Nucleic Acids Research Advance Access originally published online on November 27, 2006
Nucleic Acids Research 2006 34(22):6438-6449; doi:10.1093/nar/gkl761
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Nucleic Acids Research, 2006, Vol. 34, No. 22 6438-6449
© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Template properties of mutagenic cytosine analogues in reverse transcription
Department of Genomics and Proteomics, Okayama University Advanced Science Research Center Tsushima, Okayama 700-8530, Japan 1 School of Pharmacy, Shujitsu University Nishigawara, Okayama 703-8516, Japan 2 Division of Genetic Engineering, Research Institute for Biological Sciences OKAYAMA 7549-1, Kibichuo-Cho, Kaga-Gun, Okayama 716-1241, Japan 3 Medical Research Council, Laboratory of Molecular Biology Hills Road, Cambridge CB2 2QH, UK
*To whom correspondence should be addressed. Tel: +81 48 721 1155; Fax: +81 48 721 6718; Email: knegishi{at}nichiyaku.ac.jp
Received June 9, 2006. Revised September 25, 2006. Accepted September 26, 2006.
We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(ß-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N4-hydroxycytidine, N4-methoxycytidine, N4-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N4-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome.
Present address: Kazuo Negishi, Nihon Pharmaceutical University, 10281 Komuro, Ina, Kita-adachi-Gun, Saitama 362-0806, Japan
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