Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity.
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 10(4-) greater than 1.2 X 10(6]-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- greater than 61-fold. Finally, KF polymerizes the next correct dNTP onto a mismatch very slowly, providing a further 6- greater than 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.
Kuchta
R D
RD
Department of Chemistry, Pennsylvania State University, University Park 16802.
Benkovic
P
P
Benkovic
S J
SJ
D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004926:Escherichia coli / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V