Nucleic Acids Research, 2002, Vol. 30, No. 2 569-573
© 2002 Oxford University Press
An oxidized nucleotide affects DNA replication through activation of protein kinases in Xenopus egg lysates
Graduate School of Science and Technology and 1Department of Biological Science, Faculty of Science, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555, Japan, 2Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan, 3Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan and 4Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8555, Japan
To elucidate the response to oxidative stress in eukaryotic cells, the effect of an oxidized nucleotide, 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), generated from dGTP with an active oxygen, on DNA synthesis was studied using a cell-free DNA replication system derived from Xenopus egg lysates with a single-stranded DNA template. Amounts of newly synthesized DNA were reduced according to the increasing concentration of 8-oxo-dGTP. Pulse labeling analysis revealed that 8-oxo-dGTP could delay DNA synthesis by reducing the rate of chain elongation. This delay was recovered by addition of a protein kinase inhibitor, staurosporine or bisindolylmaleimide I. These results indicate that a staurosporine- or bisindolylmaleimide I-sensitive protein kinase, such as a protein kinase C family member, may contribute to the delay of DNA synthesis by 8-oxo-dGTP. UV-irradiated single-stranded DNA also caused a delay of DNA synthesis on the undamaged template in the lysates. However, this delay was not recovered by staurosporine or bisindolylmaleimide I. Therefore, the mechanism of delay of DNA synthesis by 8-oxo-dGTP may be different from that by UV lesions. This is the first report that demonstrates an effect of an oxidized nucleotide on DNA replication in eukaryotes.
* To whom correspondence should be addressed. Tel: +81 096 342 3435; Fax: +81 096 342 3431; Email: izuta{at}gpo.kumamoto-u.ac.jp
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