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

Abstract:

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.

Polymerases:

Topics:

Kinetic Parameters, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol alpha Kamiya H1995 KM 0.2uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Steady State
Human Pol alpha Kamiya H1995 KM 89uM Reaction: Nucleotide incorporation; Substrate: ATP analog; Technique: Steady State
Human Pol alpha Kamiya H1995 Vmax 1.1 /minute Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Steady State
Human Pol alpha Kamiya H1995 Vmax 1.7 /minute Reaction: Nucleotide incorporation; Substrate: ATP analog; Technique: Steady State
Klenow fragment Kamiya H1995 KM 0.24uM Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Steady State
Klenow fragment Kamiya H1995 KM 1.8uM Reaction: Nucleotide incorporation; Substrate: ATP analog; Technique: Steady State
Klenow fragment Kamiya H1995 Vmax 14 /minute Reaction: Nucleotide incorporation; Substrate: dNTPs; Technique: Steady State
Klenow fragment Kamiya H1995 Vmax 23 /minute Reaction: Nucleotide incorporation; Substrate: ATP analog; Technique: Steady State

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