Bypass of N²-ethylguanine by Human DNA Polymerase κ

DNA Repair (Amst). 2011 Jan 2;10(1):56-64. doi: 10.1016/j.dnarep.2010.09.007. Epub 2010 Oct 16.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biocatalysis
  • Catalytic Domain / genetics
  • DNA Adducts / chemistry
  • DNA Adducts / metabolism
  • DNA Replication
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxycytosine Nucleotides / metabolism
  • Guanine / analogs & derivatives*
  • Guanine / metabolism
  • Humans
  • Kinetics
  • Magnesium / metabolism
  • Manganese / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*

Substances

  • DNA Adducts
  • Deoxycytosine Nucleotides
  • Recombinant Fusion Proteins
  • N(2)-ethylguanine
  • 2'-deoxycytidine 5'-triphosphate
  • Manganese
  • Guanine
  • DNA-Directed DNA Polymerase
  • POLK protein, human
  • Magnesium