![]() ![]() In this model, the incoming dNTP directly pairs opposite its complementary template partner via direct hydrogen bonding and the enzyme catalyzes phosphodiester bond formation only if the two nucleobases align correctly. Replication fidelity has historically been interpreted with respect to the formation of proper hydrogen bonds between the nascent base pairs ( 2- 5). Maintaining fidelity during DNA replication is a daunting challenge since the changing nature of the heteropolymeric DNA template places strains on the unusually high demand for substrate specificity. Although the chemical mechanism of this phosphoryl transfer reaction is well-defined ( 1), several questions remain as to how most DNA polymerases maintain an incredible degree of substrate fidelity during this reaction. These biophysical differences argue against a unified mechanism of translesion DNA synthesis and suggest that polymerases employ different catalytic strategies during the misreplication of damaged DNA.ĭNA polymerases catalyze the addition of dNTPs into a growing polymer (primer) using a DNA template as a guide for directing each incorporation event. The kinetic data obtained with the Klenow fragment are compared to that of the high fidelity bacteriophage T4 DNA polymerase and reveal distinct differences in the dynamics by which these non-natural nucleotides are incorporated opposite an abasic site. This reduction reflects minimal increases in K m values coupled with large decreases in k cat. As expected, the efficiency by which these non-natural nucleotides are incorporated opposite templating nucleobases is significantly reduced. While the measured K m values depend upon the size and π-electron density of the incoming nucleotide, k cat values are surprisingly independent of both biophysical features. In general, analogs that contain large substituent groups in conjunction with significant π-electron density display the highest catalytic efficiencies (k cat/K m) forincorporation. However, several 5-substituted indolyl nucleotides lacking classical hydrogen-bonding groups are incorporated ~100-fold more efficiently than the natural nucleotide. ![]() Like most other high fidelity DNA polymerases, the Klenow fragment follows the “A-rule” of translesion DNA synthesis by preferentially incorporating dATP opposite the non-instructional lesion. In this report, the mechanism and dynamics by which the Escherichia coli Klenow fragment performs translesion DNA synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. Translesion DNA synthesis represents the ability of a DNA polymerase to incorporate and extend beyond damaged DNA. ![]()
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