Kinetics of DNA polymerase conformational changes during nucleotide binding and incorporation

DNA polymerases are enzymes responsible for replicating DNA molecules in cells. They have evolved to carry out replication efficiently and highly accurately. For example, the T7 DNA polymerase catalyzes nucleotide incorporation at a rate of 300 s-1 with an error frequency of only one per 105 -106 nucleotides incorporated. Previous studies examining the underlying mechanisms for the exceptional nucleotide selectivity have suggested an induced-fit model based on the structural and kinetic evidence. The key feature of the model is that a rate-limiting conformational change step preceding the phosphoryl transfer step controls the incorporation reaction. If the incoming nucleotide is not correctly base-paired with the template base, the conformational change proceeds at a greatly reduced rate to prevent misincorporation. However, the lack of direct measurement of the kinetics of this rate-limiting conformational change step leads to questions whether the proposed model could account for the nucleotide selectivity. I created a mutant T7 DNA polymerase labeled with an environmentally sensitive fluorophore at an amino acid residue in the recognition domain. The signal from this enzyme is sensitive to the conformational state of the polymerase and was used for studying the kinetics of the conformational changes. Results from my kinetic studies suggested the conformational change step preceding the chemistry step is at least partially rate-limiting during correct nucleotide incorporation. When a mismatched nucleotide binds to the enzyme active site, the conformational change steps not only become slow and unfavorable but also lead to misalignment of active site residues to prevent incorporation of incorrect nucleotide. The new model for nucleotide selectivity is compatible with the previously established induced-fit mechanism, but it also provides new insights on how a small free energy difference between correct and incorrect nucleotide binding leads to the remarkable selectivity the polymerase can achieve. Furthermore, I demonstrated that the fluorescence signal from this novel T7 DNA polymerase construct can be utilized to detect point mutations in DNA sequences. Other than the T7 polymerase project, my attempts to obtain an active HCV RNA polymerase are also presented in this dissertation.