Conformational coupling in DNA polymerase fidelity

The fidelity of DNA polymerases is largely attributable to a two-step nucleotide binding mechanism. In the first step, binding contacts are initially made between the template and the incoming dNTP. The selectivity of this ground-state binding is similar in magnitude to the selectivity seen in forming base pairs in solution. In the second step, a change in protein conformation occurs, which leads to rapid incorporation of the dNTP into the growing polymer. This conformational change appears to occur globally in that it is inhibited by mismatches in the dNTP or in any of the three terminal base pairs of the primer/template. The open conformation allows rapid binding of the dNTP from solution, while the closed conformation provides steric checks for the proper Watson-Crick base pair geometry. This conformational change accounts for the extraordinary fidelity of polymerization and also provides selectivity to the exonuclease by inhibiting polymerization over a mismatch in the primer/template. The overall fidelity approaches one error in 10(10) by a combination of selectivity in polymerization (10(5)-10(6)) and in proofreading (10(3)-10(4)). This paradigm provides the theoretical basis for further investigation of the structural basis for fidelity by pointing to the essential elements of the polymerization reaction that need to be examined in order to evaluate active-site-directed mutants of polymerases to test appropriate structure/function relationships.