NMR studies of a DNA containing 8-hydroxydeoxyguanosine

Oda, Yasushi; Uesugi, Seichi; Ikehara, Morio; Nishimura, Susumu; Kawase, Yasutoshi; Ishikawa, Hiroyuki; Inoue, Hideo; Ohtsuka, Eiko

doi:10.1093/nar/19.7.1407

Cited by 215 publications

(203 citation statements)

References 24 publications

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“…The incorporation of 8-OH-GTP opposite A and that opposite C seemed to occur with similar efficiencies. This result is in agreement with 9 the biophysical observations that 8-OH-Gua can pair with C and A [7][8][9][10], and suggests that 8-OH-GTP is "mutagenic". …”

supporting

confidence: 91%

“…The highly reactive hydroxy radical and guanine-selective singlet oxygen are thought to be involved in the formation of 8-OH-Gua [6]. 8-OH-Gua can pair with both cytosine and adenine [7][8][9][10]. The oxidized nucleobase in DNA seems to be an important source of mutations, and it induces G:CT:A transversions in living cells [11][12][13][14][15][16][17][18][19].…”

Section: Introductionmentioning

confidence: 99%

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Incorporation of 8-hydroxyguanosine (8-oxo-7,8-dihydroguanosine) 5′-triphosphate by bacterial and human RNA polymerases

Kamiya

Suzuki

Yamaguchi

et al. 2009

Free Radical Biology and Medicine

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Oxidized RNA precursors formed in the nucleotide pool may be incorporated into RNA. In this study, the incorporation of is one of the most abundant ([5] and references therein). The highly reactive hydroxy radical and guanine-selective singlet oxygen are thought to be involved in the formation of 8-OH-Gua [6]. 8-OH-Gua can pair with both cytosine and adenine [7][8][9][10]. The oxidized nucleobase in DNA seems to be an important source of mutations, and it induces G:CT:A transversions in living cells [11][12][13][14][15][16][17][18][19]. The oxidation of dGTP in the nucleotide pool by reactive oxygen species is another significant source in the mutation process. 8-Hydroxy-2'-deoxyguanosine 5'-triphosphate In this study, we analyzed the incorporation of 8-OH-GTP by the E. coli and human RNA pols in vitro. We found that the oxidized ribonucleotide was incorporated into RNA opposite C and A in the template DNA. This result suggests that 8-OH-GTP is incorporated into RNA, resulting in transcriptional and translational errors. In contrast,another oxidized ribonucleotide, 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP) [29], was incorporated "correctly" opposite T. Materials and methods Materials 6E. coli RNA polymerase was purchased from Sigma-Aldrich (St.Louis, MO, USA). Human RNA pol II was purified from HeLa cells expressing Flag-and His-tagged human Rpb3 (the third subunit of RNA pol II), as described previously [32]. Ribonuclease inhibitor was from Takara (Kusatsu, Japan). 8-OH-GTP and 2-OH-ATP were prepared by the oxidation of GTP and ATP, respectively, as described previously [29]. The purities of the 8-OH-GTP and 2-OH-ATP were both estimated to be more than 99%, as determined by an HPLC analysis (data not shown). The unmodified ribonucleoside 5'-triphosphates were from GE HealthcareBio-Sciences (Piscataway, NJ, USA). The Alexa Fluor 647 (Alexa 647)-modified oligoribonucleotide and the unmodified oligodeoxyribonucleotides (Table 1) were purchased from Japan Bio Services (Asaka, Japan) and Invitrogen Japan (Tokyo, Japan), respectively, in purified forms. In vitro RNA synthesisAn RNA primer-extension assay, in which the elongation complexes were assembled on nucleic acid scaffolds [33][34][35][36][37][38] Hepes-NaOH (pH 7.9), 5 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.5 U/µl ribonuclease inhibitor, 500 µM NTP and the enzyme at 30°C. In both reactions, the RNA/DNA hybrid was incubated with RNA pol for 5 min to assemble the elongation complex (preincubation), and then the substrate ribonucleotide was added to start the reactions.Reactions were stopped by the addition of a two-fold volume of termination solution (90% formamide, 50 mM EDTA). Samples were heated at 98°C for 5 min and chilled on ice, and were then applied to an 8 M urea, 20% polyacrylamide gel. Fluorograms were obtained with a Ε. coli RNA pol were carried out at 37°C for two to ten min. Results Incorporation of 8-OH-GTP by E. coli RNA polymeraseTo monitor nucleotide incorporation, we utilized a primer-extension assay, in which the elon...

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supporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Incorporation of 8-hydroxyguanosine (8-oxo-7,8-dihydroguanosine) 5′-triphosphate by bacterial and human RNA polymerases

Kamiya

Suzuki

Yamaguchi

et al. 2009

Free Radical Biology and Medicine

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“…However, in that case, it was not caused by a tautomeric shift, since it appears that this oxidized base exists, under physiological conditions, in a single tautomeric N1-H, N7-H, 6,8-dioxo form [43][44][45]. The formation of the nonmutagenic Watson-Crick-type 8-oxoG : C pair with 8-oxodG in anti conformation and the mutagenic Hoogsteen-type 8-oxoG : A pair with 8-oxodG in syn conformation was proposed on the basis of NMR spectra of designed oligodeoxynucleotide complexes [46,47]. Hence, it is very likely that neighbouring bases shift the syn-anti equilibrium of 8-oxodG in the template, resulting in a change of efficiency for incorporation of dCTP versus dATP [42].…”

Section: Figure 4 Tautomeric Forms Of Igmentioning

confidence: 99%

Neighbouring bases in template influence base-pairing of isoguanine

Maciejewska

Lichota

Kuśmierek

2003

Biochemical Journal

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Assuming that the efficiency of the incorporation of 5-methyl-2h-deoxyisocytosine-5h triphosphate (dMiCTP) and dTTP opposite isoguanine (iG) is a measure of the proportion of the keto and enol tautomers of iG in the Thermus aquaticus DNA polymerase active centre, we studied the influence of temperature and iGneighbouring bases in the template on base-pairing of iG. On the basis of experiments with four sequences (3h-TXT-5h, 3h-GXG-5h, 3h-CXC-5h, 3h-CXT-5h, where X l iG) at 37, 50, 65 and 80 mC, we found that 3h-neighbours decrease the fraction of the keto tautomer in the order C G T, whereas temperature apparently does not influence the tautomeric equilibrium of iG. The variability of the ratio of incorporation of dMiCTP versus dTTP (5-20) primarily reflects the variability of K m values, since

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“…This oxidized G base is miscoding, due to its ability to form base pairs with A in addition to C [4][5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Roles of specialized DNA polymerases in mutagenesis by 8-hydroxyguanine in human cells

Kamiya

Yamaguchi

Suzuki

et al. 2010

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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NMR studies of a DNA containing 8-hydroxydeoxyguanosine

Cited by 215 publications

References 24 publications

Incorporation of 8-hydroxyguanosine (8-oxo-7,8-dihydroguanosine) 5′-triphosphate by bacterial and human RNA polymerases

Incorporation of 8-hydroxyguanosine (8-oxo-7,8-dihydroguanosine) 5′-triphosphate by bacterial and human RNA polymerases

Neighbouring bases in template influence base-pairing of isoguanine

Roles of specialized DNA polymerases in mutagenesis by 8-hydroxyguanine in human cells

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