Degree Type

Thesis

Date of Award

2013

Degree Name

Master of Science

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Scott W. Nelson

Abstract

Members of the phylum Apicomplexa, including pathogens such as Plasmodium falciparum, Toxoplasma gondii, and Babesia bovis contain a non-photosynthetic plastid called an apicoplast. It has been implicated in the biosynthesis of fatty acids, iron-sulfur clusters, heme groups, and isoprenoid synthesis for the pathogen. This unique plastid was immediately recognized as a potential target for drug discovery as it is essential for the survival and reproduction of the parasite. With this in mind, our group has initiated characterization of the DNA replication system within the apicoplast. Here we report our efforts to determine the fidelity of the DNA polymerase (apPOL) using pre–steady state kinetics. The apPOL exhibits error rates typical of a high fidelity family A polymerase in the 104 to the 106 range and has an average processivity of approximately 2 nucleotides incorporated per binding event. The data indicates that the rate of chemistry or a conformational change preceding the chemical step is the rate limiting step for sequential polymerization as inclusion of a translocation step was not necessary to explain the product time courses. Additionally, with some mismatches, stalling of the primer extension reactions was observed, indicating that like the misincorporation reaction, the degree of mismatch extension is highly variable. This stalling may indicate that severe distortions of the active site following misincorporation significantly slows mismatch extension, which could increase the excision of the mismatch by the 3'–5' exonuclease activity of the wild–type apPOL and increase overall fidelity. Finally, it was observed that the apPOL will catalyze non–template nucleotide additions to the primer strand with a preference for dGTP, followed by dATP, dCTP, and dTTP.

Copyright Owner

Eric Evan Parrott

Language

en

File Format

application/pdf

File Size

52 pages

Included in

Biochemistry Commons

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