Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA Adduct, during translesion synthesis catalyzed by human DNA polymerases |
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Authors: | Yasui Manabu Suenaga Emi Koyama Naoki Masutani Chikahide Hanaoka Fumio Gruz Petr Shibutani Shinya Nohmi Takehiko Hayashi Makoto Honma Masamitsu |
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Institution: | 1 Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan 2 Division of Pharmacognosy, Phytochemistry and Narcotics, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan 3 Cellular Biology Laboratory, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan 4 Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA |
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Abstract: | Chronic inflammation involving constant generation of nitric oxide ( NO) by macrophages has been recognized as a factor related to carcinogenesis. At the site of inflammation, nitrosatively deaminated DNA adducts such as 2′-deoxyinosine (dI) and 2′-deoxyxanthosine are primarily formed by NO and may be associated with the development of cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol α was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol η and pol κΔC (a truncated form of pol κ) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol α, pol η, and pol κΔC, promoted preferential incorporation of 2′-deoxycytidine monophosphate (dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of 2′-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol α, pol η, and pol κΔC indicated that dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A → G transition. This NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis. |
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Keywords: | els-cdn NO" target="_blank">com/sd/entities/rad" class="glyphImg">NO nitric oxide dI 2&prime -deoxyinosine dX 2&prime -deoxyxanthosine dA 2&prime -deoxyadenosine 8-OxodG 8-oxo-2&prime -deoxyguanosine dNTP 2&prime -deoxynucleoside triphosphate Alexa546 Alexa Fluor 546 dye pol α human DNA polymerase α pol η human DNA polymerase η pol κ human DNA polymerase κ pol κΔC a truncated form of pol κ Fins frequency of insertion Fext frequency of extension 8-NO2-dG 8-nitro-2&prime -deoxyguanosine dCMP 2&prime -deoxycytidine monophosphate dTMP 2&prime -deoxythymidine monophosphate dTTP 2&prime -deoxythymidine triphosphate dCTP 2&prime -deoxycytidine triphosphate dGTP 2&prime -deoxyguanosine triphosphate Cy3 Cyanin 3 endo V endonuclease V |
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