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 X 104→ 1.2 X 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’→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/→5/-exonuclease 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.
|Original language||English (US)|
|Number of pages||10|
|State||Published - Sep 1 1988|
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