Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity

Abstract

A complete kinetic scheme describing the polymerization of correct and incorrect dNTPs by the Klenow fragment (KF) of DNA polymerase I has been developed by using short DNA oligomers of defined sequence. The high fidelity arises from a three-stage mechanism. The first stage of discrimination [(1.1 .times. 10-4->1.2 .times. 106)-fold] comes primarily from a dramatically reduced rate of phosphodiester bond formation for incorrect nucleotides, but it also gains a smaller contribution from selective dNTP binding. After phosphodiester bond formation, a conformational change slows dissociation of the incorrect DNA products from KF and, in conjunction with editing by the 3''.fwdarw.5''-exonuclease, increases fidelity 4->61-fold. Finally, KF polymerizes the next correct dNTP onto a mismatch very slowly, providing a further 6->340-fold increase in fidelity. Surprisingly, the 3''.fwdarw.5''-exonulcease did not in its hydrolysis reaction differentiate between correctly and incorrectly base-paired nucleotides; rather, an increased lifetime of the enzyme-DNA complex containing the misincorporated base is responsible for discrimination.