Open Access Dissertation
Date of Award
Loui J. Romano
DNA polymerases maintain the genome integrity from one generation to the next by faithfully synthesizing new DNA and by participating in DNA repair processes. The Klenow fragment of E. coli DNA polymerase I has served as a model polymerase for decades because it is straightforward to purify, well-characterized kinetically, and it is able to carry out DNA synthesis and proofreading. In 2009, Christian, et al. developed a single molecule Förster resonance energy transfer (smFRET) approach to monitor the polymerase position on the DNA with single base pair resolution.
We have worked to optimize nearly every aspect of that approach and to apply the technique to characterize DNA polymerase dynamics on the DNA with unprecedented detail. First, we improved the cysteine-labeling protocol to ensure the labeling was site-specific and efficient. Second, we applied a new single molecule fluorescence technique, single molecule protein induced fluorescence enhancement (smPIFE), to our system to accurately measure binding dynamics and to strengthen our interpretation of the polymerase position on the DNA. Third, determined how carcinogenic DNA adducts disrupt polymerase binding on the DNA by using smFRET and smPIFE. Accordingly, we designed the experiment to distinguish between pol and exo site binding in real-time, and we identified a novel intermediate state in the proofreading mechanism. Finally, we tracked individual polymerases as they incorporated bases on the DNA. These experiments support the existence of a fidelity-checking step following each incorporation. The assays and applications described herein lay the groundwork for future DNA polymerase mechanistic studies.
Vrtis, Kyle, "Unveiling Dna Polymerase Synthesis And Proofreading Activities - One Molecule At A Time" (2013). Wayne State University Dissertations. 862.