Date of Award
Spring 2013
Document Type
Thesis
Degree Name
Bachelor of Science
Department
Biochemistry & Molecular Biol.
First Advisor
Dr. Eugene Wu
Second Advisor
Dr. Michelle Hamm
Abstract
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.
Recommended Citation
Kornberg, Emily P., "The inner workings of the DNA copying nanomachine : kinetic studies of DNA polymerase I from the thermophilic bacterium Rhodothermus marinus" (2013). Honors Theses. 55.
https://scholarship.richmond.edu/honors-theses/55