Date of Award

Spring 2012

Document Type


Degree Name

Bachelor of Science


Biochemistry & Molecular Biol.

First Advisor

Dr. Eugene Wu

Second Advisor

Dr. Michelle Hamm


DNA polymerase I employs a multistep mechanism for sorting correctly paired nucleotides from mismatches. We aim to characterize reaction intermediates during nucleotide selection to better understand how this class of enzymes achieves high DNA replication fidelity. DNA polymerase I from R. marinus contains an unusual and disruptive proline in the mobile O helix near the active site. To characterize this enzyme, the structure of the large (5´-to-3´ exo-deficient) fragment of the R. marinus DNA polymerase I (RF) was solved to 2.95 Å (R = 0.234) using multi-wavelength anomalous dispersion. Alignment with homologous Escherichia coli Klenow Fragment (KF) DNA polymerase I confirmed that the active sites of each structural domain were conserved. In order to study the polymerase activity in isolation, 3’-to-5’ exonuclease activity was eliminated using site-directed mutagenesis of an aspartic acid involved in binding DNA (D497A). Unexpectedly, mutation of an aspartic acid involved in binding a catalytic magnesium ion (D421A in RF) failed to abolish exonuclease activity despite its ability to do so in the KF (D424A). Future structural studies will include crystallization of the polymerase in both its binary and ternary complex to study the conformational changes associated with the process of nucleotide selection as well as examination of the exonuclease domain to characterize its unusual activity in this enzyme.