Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP

Fujikawa, Katsuyoshi; Kamiya, Hiroyuki; Yakushiji, Hiroyuki; Nakabeppu, Yusaku; Kasai, Hiroshi

doi:10.1093/nar/29.2.449

Cited by 119 publications

(92 citation statements)

References 23 publications

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“…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 To obtain steady-state kinetic parameters, reactions catalyzed by the Ε. coli RNA pol were carried out at 37°C for two to ten min.…”

Section: Methodsmentioning

confidence: 99%

“…Last, we examined the incorporation of another oxidized ribonucleotide, 2-OH-ATP [29]. 2-OH-ATP (500 µM) was added to the reaction mixture containing the primed template and RNA pol.…”

Section: Incorporation Of 2-oh-atp By Rna Polymerasesmentioning

confidence: 99%

“…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. …”

mentioning

confidence: 99%

“…Previously, Taddei 8-oxo-7,8-dihydroguanosine 5'-triphosphate), was incorporated into RNA [28] and concluded that 8-OH-GTP induced translational errors. The findings that the E. coli MutT, and mammalian MTH1 (NUDT1) and NUDT5 proteins catalyze the hydrolysis of oxidized RNA precursors suggest the importance of the hydrolytic elimination of oxidized ribonucleotides [28][29][30]. Moreover, disruption of the transcription factor S-II, which enhances the excision of ribonucleotides misincorporated by RNA polymerase (pol) II in vitro, confers sensitivity to oxidants together with low transcriptional fidelity in yeast, and antioxidant treatment relieves this low transcriptional fidelity [31].…”

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confidence: 99%

“…another oxidized ribonucleotide, 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP) [29], was incorporated "correctly" opposite T.…”

mentioning

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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Section: Methodsmentioning

confidence: 99%

“…Last, we examined the incorporation of another oxidized ribonucleotide, 2-OH-ATP [29]. 2-OH-ATP (500 µM) was added to the reaction mixture containing the primed template and RNA pol.…”

Section: Incorporation Of 2-oh-atp By Rna Polymerasesmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

“…another oxidized ribonucleotide, 2-hydroxyadenosine 5'-triphosphate (2-OH-ATP) [29], was incorporated "correctly" opposite T.…”

mentioning

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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Ein metallorganischer Inhibitor des humanen Reparaturenzyms 7,8‐Dihydro‐8‐oxoguanosin‐Triphosphatase

et al. 2013

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[5]Als Ausgangspunkt unserer Untersuchungen wurde der Organoruthenium-Halbsandwichkomplex 1 konzipiert, der zusätzlich zu dem zweizähnigen 8-(Pyridin-2-yl)adenin-Liganden eine h 5 -koordinierenden Cyclopentadienylgruppe und einen CO-Liganden enthält (Abbildung 1). Wir gingen dabei davon aus, dass die Organometallverbindung 1 als Sonde für Adeninnukleotid-bindende Proteine dienen kann, da sie sowohl eine Ruthenium-koordinierte Adenin-Nukleobase enthält, als auch durch ihre Molekülstruktur die dreidimensionale Form von Nukleotiden nachahmt. Zur Verifizierung dieser Hypothese wurde eine Technik verwendet, bei der ein biotinyliertes Acylphosphatderivat von ATP und ADP zum Einsatz kommt. Dieses kann mit konservierten LysinResten in den Bindungstaschen ATP-bindender Proteine irreversibel reagieren und ermçglicht damit die Bestimmung der direkten Konkurrenz zwischen der Bindung eine Inhibitors und der reaktiven Acylphosphatsonde und zwar in einer frei wählbaren biologischen Probe.[6] Das entsprechende Profiling der metallorganischen Verbindung 1 (100 mm) gegen

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An Organometallic Inhibitor for the Human Repair Enzyme 7,8‐Dihydro‐8‐oxoguanosine Triphosphatase

et al. 2013

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The probe‐based discovery of the first small‐molecule inhibitor of the repair enzyme 8‐oxo‐dGTPase (MTH1) is presented, which is an unconventional cyclometalated ruthenium half‐sandwich complex. The organometallic inhibitor with low‐nanomolar activity displays astonishing specificity, as verified in tests with an extended panel of protein kinases and other ATP binding proteins. The binding of the organometallic inhibitor to MTH1 is investigated by protein crystallography.

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Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP

Cited by 119 publications

References 23 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

Ein metallorganischer Inhibitor des humanen Reparaturenzyms 7,8‐Dihydro‐8‐oxoguanosin‐Triphosphatase

An Organometallic Inhibitor for the Human Repair Enzyme 7,8‐Dihydro‐8‐oxoguanosine Triphosphatase

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