Formation of 2-hydroxydeoxyadenosine triphosphate, an oxidatively damaged nucleotide, and its incorporation by DNA polymerases. Steady-state kinetics of the incorporation

J Biol Chem. 1995 Aug 18;270(33):19446-50. doi: 10.1074/jbc.270.33.19446.


We found that hydroxylation occurs at the C-2 position of adenine by oxygen radical treatment (Fe2+-EDTA) of dA, dATP, and single- and double-stranded DNA. This oxidatively damaged base, 2-hydroxyadenine, was produced 3-6-fold and 40-fold less than 8-hydroxyguanine when monomers and polynucleotides, respectively, were treated. To determine whether the damaged nucleotide, 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP), is incorporated into a growing DNA, and to reveal the kinds of nucleotides opposite which 2-OH-dATP is incorporated, calf thymus DNA polymerase alpha and the Klenow fragment of Escherichia coli DNA polymerase I were used in vitro DAN synthesis in the presence of 2-OH-dATP. DNA polymerase alpha incorporated the nucleotide opposite T and C in the DNA template. On the other hand, in an experiment using the Klenow fragment, incorporation of 2-OH-dATP was observed only opposite T. Steady-state kinetic studies indicated that incorporation of 2-OH-dATP by DNA polymerase alpha opposite T was favored over that opposite C by a factor of only 4.5. These results indicate that 2-OH-dATP, an oxidatively damaged nucleotide, is a substrate for DNA polymerases and is incorporated incorrectly by the replicative DNA polymerase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / metabolism
  • Base Sequence
  • DNA / biosynthesis
  • DNA Polymerase II / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Edetic Acid / metabolism
  • Guanine / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Oxidative Stress*


  • 2-hydroxydeoxyadenosine triphosphate
  • Deoxyadenine Nucleotides
  • Guanine
  • 2'-deoxy-5'-adenosine monophosphate
  • Adenosine Triphosphate
  • DNA
  • Edetic Acid
  • isoguanine
  • DNA Polymerase II