Catherine M Joyce DPhil
Senior Research Scientist in Molecular Biophysics and Biochemistry; Senior Research Scientist/Scho
DNA polymerase pathway; Dbh polymerase
Structure and mechanism of DNA polymerases
We are using simple DNA polymerases of known structures as model systems to explore the molecular details of DNA replication. In addition to defining the sequence of chemical events involved in the DNA synthesis reaction, we are interested in the molecular processes that ensure polymerase accuracy and the mechanism by which an enzyme can act processively on a macromolecular substrate. Much of our work has focused on DNA polymerase I of E. coli. Based on the wealth of structural information (including a variety of enzyme-substrate complexes) for this polymerase and its homologues, we have used site-directed mutagenesis to make changes in the polymerase active site region. Kinetic studies of the resulting mutant proteins have allowed us to identify protein side chains that recognize the sugar structure of the incoming nucleotide and that influence polymerase accuracy. To learn more about the DNA polymerase reaction pathway, we have started to use fluorescence and EPR techniques to investigate the conformational transitions that take place during the reaction. In recent years, we have expanded our studies to include the Dbh polymerase, a member of the newly discovered family of lesion bypass DNA polymerases. By comparing the highly accurate DNA polymerase I with the extremely inaccurate Dbh, we hope to gain additional insights into those polymerase characteristics that determine fidelity.