DNA-Binding Studies of Taq Polymerase

技术MCB-9904680 LICATA该项目将提供水产黄热菌DNA聚合酶(Taq聚合酶)的详细生物热力学和生化研究，以确定和表征使该聚合酶在750℃的最佳温度下发挥作用的热力学行为和非共价驱动力。这些研究将主要集中在DNA复制的第一步：聚合酶与DNA的结合。本研究的具体目的是：1)定量测定Taq聚合酶与DNA结合的热力学，以及结合过程中结合水量的变化。聚合酶与短的、已定义的DNA片段(AG、AH、AS)结合的热力学将使用量热法和分光光度直接结合分析法来测量，并通过过滤结合和凝胶位移分析等“分离”分析来加强。将测量结合对渗透应力的依赖关系，并用于计算结合时结合水的变化。2)研究Taq聚合酶和Pol I聚合酶与同一DNA结合的温度、pH和离子依赖性。聚合酶的结合将随着温度的变化而定量，以计算与结合反应相关的热容(ACP)的变化；结合将作为不同离子条件和pH的函数进行检查，以定量连接离子和质子的参与。3)确定DNA序列对全球结合和水合热力学的贡献。序列相关性研究将包括与含有均聚体模板区和/或引物区的模板-引物的结合，以便在简化的上下文中建立每个碱基类型对结合的贡献的参考值。紧随其后的是这些区域内越来越复杂的碱基序列变化，以评估碱基位置的贡献以及多个序列变化的可加性或非可加性水平。4)定量测定Taq和E.ColiPol I聚合酶的去折叠热力学，并测定聚合酶结合时DNA熔融热力学的变化。非技术性本项目将首次提供嗜热DNA聚合酶的详细功能、生化和生物能量学研究。将使用Thermus Aquaticus DNA聚合酶(Taq聚合酶)进行研究，以确定和表征使该聚合酶在75℃的最佳温度下发挥作用的热力学行为和非共价驱动力。这些研究将主要集中在DNA复制的第一步：聚合酶与DNA的结合。使用一系列生物物理和生化技术，将在不同的条件下确定结合反应，并根据这些溶液依赖性确定聚合酶-DNA相互作用的连接离子、连接质子和连接水。还将评估结合的DNA序列依赖性。此外，该项目将首次对Pol I和Taq聚合酶的温度稳定性进行定量热力学表征。由于其在聚合酶链式反应(PCR)中的应用，Taq聚合酶是当今使用的最重要的生物技术试剂之一，但其生物化学和功能生物物理特性几乎完全不为人所知。这个项目将扩大我们对聚合酶结合DNA的一般要求、特征、主要作用力和主要的非共价相互作用的基本理解。提高我们对DNA-蛋白质相互作用的理解也与生物技术的许多方面直接相关，如生物过程开发。此外，该项目将开始提供一种理解，即必须大力调整DNA结合和复制这一相同的基本过程，以便在地球生物圈的广泛环境范围内运作。

Technical MCB-9904680 LiCata This project will provide detailed biothermodynamic and biochemical studies of Thermus aquaticus DNA polymerase (Taq polymerase) in order to define and characterize the thermodynamic behavior and non-covalent driving forces that allow the polymerase to function at a temperature optimum of 750C. These studies will primarily focus on the initial step of DNA replication: binding of the polymerase to DNA. The specific aims of this study are: 1) To quantitate the DNA binding thermodynamics of Taq polymerase, and quantitate the change in the amount of associated water during binding. The thermodynamics of binding (AG, AH, AS) of the polymerase to short, defined pieces of DNA will be measured using calorimetric and spectrophotometric direct binding assays, augmented by "separation" assays such as filter binding and gel shift assays. The dependence of binding on osmotic stress will be measured, and used to calculate the change in associated water upon binding. 2) To characterize the temperature, pH, and ionic dependencies of Taq polymerase and Pol I polymerase binding to the same DNA. Binding of the polymerases will be quantitated versus temperature in order to calculate the change in heat capacity (ACp) associated with the binding reaction; and binding will be examined as a function of different ionic conditions and pH in order to quantitate the participation of linked ions and protons. 3) To determine the contributions of the DNA sequence to the global thermodynamics of binding and hydration. Sequence dependence studies will include binding to template-primers containing homopolymeric template regions and/or primer regions, in order to establish reference values for each base type's contribution to binding within a simplified context. This will be followed by increasingly more complex base sequence changes within these regions to assess base position contributions and the level of additivity or nonadditivity of multiple sequence changes. 4) To quantitatively determine the thermodynamics of unfolding of Taq and E. coli Pol I polymerases; and determine the change in DNA melting thermodynamics when polymerase binds.Non-TechnicalThis project will provide the first detailed functional biochemical and bioenergetic studies of a thermophilic DNA polymerase. Studies will be carried out with Thermus aquaticus DNA polymerase (Taq polymerase) in order to define and characterize the thermodynamic behavior and non-covalent driving forces that allow this polymerase to function at a temperature optimum of 75C. These studies will focus primarily on the initial step of DNA replication: binding of the polymerase to DNA. Using an array of biophysical and biochemical techniques, the binding reaction will be determined under different conditions, and the linked ions, linked protons, and linked waters of the polymerase-DNA interaction will be determined from these solution dependencies. The DNA sequence dependence of the binding will also be assessed. Further, this project will provide the first quantitative thermodynamic characterization of the temperature stability of Pol I and Taq polymerases. Due to its use in the polymerase chain reaction (PCR), Taq polymerase is one of the most important biotechnological reagents in use today, yet its biochemistry and functional biophysics remain almost completely uncharacterized. This project will extend our fundamental understanding of the general requirements, characteristics, primary forces, and predominant non-covalent interactions involved in the binding of DNA by polymerases. Advancing our understanding of DNA-protein interactions is also directly relevant to many aspects of biotechnology such as bioprocess development. Further, this project will begin to provide an understanding of how the same essential process of DNA binding and replication must be energetically fine-tuned in order to operate across the broad environmental range of Earth's biosphere.