Thermodynamic dissection of the polymerizing and editing modes of a DNA polymerase Academic Article uri icon

publication date

  • 2004


  • DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5'-3' polymerization or 3'-5' editing of a nascent DNA chain. Thermodynamic measurements designed to quantify the energetic contributions of individual DNA-protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA-protein binding assays. To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase-DNA system. Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes. From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally. This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5'-3' polymerase and 3'-5' exonuclease sites of KF. It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein. While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.