校对率通过调节位点转换动力学。

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.