DNA replication fidelity: kinetics and thermodynamics.

Mechanisms that control the fidelity of DNA replication are discussed. Data are reviewed for 3 steps in a fidelity pathway: nucleotide insertion, exonucleolytic proofreading, and extension from matched and mismatched 3'-primer termini. Fidelity mechanisms that involve predominantly Km discrimination, Vmax discrimination, or a combination of the two are analyzed in the context of a simple model for fidelity. Each fidelity step is divided into 2 components, thermodynamic and kinetic. The thermodynamic component, which relates to free-energy differences between right and wrong base pairs, is associated with a Km discrimination mechanism for polymerase. The kinetic component, which represents the enzyme's ability to select bases for insertion and excision to achieve fidelity greater than that available from base pairing free-energy differences, is associated with a Vmax discrimination mechanism for polymerase. Currently available fidelity data for nucleotide insertion and primer extension in the absence of proofreading appears to have relatively large Km and small Vmax components. An important complication can arise when analyzing data from polymerases containing an associated 3'-exonuclease activity. In the presence of proofreading, a Vmax discrimination mechanism is likely to occur, but this may be the result of two Km discrimination mechanisms acting serially, one for nucleotide insertion and the other for excision. Possible relationships between base pairing free energy differences measured in aqueous solution and those defined within the polymerase active cleft are considered in the context of the enzyme's ability to exclude water, at least partially, from the vicinity of its active site.