Formation of 2-hydroxydeoxyadenosine triphosphate, an oxidatively damaged nucleotide, and its incorporation by DNA polymerases. Steady-state kinetics of the incorporation.
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.
Kamiya
H
H
Department of Environmental Oncology, University of Occupational and Environmental Health, Kitakyushu, Japan.
Kasai
H
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D018384:Oxidative Stress
C095141:2-hydroxydeoxyadenosine triphosphate; D003838:Deoxyadenine Nucleotides; D006147:Guanine; C012873:2'-deoxy-5'-adenosine monophosphate; D000255:Adenosine Triphosphate; D004247:DNA; D004492:Edetic Acid; C008183:isoguanine; D004257:DNA Polymerase II