Bypass of N²-ethylguanine by human DNA polymerase ?.

ABSTRACT: The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) ? past N(2)-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol ? 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 ? at the insertion and extension steps of N(2)-ethyl-Gua bypass. The DNA pol ? 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 ?-catalyzed N(2)-ethyl-Gua bypass is Mg(2+)-dependent. Strikingly, Mn(2+) activation of DNA pol ? 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 ? preferentially utilizes Mg(2+) activation. These kinetic data support a DNA pol ? wobble base pairing mechanism for dCTP incorporation opposite N(2)-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols ? and ? in the presence of Mn(2+) suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis.

SUBMITTER: Pence MG 

PROVIDER: S-EPMC3010520 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature