DNA Binding Studies of Extremophilic DNA Polymerases

Louisiana State University, Baton Rouge LA

Investigators

Abstract

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.