Base Selectivity Is Impaired by Mutants that Perturb Hydrogen Bonding Networks in the RB69 DNA Polymerase Active Site

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Abstract
To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the “floor” of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dNTPs by the exo- form of RB69 pol and its mutants. We found that the Y567A mutant had the same Kd and kpol values for incorporation of C versus G as the wild-type exo- enzyme; however, the kpol/Kd ratio for G versus G incorporation with the Y567A mutant was 10 times higher than the kpol/Kd efficiency of G versus G incorporation using the exo- RB69 pol. The reduced level of discrimination by the Y567A mutant against incorporation of mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence was also employed to monitor rates of putative conformational changes with the exo- RB69 pol and its mutants using a primer−template complex containing 2-aminopurine. The rates of fluorescence changes were equal to or greater than the rates of the rapid chemical quench, indicating that we were monitoring a process occurring before or during the phosphoryl transfer reaction. We have interpreted our results within the context of the crystal structure of the RB69 pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657−667].

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