Effects of 8-hydroxy-GTP and 2-hydroxy-ATP on in vitro transcription

Kamiya, Hiroyuki; Suzuki, Akihiro; Kawai, Kazuaki; Kasai, Hiroshi; Harashima, Hideyoshi

doi:10.1016/j.freeradbiomed.2007.05.034

Cited by 9 publications

(5 citation statements)

References 46 publications

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“…Conditions of oxidative stress induced by 1 mM L-ascorbic acid and 10 µM copper sulfate are sufficient to induce formation of oxo 8 GTP without oxidation of the Gua incorporated in DNA [13]. These results support a growing body of evidence that oxo 8 GTP is a modulator of cellular targets of GTP including GTP-binding proteins, GTP cyclohydrolase, and RNA production [17,18,31,32]. …”

Section: Discussionmentioning

confidence: 55%

“…Recent studies have shown that addition of oxo 8 GTP to in vitro transcription reactions reduces the total amount of mRNA synthesized as well as increases the number of mutations in the mRNA produced [17]. Additionally, oxo 8 GTP was shown to increase the activity of the GTP-binding protein Ras as well as downstream activators of the Ras-ERK pathway in HEK293T cells, while the opposite effect was observed with the GTP-binding protein Rac1 [18].…”

Section: Introductionmentioning

confidence: 99%

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Characterization of oxidized guanosine 5′-triphosphate as a viable inhibitor of soluble guanylyl cyclase

Bolin

Cardozo-Pelaez

2009

Free Radical Biology and Medicine

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The guanine base is liable for oxidation by free radicals regardless of the cellular moiety it is bound to. However, under conditions of oxidative stress, oxo8GTP formation has been shown to occur without oxidation to the guanine base in DNA. In vitro studies have suggested that oxo8GTP could impact G-protein signaling and RNA synthesis. Whether increased levels of oxo8GTP translate into cellular malfunction is unknown. Data presented herein shows that oxo8GTP is formed in cell-free preparations as well as in PC12 cells after exposure to physiologically relevant oxidative conditions generated with 10 µM copper sulphate and 1 mM L-Ascorbic Acid (Cu/Asc). We also determined that oxo8GTP has biological activity as a potent inhibitor of nitric-oxide stimulated soluble guanylyl cyclase (sGC). The increase in oxo8GTP formation in purified GTP and PC12 cells exposed to Cu/Asc caused a significant reduction in the product of sGC activity, cGMP. This oxidation of GTP was attenuated by addition of reduced glutathione under these same Cu/Asc conditions thus preventing the decrease in sGC activity. This suggests that oxo8GTP is produced by free radicals in vivo and could have significant impact on cell functions regulated by sGC activity such as synaptic plasticity in the central nervous system.

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Section: Discussionmentioning

confidence: 55%

Section: Introductionmentioning

confidence: 99%

Characterization of oxidized guanosine 5′-triphosphate as a viable inhibitor of soluble guanylyl cyclase

Bolin

Cardozo-Pelaez

2009

Free Radical Biology and Medicine

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“…However, the reduction of intracellular ATP levels after exposure to 2-Cl-Ado is caused by not only consumption of ATP itself but also deamination of AMP 6 , and 8-Cl-ATP may inhibit mitochondrial ATP synthase via binding to the α tp β tp site of the enzyme 26 . It has been reported that 2-oxo-ATP reduces the amount of RNA synthesized in vitro and increases base substitutions 30 , suggesting that 2-oxo-ATP decreases transcription levels with increased production of abnormal proteins in vivo . In the present study, we demonstrated that exposure of T5v cells to 2-oxo-Ado significantly increased the 2-oxo-Ado level in cellular RNA, which was efficiently suppressed by overexpression of hMTH1 (Fig.…”

Section: Discussionmentioning

confidence: 99%

2-Oxoadenosine induces cytotoxicity through intracellular accumulation of 2-oxo-ATP and depletion of ATP but not via the p38 MAPK pathway

Asada

Ohta

Akimoto

et al. 2017

Sci Rep

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2-Oxoadenosine (2-oxo-Ado), an oxidized form of adenosine, is cytotoxic and induces growth arrest and cell death, which has potential as an anti-cancer drug. However, it is not well understood how 2-oxo-Ado exerts its cytotoxicity. We examined the effects of 2-oxo-Ado on non-tumour cells, namely immortalized mouse embryonic fibroblast lines, and investigated mechanisms by which 2-oxo-Ado exerts its cytotoxicity. We found that cell death induced by 2-oxo-Ado is classical caspase-dependent apoptosis, and requires its sequential intracellular phosphorylation catalysed by adenosine kinase (ADK) and adenylate kinase 2, resulting in intracellular accumulation of 2-oxo-ATP accompanied by accumulation of 2-oxo-Ado in RNA and depletion of ATP. Moreover, we showed that overexpression of MTH1, an oxidized purine nucleoside triphosphatase, prevents 2-oxo-Ado-induced cytotoxicity accompanied by suppression of accumulation of both intracellular 2-oxo-ATP and 2-oxo-Ado in RNA and recovery of ATP levels. We also found that 2-oxo-Ado activates the p38 MAPK pathway. However, siRNAs against Mkk3 and Mkk6, or treatment with several p38 MAPK inhibitors, except SB203580, did not prevent the cytotoxicity. SB203580 prevented intracellular phosphorylation of 2-oxo-Ado to 2-oxo-AMP, and an in vitro ADK assay revealed that SB203580 directly inhibits ADK activity, suggesting that some of the effects of SB203580 may depend on ADK inhibition.

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“…8-OH-GTP was previously used in in vitro transcription reactions conducted by RNA pols from phage T7, E. coli, and calf thymus [28,56,57]. Poly(dA-dT), E. coli and calf thymus DNAs, and linearized plasmid DNA were employed as templates in the previous studies, and thus a detailed analysis of 8-OH-GTP incorporation was difficult.…”

Section: Discussionmentioning

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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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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Effects of 8-hydroxy-GTP and 2-hydroxy-ATP on in vitro transcription

Abstract: Oxidation of RNA precursors may disturb genetic information. In this study, the effects of oxidized RNA precursors on in vitro transcription

Cited by 9 publications

References 46 publications

Characterization of oxidized guanosine 5′-triphosphate as a viable inhibitor of soluble guanylyl cyclase

Characterization of oxidized guanosine 5′-triphosphate as a viable inhibitor of soluble guanylyl cyclase

2-Oxoadenosine induces cytotoxicity through intracellular accumulation of 2-oxo-ATP and depletion of ATP but not via the p38 MAPK pathway

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

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