Bypass of N2-ethylguanine by human DNA polymerase κ

Abstract

The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N 2-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3′ termini opposite N 2-ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N 2-ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn 2+ compared to Mg 2+. In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N 2-ethyl-Gua bypass is Mg 2+-dependent. Strikingly, Mn 2+ activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N 2-ethyl-Gua and Gua compared to that detected upon Mg 2+ activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high K m values for dCTP insertion opposite N 2-ethyl-Gua and Gua with Mn 2+ activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn 2+ compared to Mg 2+ indicating that DNA pol κ preferentially utilizes Mg 2+ activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N 2-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn 2+ suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis.