DNA Binding Studies of Extremophilic DNA Polymerases

本研究以来自嗜热菌Thermus aquaticus的Taq聚合酶为研究对象，探讨其与DNA结合的功能和结构特征。由于Taq聚合酶在聚合酶链反应中的应用，Taq聚合酶是当今世界上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学的研究才刚刚开始。这项工作的中心假设是Taq和E.大肠杆菌聚合酶在许多重要的功能和热力学方面存在差异，Taq和E.大肠杆菌聚合酶的研究将有助于深入了解它们生理作用的重叠和差异。该项目将定量表征不同种类聚合酶的DNA结构结合偏好和热力学。它还将使用几种溶液结构技术表征结合的DNA的结构拓扑。还将表征不同聚合酶的DNA结合动力学。这项工作的智力价值包括这些重要酶急需的功能和结构特征。只有随着对这些极端聚合酶的知识基础的扩展，才有可能完全理解构成和控制整个生物体范围内DNA复制过程的所有参数。此外，Taq聚合酶的数据经常被用来回答有关E。coli Pol 1，反之亦然，因为它们都是1型聚合酶。初步数据显示，这种推断是有风险的。对Taq的进一步了解也将对生物技术领域产生广泛的科学影响。这项工作的教育和广泛的影响目标包括：1）在路易斯安那州立大学州立大学的现有课程中引入更多的实践活动和当前的研究; 2）培训新的研究生使用同步加速器小角度X射线散射方法来解决生物学问题; 3）为所有年龄段的观众编写一系列具有科学内容的戏剧，并追求这些戏剧的最终生产。

This project will examine functional and structural characteristics of DNA binding by extremophilic DNA polymerases, focusing on Taq polymerase from the thermophilic bacterium Thermus aquaticus .Due to its use in the polymerase chain reaction, Taq is one of the most important biotechnological reagents in use in the world today, yet its biochemistry and functional biophysics have only just begun to be characterized. The central hypothesis of this work is that Taq and E. coli polymerases differ in a number of significant functional and thermodynamic ways, and that the differences and similarities between Taq and E. coli polymerases will provide insight into the overlaps and differences in their physiological roles. This project will quantitatively characterize the DNA structural binding preferences and thermodynamics of the different species of polymerase. It will also characterize the structural topology of the bound DNA using several solution structural techniques. The kinetics of DNA binding by the different polymerases will also be characterized. The intellectual merits of this work include the much needed functional and structural characterizations of these important enzymes. Only as the knowledge base on these extremophilic polymerases expands will it be possible to fully understand all the parameters that comprise and control the process of DNA replication across the entire spectrum of living organisms. In addition, data on Taq polymerase is often used to answer questions about E. coli Pol 1,and vice versa, since they are both Type 1 polymerases. Preliminary data show that such extrapolation is risky. Further understanding of Taq will have broad scientific impact on the biotechnological field as well. The educational and broad impact goals of this work include: 1) introducing more hands-on activities and current research into current courses at Louisiana State University; 2) training new graduate students in the use of synchrotron small angle X-ray scattering methods for addressing biological questions; 3) writing a series of plays with scientific content for all-age audiences, and pursuing eventual production of these plays.