Bypass of N-2-ethylguanine by human DNA polymerase kappa

作者：Pence Matthew G; Blans Patrick; Zink Charles N; Fishbein James C; Perrino Fred W^*

来源：DNA Repair, 2011, 10(1): 56-64.

DOI：10.1016/j.dnarep.2010.09.007

• Y-family DNA polymerase
• Translesion synthesis
• Alkylation DNA damage
• Metal ion activation
• Rapid kinetic analysis
• DNA polymerase kappa

The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) kappa past N-2-ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol kappa 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 kappa at the insertion and extension steps of N-2-ethyl-Gua bypass. The DNA pol K was maximally activated to similar levels by a twenty-fold lower concentration of Mg2+ compared to Mg2+. In addition, the steady state analysis indicated that high fidelity DNA pol kappa-catalyzed N-2-ethyl-Gua bypass is Mg2+-dependent. Strikingly, Mn2+ activation of DNA pol kappa resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N-2-ethyl-Gua and Gua compared to that detected upon Mg2+ 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 Mg2+ activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn2+ compared to Mg2+ indicating that DNA pol K preferentially utilizes Mg2+ activation. These kinetic data support a DNA pol kappa wobble base pairing mechanism for dCTP incorporation opposite N-2-ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols kappa and iota in the presence of Mg2+ suggest a metal ion-dependent regulation in coordinating the activities of these DNA pots during translesion synthesis.

• 出版日期2011-1-2