Date of Award

Spring 2013

Document Type


Degree Name

Bachelor of Science


Biochemistry & Molecular Biol.

First Advisor

Dr. Eugene Wu

Second Advisor

Dr. Michelle Hamm


DNA polymerase I from Rhodothermus marinus is a high-fidelity DNA polymerase capable of operating at high temperatures and incorporating 2´,3´-dideoxynucleotides. The R. marinus DNA Polymerase I active site contains an unusual proline in the middle of a mobile “O helix.” This proline residue is hypothesized to decrease the relative free energy of a kinetic checkpoint termed the ajar conformation, and thereby slow the incorporation of incorrect nucleotides. We aim to test the accuracy of a newly developed nucleotide incorporation model, in which the enzyme allows the template to interact with the bound dNTP in the ajar conformation, whether correct or incorrect, prior to catalyzing phosphodiester bond formation. The active site proline 760 in R. marinus DNA Pol I was mutated and error rates were determined to test this new model of nucleotide selection. Our studies have further characterized a “missing link” in the mechanism for nucleotide discrimination in high-fidelity DNA polymerases.