Dynamic Fluorescence Studies of DNA-Protein Complexes

描述(申请人提供)：本项目的目标是了解DNA聚合酶实现高保真DNA复制的机制，使用大肠杆菌的Kienow片段(polI家族)和RB69噬菌体DNA聚合酶(p01alphaI家族)作为模型系统。将开发单分子荧光方法来监测核苷酸选择和核外溶解校对过程中蛋白质结构域和DNA底物的大规模运动。对DNA聚合酶构象动力学的详细描述将有助于更深入地理解这些酶是如何调节和协调它们的不同活性以实现高保真DNA复制的。 我们将研究在校对过程中，Kienow片段和RB69聚合酶中不同的结构域排列如何决定DNA在聚合酶和核酸外切酶活性位点之间转移的途径。突变将被引入拇指亚域的尖端和底部，以确定该区域是否有助于在两个位置之间引导DNA底物。双链熔化在活性中心转换中的作用将对这两种聚合酶进行研究。 在核苷酸底物存在和不存在的情况下，将观察Finger亚域的构象动力学，以阐明Finger在核苷酸选择中的作用。将测量模板底座翻转的速率，以确定这些转换是否由闭合手指触发。我们还将检验这样的假设，即指域移动通过调节位点切换动力学来控制核外溶解校对的速度。

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.