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  • 93
    Jena Bioscience 8 oxo dgtp
    (A) Ribbon diagram of the Vc MutT X-ray structure with β-strands in firebrick red, α-helices in brown and loops in grey. (B) Homology model of Vc MutT-closed (grey) made from Ec <t>MutT-8-oxo-dGTP-Mn</t> superimposed on Vc MutT (firebrick red). 8-oxo-dGMP and Mn 2+ are modelled from the Ec MutT-8-oxo-dGTP-Mn structure. (C) Hydrogen bonding interactions between 8-oxo-dGMP and Vc MutT (crystal structure in firebrick red and closed homology model in grey). The super imposed As MutT models (closed in dark-grey and open in sky-blue) are also shown. Residues involved in Mn 2+ coordination and residues promoting conformational stabilization to the enzymes structure are indicated. (D) Hydrogen bonds between the Vc MutT-closed model and 8-oxo-dGMP. Electrostatic surface of the homology model of (E) Vc MutT-closed and (F) As MutT-closed with important residue differences highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    8 Oxo Dgtp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs 7 deaza dgtp
    Hybrid G-quadruplexes form during transcription and are dependent on CSB II. ( A ) In vitro transcription using the mitochondrial transcription apparatus on supercoiled templates containing either wildtype or mutant CSB II. Transcription reactions were divided into three parts, and either not treated or treated with RNase A and/or hRNaseH1, as indicated. RNase A-treatment reveals a non-degradable product of 45–50 bp (marked by asterisks) that is dependent on the CSB II sequence and Hoogsteen base pairing. ( B ) The labeled CSB II RNA oligonucleotide was allowed to form G-quadruplexes alone (lanes 1–3), with wildtype CSB II DNA oligonucleotide of identical sequence (lanes 4–6), or with a reverse complement DNA oligonucleotide (lanes 7–9), and then subjected to treatment with RNase A or hRNaseH1 in order to show that the G-quadruplex hybrid is resistant to hRNaseH1 (lane 6). The Watson–Crick basepaired hybrid with reverse complement DNA was degraded by hRNaseH1 as expected (lane 9). M, marker lane. ( C ) Transcription on templates encompassing base positions 1–477 of human mtDNA and containing either <t>dGTP</t> or <t>7-deaza-dGTP</t> (lanes 1 and 2). After transcription, part of each reaction was treated with RNaseA (lanes 3 and 4). The RNaseA-resistant species of ∼50 bp (indicated by black bar) are absent when G-quadruplex formation with the template is eliminated (lane 3). M, marker lane.
    7 Deaza Dgtp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dgtp
    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 <t>P]dGTP</t> (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released <t>dGMP</t> from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
    Dgtp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer dgtp
    A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing <t>telomerase</t> reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 <t>P-dGTP</t> for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.
    Dgtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 2794 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TriLink o6 methyl dgtp
    MTH1 is an efficient catalyst of <t>O6-methyl-dGTP</t> hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
    O6 Methyl Dgtp, supplied by TriLink, used in various techniques. Bioz Stars score: 99/100, based on 405 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore dgtp
    MTH1 is an efficient catalyst of <t>O6-methyl-dGTP</t> hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM <t>O6-methyl-GTP</t> and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.
    Dgtp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 1005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dgtp  (TaKaRa)
    97
    TaKaRa dgtp
    The PCR product of a foreign gene was amplified by <t>T4</t> DNA polymerase and <t>dGTP,</t> and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.
    Dgtp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare 7 deaza dgtp
    The PCR product of a foreign gene was amplified by <t>T4</t> DNA polymerase and <t>dGTP,</t> and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.
    7 Deaza Dgtp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 716 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TriLink 8 oxo dgtp
    NUDT5 is a key regulator of ADP-ribose metabolism. a Hydrolysis of potential oxidized nucleotides and nucleotide-sugar substrates by MTH1 (blue) and NUDT5 (red), as measured by the enzyme-coupled malachite green assay (MG assay), at pH 7.5. A representative experiment (of n = 2) with mean ± SD and individual values of quadruplicate replicates is shown. b Representative HPLC traces of NUDT5-mediated ADPR (red) and <t>8-oxo-dGDP</t> (blue) hydrolysis to AMP or 8-oxo-dGMP, respectively ( n = 2). c Top: Representative, pseudo-colored immunofluorescence stainings for γH2A.X, chromatin-bound RPA, and 53BP1 in U-2 OS cells following treatment with negative control siRNA (siNeg) or NUDT5 siRNA (siNUDT5 #1, siNUDT5 #7) for 72 h. Scale bar = 20 µm. Bottom: Quantification of γH2A.X, chromatin-bound RPA, and 53BP1 staining intensity in cells representing three independent experiments (cells scored: γH2A.X, n = 871; RPA, n = 463; 53BP1, n = 981). Lines represent median fluorescence (and interquartile range for 53BP1); a.u. arbitrary units. NS not significant, *** p
    8 Oxo Dgtp, supplied by TriLink, used in various techniques. Bioz Stars score: 92/100, based on 214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega set of datp dctp dgtp dttp
    NUDT5 is a key regulator of ADP-ribose metabolism. a Hydrolysis of potential oxidized nucleotides and nucleotide-sugar substrates by MTH1 (blue) and NUDT5 (red), as measured by the enzyme-coupled malachite green assay (MG assay), at pH 7.5. A representative experiment (of n = 2) with mean ± SD and individual values of quadruplicate replicates is shown. b Representative HPLC traces of NUDT5-mediated ADPR (red) and <t>8-oxo-dGDP</t> (blue) hydrolysis to AMP or 8-oxo-dGMP, respectively ( n = 2). c Top: Representative, pseudo-colored immunofluorescence stainings for γH2A.X, chromatin-bound RPA, and 53BP1 in U-2 OS cells following treatment with negative control siRNA (siNeg) or NUDT5 siRNA (siNUDT5 #1, siNUDT5 #7) for 72 h. Scale bar = 20 µm. Bottom: Quantification of γH2A.X, chromatin-bound RPA, and 53BP1 staining intensity in cells representing three independent experiments (cells scored: γH2A.X, n = 871; RPA, n = 463; 53BP1, n = 981). Lines represent median fluorescence (and interquartile range for 53BP1); a.u. arbitrary units. NS not significant, *** p
    Set Of Datp Dctp Dgtp Dttp, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega dgtp
    NUDT5 is a key regulator of ADP-ribose metabolism. a Hydrolysis of potential oxidized nucleotides and nucleotide-sugar substrates by MTH1 (blue) and NUDT5 (red), as measured by the enzyme-coupled malachite green assay (MG assay), at pH 7.5. A representative experiment (of n = 2) with mean ± SD and individual values of quadruplicate replicates is shown. b Representative HPLC traces of NUDT5-mediated ADPR (red) and <t>8-oxo-dGDP</t> (blue) hydrolysis to AMP or 8-oxo-dGMP, respectively ( n = 2). c Top: Representative, pseudo-colored immunofluorescence stainings for γH2A.X, chromatin-bound RPA, and 53BP1 in U-2 OS cells following treatment with negative control siRNA (siNeg) or NUDT5 siRNA (siNUDT5 #1, siNUDT5 #7) for 72 h. Scale bar = 20 µm. Bottom: Quantification of γH2A.X, chromatin-bound RPA, and 53BP1 staining intensity in cells representing three independent experiments (cells scored: γH2A.X, n = 871; RPA, n = 463; 53BP1, n = 981). Lines represent median fluorescence (and interquartile range for 53BP1); a.u. arbitrary units. NS not significant, *** p
    Dgtp, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Jena Bioscience dgtp
    Binding of mant-deoxyguanine nucleotides to EFL1. Mant fluorescence was excited by FRET from the intrinsic tryptophan residues in EFL1. Concentrations after mixing consisted of 4 μ m EFL1, 50 μ m <t>mant-deoxy-GDP/deoxy-GTP,</t> and 5 m m Mg 2+ as
    Dgtp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare dgtp
    Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , <t>ATP;</t> lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , <t>dGTP,</t> lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.
    Dgtp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 1730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dgtp bigdye terminator v3 0 ready reaction cycle sequencing kit
    Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , <t>ATP;</t> lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , <t>dGTP,</t> lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.
    Dgtp Bigdye Terminator V3 0 Ready Reaction Cycle Sequencing Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Meridian Life Science dgtp
    Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , <t>ATP;</t> lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , <t>dGTP,</t> lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.
    Dgtp, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 92/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare α 32 p dgtp
    Fig. 3. Comparison of native and recombinant telomerase. ( A ) Direct telomerase assay. Affinity-purified telomerase was incubated with (TTAGGG) 3 , dATP, dTTP and [α- 32 <t>P]dGTP</t> in presence of different concentrations of KCl. Telomerase products were separated on a sequencing gel. ( B ) Analysis of the native molecular mass of the telomerase RNP. Affinity-purified telomerase was fractionated by gel filtration on a Superose 6 column in running buffer containing 500 mM KCl and the activity of the fractions was determined using the TRAP assay. The void volume of the column and the elution volume of three marker proteins are indicated, together with the respective molecular masses and Stokes radii (in brackets).
    α 32 P Dgtp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 205 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Boehringer Mannheim 7 deaza dgtp
    Fig. 3. Comparison of native and recombinant telomerase. ( A ) Direct telomerase assay. Affinity-purified telomerase was incubated with (TTAGGG) 3 , dATP, dTTP and [α- 32 <t>P]dGTP</t> in presence of different concentrations of KCl. Telomerase products were separated on a sequencing gel. ( B ) Analysis of the native molecular mass of the telomerase RNP. Affinity-purified telomerase was fractionated by gel filtration on a Superose 6 column in running buffer containing 500 mM KCl and the activity of the fractions was determined using the TRAP assay. The void volume of the column and the elution volume of three marker proteins are indicated, together with the respective molecular masses and Stokes radii (in brackets).
    7 Deaza Dgtp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 91/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PerkinElmer biotin 11 dgtp
    Fig. 3. Comparison of native and recombinant telomerase. ( A ) Direct telomerase assay. Affinity-purified telomerase was incubated with (TTAGGG) 3 , dATP, dTTP and [α- 32 <t>P]dGTP</t> in presence of different concentrations of KCl. Telomerase products were separated on a sequencing gel. ( B ) Analysis of the native molecular mass of the telomerase RNP. Affinity-purified telomerase was fractionated by gel filtration on a Superose 6 column in running buffer containing 500 mM KCl and the activity of the fractions was determined using the TRAP assay. The void volume of the column and the elution volume of three marker proteins are indicated, together with the respective molecular masses and Stokes radii (in brackets).
    Biotin 11 Dgtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher dgtp bigdye terminator chemistry
    Fig. 3. Comparison of native and recombinant telomerase. ( A ) Direct telomerase assay. Affinity-purified telomerase was incubated with (TTAGGG) 3 , dATP, dTTP and [α- 32 <t>P]dGTP</t> in presence of different concentrations of KCl. Telomerase products were separated on a sequencing gel. ( B ) Analysis of the native molecular mass of the telomerase RNP. Affinity-purified telomerase was fractionated by gel filtration on a Superose 6 column in running buffer containing 500 mM KCl and the activity of the fractions was determined using the TRAP assay. The void volume of the column and the elution volume of three marker proteins are indicated, together with the respective molecular masses and Stokes radii (in brackets).
    Dgtp Bigdye Terminator Chemistry, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dntps
    Fig. 3. Comparison of native and recombinant telomerase. ( A ) Direct telomerase assay. Affinity-purified telomerase was incubated with (TTAGGG) 3 , dATP, dTTP and [α- 32 <t>P]dGTP</t> in presence of different concentrations of KCl. Telomerase products were separated on a sequencing gel. ( B ) Analysis of the native molecular mass of the telomerase RNP. Affinity-purified telomerase was fractionated by gel filtration on a Superose 6 column in running buffer containing 500 mM KCl and the activity of the fractions was determined using the TRAP assay. The void volume of the column and the elution volume of three marker proteins are indicated, together with the respective molecular masses and Stokes radii (in brackets).
    Dntps, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 52680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (A) Ribbon diagram of the Vc MutT X-ray structure with β-strands in firebrick red, α-helices in brown and loops in grey. (B) Homology model of Vc MutT-closed (grey) made from Ec MutT-8-oxo-dGTP-Mn superimposed on Vc MutT (firebrick red). 8-oxo-dGMP and Mn 2+ are modelled from the Ec MutT-8-oxo-dGTP-Mn structure. (C) Hydrogen bonding interactions between 8-oxo-dGMP and Vc MutT (crystal structure in firebrick red and closed homology model in grey). The super imposed As MutT models (closed in dark-grey and open in sky-blue) are also shown. Residues involved in Mn 2+ coordination and residues promoting conformational stabilization to the enzymes structure are indicated. (D) Hydrogen bonds between the Vc MutT-closed model and 8-oxo-dGMP. Electrostatic surface of the homology model of (E) Vc MutT-closed and (F) As MutT-closed with important residue differences highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Journal: FEBS Open Bio

    Article Title: MutT from the fish pathogen Aliivibrio salmonicida is a cold-active nucleotide-pool sanitization enzyme with unexpectedly high thermostability

    doi: 10.1016/j.fob.2015.01.006

    Figure Lengend Snippet: (A) Ribbon diagram of the Vc MutT X-ray structure with β-strands in firebrick red, α-helices in brown and loops in grey. (B) Homology model of Vc MutT-closed (grey) made from Ec MutT-8-oxo-dGTP-Mn superimposed on Vc MutT (firebrick red). 8-oxo-dGMP and Mn 2+ are modelled from the Ec MutT-8-oxo-dGTP-Mn structure. (C) Hydrogen bonding interactions between 8-oxo-dGMP and Vc MutT (crystal structure in firebrick red and closed homology model in grey). The super imposed As MutT models (closed in dark-grey and open in sky-blue) are also shown. Residues involved in Mn 2+ coordination and residues promoting conformational stabilization to the enzymes structure are indicated. (D) Hydrogen bonds between the Vc MutT-closed model and 8-oxo-dGMP. Electrostatic surface of the homology model of (E) Vc MutT-closed and (F) As MutT-closed with important residue differences highlighted. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Article Snippet: The results ( ) show that both As MutT and Vc MutT possess activity for 8-oxo-dGTP and can thus be considered as MutT enzymes.

    Techniques:

    Hybrid G-quadruplexes form during transcription and are dependent on CSB II. ( A ) In vitro transcription using the mitochondrial transcription apparatus on supercoiled templates containing either wildtype or mutant CSB II. Transcription reactions were divided into three parts, and either not treated or treated with RNase A and/or hRNaseH1, as indicated. RNase A-treatment reveals a non-degradable product of 45–50 bp (marked by asterisks) that is dependent on the CSB II sequence and Hoogsteen base pairing. ( B ) The labeled CSB II RNA oligonucleotide was allowed to form G-quadruplexes alone (lanes 1–3), with wildtype CSB II DNA oligonucleotide of identical sequence (lanes 4–6), or with a reverse complement DNA oligonucleotide (lanes 7–9), and then subjected to treatment with RNase A or hRNaseH1 in order to show that the G-quadruplex hybrid is resistant to hRNaseH1 (lane 6). The Watson–Crick basepaired hybrid with reverse complement DNA was degraded by hRNaseH1 as expected (lane 9). M, marker lane. ( C ) Transcription on templates encompassing base positions 1–477 of human mtDNA and containing either dGTP or 7-deaza-dGTP (lanes 1 and 2). After transcription, part of each reaction was treated with RNaseA (lanes 3 and 4). The RNaseA-resistant species of ∼50 bp (indicated by black bar) are absent when G-quadruplex formation with the template is eliminated (lane 3). M, marker lane.

    Journal: Nucleic Acids Research

    Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

    doi: 10.1093/nar/gks802

    Figure Lengend Snippet: Hybrid G-quadruplexes form during transcription and are dependent on CSB II. ( A ) In vitro transcription using the mitochondrial transcription apparatus on supercoiled templates containing either wildtype or mutant CSB II. Transcription reactions were divided into three parts, and either not treated or treated with RNase A and/or hRNaseH1, as indicated. RNase A-treatment reveals a non-degradable product of 45–50 bp (marked by asterisks) that is dependent on the CSB II sequence and Hoogsteen base pairing. ( B ) The labeled CSB II RNA oligonucleotide was allowed to form G-quadruplexes alone (lanes 1–3), with wildtype CSB II DNA oligonucleotide of identical sequence (lanes 4–6), or with a reverse complement DNA oligonucleotide (lanes 7–9), and then subjected to treatment with RNase A or hRNaseH1 in order to show that the G-quadruplex hybrid is resistant to hRNaseH1 (lane 6). The Watson–Crick basepaired hybrid with reverse complement DNA was degraded by hRNaseH1 as expected (lane 9). M, marker lane. ( C ) Transcription on templates encompassing base positions 1–477 of human mtDNA and containing either dGTP or 7-deaza-dGTP (lanes 1 and 2). After transcription, part of each reaction was treated with RNaseA (lanes 3 and 4). The RNaseA-resistant species of ∼50 bp (indicated by black bar) are absent when G-quadruplex formation with the template is eliminated (lane 3). M, marker lane.

    Article Snippet: The fragment was PCR amplified either in the presence of dGTP or in the presence of 7-deaza-dGTP.

    Techniques: In Vitro, Mutagenesis, Sequencing, Labeling, Marker

    G-quadruplex formation in nascent RNA, but not in template DNA, causes premature termination of transcription. In vitro transcription with the mitochondrial transcription apparatus was carried out in the presence or absence of 7-deaza-GTP to monitor effects of G-quadruplex formation in RNA. To address the effects of G-quadruplex formation in DNA, the DNA templates used were produced by PCR amplification of positions 1–512 of human mtDNA in the presence or absence of 7-deaza-dGTP.

    Journal: Nucleic Acids Research

    Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

    doi: 10.1093/nar/gks802

    Figure Lengend Snippet: G-quadruplex formation in nascent RNA, but not in template DNA, causes premature termination of transcription. In vitro transcription with the mitochondrial transcription apparatus was carried out in the presence or absence of 7-deaza-GTP to monitor effects of G-quadruplex formation in RNA. To address the effects of G-quadruplex formation in DNA, the DNA templates used were produced by PCR amplification of positions 1–512 of human mtDNA in the presence or absence of 7-deaza-dGTP.

    Article Snippet: The fragment was PCR amplified either in the presence of dGTP or in the presence of 7-deaza-dGTP.

    Techniques: In Vitro, Produced, Polymerase Chain Reaction, Amplification

    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Article Snippet: As standards, unlabeled dGMP (Sigma) and dGTP (Invitrogen) were spiked into each sample and run concurrently as markers.

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

    Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Article Snippet: As standards, unlabeled dGMP (Sigma) and dGTP (Invitrogen) were spiked into each sample and run concurrently as markers.

    Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

    Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Article Snippet: As standards, unlabeled dGMP (Sigma) and dGTP (Invitrogen) were spiked into each sample and run concurrently as markers.

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker

    A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing telomerase reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 P-dGTP for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.

    Journal: The EMBO Journal

    Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

    doi: 10.1038/emboj.2011.363

    Figure Lengend Snippet: A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing telomerase reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 P-dGTP for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.

    Article Snippet: In brief, 2–3 μl of in vitro reconstituted telomerase was assayed in a 10-μl reaction containing 1 × telomerase reaction buffer, 1 mM dTTP, 1 mM dATP, 2 μM dGTP, 0.165 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 1 μM (TTAGGG)3 DNA primer.

    Techniques: Pulse Chase, Generated

    Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.

    Journal: Scientific Reports

    Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol

    doi: 10.1038/s41598-018-37439-0

    Figure Lengend Snippet: Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.

    Article Snippet: Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dATP and [α-32 P]dGTP (3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA).

    Techniques: Variant Assay, Autoradiography, Activity Assay, Labeling, Concentration Assay, Synthesized

    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Activity Assay, Software, Two Tailed Test, Produced, Concentration Assay

    Activity of MTH1 with O6-methyl-dGTP has been conserved through evolution. ( A ) Comparison of activities of MTH1 (NUDT1) (1.5 nM) from different species with 75 μM 8-oxo-dGTP and 75 μM O6-methyl-dGTP (hsNUDT1, human NUDT1; mmNUDT1, mouse NUDT1; rnNUDT1, rat NUDT1; ssNUDT1, pig NUDT1; clNUDT1, dog NUDT1; atNUDX1, Arabidopsis thaliana NUDT1; zfNUDT1, zebrafish MTH1; MutT, E. coli MutT). Reaction was performed in MTH1 reaction buffer and reaction time was 15 min. Formed PPi was detected using PPiLight Inorganic Pyrophosphate Assay kit from Lonza. Data points were recorded in triplicate. ( B ) Ratio between activities with O6-methyl-dGTP and 8-oxo-dGTP for MTH1 from different species.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Activity of MTH1 with O6-methyl-dGTP has been conserved through evolution. ( A ) Comparison of activities of MTH1 (NUDT1) (1.5 nM) from different species with 75 μM 8-oxo-dGTP and 75 μM O6-methyl-dGTP (hsNUDT1, human NUDT1; mmNUDT1, mouse NUDT1; rnNUDT1, rat NUDT1; ssNUDT1, pig NUDT1; clNUDT1, dog NUDT1; atNUDX1, Arabidopsis thaliana NUDT1; zfNUDT1, zebrafish MTH1; MutT, E. coli MutT). Reaction was performed in MTH1 reaction buffer and reaction time was 15 min. Formed PPi was detected using PPiLight Inorganic Pyrophosphate Assay kit from Lonza. Data points were recorded in triplicate. ( B ) Ratio between activities with O6-methyl-dGTP and 8-oxo-dGTP for MTH1 from different species.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Activity Assay, Pyrophosphate Assay

    Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Activity Assay

    Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Article Snippet: Some hydrolysis of O6-methyl-dGTP was also observed by NUDT17 and NUDT18 but only when using 200 nM enzyme in the assay (Figure ).

    Techniques: Incubation, Software, Injection, Fluorescence In Situ Hybridization, Inhibition, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: MTH1 is an efficient catalyst of O6-methyl-dGTP hydrolysis. ( A ) Activity assessment of MTH1 with O6-methyl-dGTP and N2-methyl-dGTP in comparison to dGTP and 8-oxo-dGTP. Activity of 5 nM MTH1 was tested with 50 μM substrate in MTH1 reaction buffer with PPase (0.2 U/ml). Formed Pi was detected using the malachite green reagent. Activity differences between samples in quadruplicate were found to be statistically significant by multiple comparisons using One way Anova in the GraphPad Prism 6.0 software. ( B ) Activity of MTH1 (45 nM) with 50 μM O6-methyl-GTP and GTP was measured as in (A) with samples in quadruplicate. Statistic significance was analysed using Paired Two-tailed T-test using the GraphPad Prism 6.0 software. ( C ) Saturation curve for MTH1 with O6-methyl-dGTP were produced by determining initial rates using 1.25 nM MTH1 and O6-methyl-dGTP ranging in concentration between 0 and 40 μM. ( D ) Saturation curve for MTH1 with O6-methyl-GTP. 50 nM MTH1 and O6-methyl-GTP ranging from 0 to 400 μM were used. Shown are representative saturation curves out of two independent experiments for each substrate with data points recorded in duplicate. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: The nucleotides included in the substrate screen were: dATP (Sigma Aldrich, DNTP-100), ATP (Sigma Aldrich, A26209), N1-methyl-ATP (Jena Biosciences NU-1027), N6-methyl-ATP (Jena Biosciences, NU-1101), UTP (Sigma Aldrich, U1006), 5-methyl-UTP (Jena Biosciences, NU-880), dCTP (Sigma Aldrich, DNTP-100), 5-methyl-dCTP (TriLink Biotechnologies, N-2026), 5-methyl-CTP (Jena Biosciences, NU-1138), GTP (Sigma Aldrich, G3776), 7-methyl-GTP (Sigma Aldrich, M6133), O6-methyl-GTP (TriLink Biotechnologies, N-1031), dGTP (Sigma Aldrich, 27-1870-04) and for comparison O6-methyl-dGTP (TriLink Biotechnologies, N-2027).

    Techniques: Activity Assay, Software, Two Tailed Test, Produced, Concentration Assay

    Activity of MTH1 with O6-methyl-dGTP has been conserved through evolution. ( A ) Comparison of activities of MTH1 (NUDT1) (1.5 nM) from different species with 75 μM 8-oxo-dGTP and 75 μM O6-methyl-dGTP (hsNUDT1, human NUDT1; mmNUDT1, mouse NUDT1; rnNUDT1, rat NUDT1; ssNUDT1, pig NUDT1; clNUDT1, dog NUDT1; atNUDX1, Arabidopsis thaliana NUDT1; zfNUDT1, zebrafish MTH1; MutT, E. coli MutT). Reaction was performed in MTH1 reaction buffer and reaction time was 15 min. Formed PPi was detected using PPiLight Inorganic Pyrophosphate Assay kit from Lonza. Data points were recorded in triplicate. ( B ) Ratio between activities with O6-methyl-dGTP and 8-oxo-dGTP for MTH1 from different species.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Activity of MTH1 with O6-methyl-dGTP has been conserved through evolution. ( A ) Comparison of activities of MTH1 (NUDT1) (1.5 nM) from different species with 75 μM 8-oxo-dGTP and 75 μM O6-methyl-dGTP (hsNUDT1, human NUDT1; mmNUDT1, mouse NUDT1; rnNUDT1, rat NUDT1; ssNUDT1, pig NUDT1; clNUDT1, dog NUDT1; atNUDX1, Arabidopsis thaliana NUDT1; zfNUDT1, zebrafish MTH1; MutT, E. coli MutT). Reaction was performed in MTH1 reaction buffer and reaction time was 15 min. Formed PPi was detected using PPiLight Inorganic Pyrophosphate Assay kit from Lonza. Data points were recorded in triplicate. ( B ) Ratio between activities with O6-methyl-dGTP and 8-oxo-dGTP for MTH1 from different species.

    Article Snippet: The nucleotides included in the substrate screen were: dATP (Sigma Aldrich, DNTP-100), ATP (Sigma Aldrich, A26209), N1-methyl-ATP (Jena Biosciences NU-1027), N6-methyl-ATP (Jena Biosciences, NU-1101), UTP (Sigma Aldrich, U1006), 5-methyl-UTP (Jena Biosciences, NU-880), dCTP (Sigma Aldrich, DNTP-100), 5-methyl-dCTP (TriLink Biotechnologies, N-2026), 5-methyl-CTP (Jena Biosciences, NU-1138), GTP (Sigma Aldrich, G3776), 7-methyl-GTP (Sigma Aldrich, M6133), O6-methyl-GTP (TriLink Biotechnologies, N-1031), dGTP (Sigma Aldrich, 27-1870-04) and for comparison O6-methyl-dGTP (TriLink Biotechnologies, N-2027).

    Techniques: Activity Assay, Pyrophosphate Assay

    Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Hydrolysis activity of MTH1 with O6-methyl-dGTP is exclusive among NUDIX hydrolases. Activity screen of human NUDIX proteins with 50 μM O6-methyl-dGTP was tested using 0, 5, or 200 nM NUDIX enzyme in presence of an excess of PPase ( A ) monitoring formation of Pi and PPi, or without coupled enzyme ( B ) detecting formation of Pi. Pi was detected using malachite green reagent and measurement of absorbance at 630 nm. Data points were recorded in triplicate. Statistically significant differences in activity compared to the mock control was assessed using multiple comparison and two-way ANOVA in GraphPad Prism 6.0. P ≤ 0.05 are considered to be statistically significant and are indicated by *, P ≤ 0.01 are indicated by **, P ≤ 0.001 are indicated by *** and P ≤ 0.0001 are indicated by ****.

    Article Snippet: The nucleotides included in the substrate screen were: dATP (Sigma Aldrich, DNTP-100), ATP (Sigma Aldrich, A26209), N1-methyl-ATP (Jena Biosciences NU-1027), N6-methyl-ATP (Jena Biosciences, NU-1101), UTP (Sigma Aldrich, U1006), 5-methyl-UTP (Jena Biosciences, NU-880), dCTP (Sigma Aldrich, DNTP-100), 5-methyl-dCTP (TriLink Biotechnologies, N-2026), 5-methyl-CTP (Jena Biosciences, NU-1138), GTP (Sigma Aldrich, G3776), 7-methyl-GTP (Sigma Aldrich, M6133), O6-methyl-GTP (TriLink Biotechnologies, N-1031), dGTP (Sigma Aldrich, 27-1870-04) and for comparison O6-methyl-dGTP (TriLink Biotechnologies, N-2027).

    Techniques: Activity Assay

    Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Journal: Nucleic Acids Research

    Article Title: MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

    doi: 10.1093/nar/gky896

    Figure Lengend Snippet: Active zfMTH1 is crucial for zebrafish embryo survival after O6-methyl-dGTP exposure. ( A ) The zfMTH1 enzyme catalyzes the hydrolysis of O6-methyl-dGTP efficiently. 100 μM dGTP or O6-methyl-dGTP was incubated with 5 nM zfMTH1 or hMTH1 for 20 min. Formed PPi was converted to Pi by using an excess of E. coli PPase and Pi was detected using malachite green reagent. Statistical significance was determined using multiple comparison and Two way Anova using the GraphPad Prism 6.0 software. ( B ) O6-methyl-dGTP (150 μM) was injected into fertilized zebra fish eggs followed by treatment with DMSO, TH588 (1.5 μM) or TH1579 (1.5 μM). Picture shows zebrafish embryos from a representative experiment. ( C ) Quantification of zebrafish survival. Inhibition of zfMTH1 in combination with microinjecting O6-methyl-dGTP in zebrafish is clearly toxic to fish embryos. Graph shows average and standard deviations from three independent experiments. ( D ) Levels of O6-methyl-dG per million dN in DNA as measured by LC–MS/MS. DNA was extracted from DMSO or TH588 treated zeb rafish embryos, zebrafish embryos microinjected with O6-methyl-dGTP or from O6-methyl-dGTP microinjected and TH588 treated zebrafish embryos. Graph shows mean and SEM from two independent experiments. Statistic significance in C and D was tested using multiple comparisons and One way Anova, P ≤ 0.05 are indicated by *. ( E ) Percentage dead embryos after microinjection of O6-methyl-dGTP and inhibition of zfMTH1 and MGMT through treatment with TH588 (1.5 μM) and Lomeguatrib (10 μM), alone and in combination, compared to untreated zebrafish embryos. Graph shows average ± SD from three independent experiments. ( F ) Co-treatment of O6-methyl-dGTP injected zebrafish eggs with TH588 and Lomeguatrib significantly decreases the survival of zebrafish embryos compared to the effects of the combined individual treatments.

    Article Snippet: The nucleotides included in the substrate screen were: dATP (Sigma Aldrich, DNTP-100), ATP (Sigma Aldrich, A26209), N1-methyl-ATP (Jena Biosciences NU-1027), N6-methyl-ATP (Jena Biosciences, NU-1101), UTP (Sigma Aldrich, U1006), 5-methyl-UTP (Jena Biosciences, NU-880), dCTP (Sigma Aldrich, DNTP-100), 5-methyl-dCTP (TriLink Biotechnologies, N-2026), 5-methyl-CTP (Jena Biosciences, NU-1138), GTP (Sigma Aldrich, G3776), 7-methyl-GTP (Sigma Aldrich, M6133), O6-methyl-GTP (TriLink Biotechnologies, N-1031), dGTP (Sigma Aldrich, 27-1870-04) and for comparison O6-methyl-dGTP (TriLink Biotechnologies, N-2027).

    Techniques: Incubation, Software, Injection, Fluorescence In Situ Hybridization, Inhibition, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Journal: Nucleic Acids Research

    Article Title: A novel and simple method for construction of recombinant adenoviruses

    doi: 10.1093/nar/gkl449

    Figure Lengend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Article Snippet: The amplified fragments were incubated with 0.5 U of T4 DNA polymerase and 4 mM dGTP (TaKaRa) at 12°C for 45 min, as described previously ( , ).

    Techniques: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay

    NUDT5 is a key regulator of ADP-ribose metabolism. a Hydrolysis of potential oxidized nucleotides and nucleotide-sugar substrates by MTH1 (blue) and NUDT5 (red), as measured by the enzyme-coupled malachite green assay (MG assay), at pH 7.5. A representative experiment (of n = 2) with mean ± SD and individual values of quadruplicate replicates is shown. b Representative HPLC traces of NUDT5-mediated ADPR (red) and 8-oxo-dGDP (blue) hydrolysis to AMP or 8-oxo-dGMP, respectively ( n = 2). c Top: Representative, pseudo-colored immunofluorescence stainings for γH2A.X, chromatin-bound RPA, and 53BP1 in U-2 OS cells following treatment with negative control siRNA (siNeg) or NUDT5 siRNA (siNUDT5 #1, siNUDT5 #7) for 72 h. Scale bar = 20 µm. Bottom: Quantification of γH2A.X, chromatin-bound RPA, and 53BP1 staining intensity in cells representing three independent experiments (cells scored: γH2A.X, n = 871; RPA, n = 463; 53BP1, n = 981). Lines represent median fluorescence (and interquartile range for 53BP1); a.u. arbitrary units. NS not significant, *** p

    Journal: Nature Communications

    Article Title: Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells

    doi: 10.1038/s41467-017-02293-7

    Figure Lengend Snippet: NUDT5 is a key regulator of ADP-ribose metabolism. a Hydrolysis of potential oxidized nucleotides and nucleotide-sugar substrates by MTH1 (blue) and NUDT5 (red), as measured by the enzyme-coupled malachite green assay (MG assay), at pH 7.5. A representative experiment (of n = 2) with mean ± SD and individual values of quadruplicate replicates is shown. b Representative HPLC traces of NUDT5-mediated ADPR (red) and 8-oxo-dGDP (blue) hydrolysis to AMP or 8-oxo-dGMP, respectively ( n = 2). c Top: Representative, pseudo-colored immunofluorescence stainings for γH2A.X, chromatin-bound RPA, and 53BP1 in U-2 OS cells following treatment with negative control siRNA (siNeg) or NUDT5 siRNA (siNUDT5 #1, siNUDT5 #7) for 72 h. Scale bar = 20 µm. Bottom: Quantification of γH2A.X, chromatin-bound RPA, and 53BP1 staining intensity in cells representing three independent experiments (cells scored: γH2A.X, n = 871; RPA, n = 463; 53BP1, n = 981). Lines represent median fluorescence (and interquartile range for 53BP1); a.u. arbitrary units. NS not significant, *** p

    Article Snippet: Activity of NUDT5 and MTH1 with a panel of potential substrates [2-OH-ATP, 2-OH-dATP, 8-oxo-dGDP, GDP and 8-oxo-GTP (Jena Biosciences), 8-oxo-dGTP (TriLink Biotechnologies), dATP (ThermoFisher Scientific), dGTP, GTP and ADPR (Sigma-Aldrich)] was assessed in reaction buffer (100 mM Tris Acetate pH 7.5, 40 mM NaCl, 10 mM Mg Acetate) at 22 °C.

    Techniques: Malachite Green Assay, High Performance Liquid Chromatography, Immunofluorescence, Recombinase Polymerase Amplification, Negative Control, Staining, Fluorescence

    Binding of mant-deoxyguanine nucleotides to EFL1. Mant fluorescence was excited by FRET from the intrinsic tryptophan residues in EFL1. Concentrations after mixing consisted of 4 μ m EFL1, 50 μ m mant-deoxy-GDP/deoxy-GTP, and 5 m m Mg 2+ as

    Journal: The Journal of Biological Chemistry

    Article Title: Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein *

    doi: 10.1074/jbc.M114.626275

    Figure Lengend Snippet: Binding of mant-deoxyguanine nucleotides to EFL1. Mant fluorescence was excited by FRET from the intrinsic tryptophan residues in EFL1. Concentrations after mixing consisted of 4 μ m EFL1, 50 μ m mant-deoxy-GDP/deoxy-GTP, and 5 m m Mg 2+ as

    Article Snippet: The fluorescent mant-derivatives of GDP, GTP, dGDP, dGTP, and GppNHp were obtained from Jena Biosciences (Jena, Germany).

    Techniques: Binding Assay, Fluorescence

    Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , ATP; lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , dGTP, lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.

    Journal: Leukemia research

    Article Title: Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues

    doi: 10.1016/j.leukres.2008.03.010

    Figure Lengend Snippet: Substrate specificity of yPAP toward various purine triphosphate analogues (A) Elongation of RNA primer 5′ UGU GCC CGA 3′ by yPAP. A mixture containing 200 nM 5′- 32 P-radiolabeled RNA primer, 4 U/μL yPAP, 250 μM analogue triphosphate, 20 mM Tris-HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl 2 , 0.2 mM EDTA, 100 μg/ml acetylated BSA, and 10% glycerol was incubated at 37 °C for 1 h. The products were analyzed by 20% dPAGE. Lane 1 , radiolabeled 10-bp DNA ladder; lane 2 , 5′- 32 P-radiolabeled unextended primer (no NTP); lane 3 , ATP; lane 4 , 2′-dATP; lane 5 , 3′-dATP; lane 6 , 2-Cl-ATP; lane 7 , 2-Cl-dATP; lane 8 , ara-ATP; lane 9 , F-ara-ATP; lane 10 , Cl-F-ara-ATP; lane 11 , Cl-F-dATP, lane 12 , GTP; lane 13 , dGTP, lane 14 , ara-GTP. (B) Graphical representation of RNA primer extension by yPAP with various modified triphosphates. Shown is the distribution of single extension products (white) and full extension products beyond first incorporation (hatched) as a percentage of the total counts in each lane, as determined using ImageQuant software. These experiments were conducted in triplicate with similar results.

    Article Snippet: ATP, 2′-dATP, GTP, and dGTP were obtained from Amersham Biosciences (Piscataway, NJ), and ara-ATP was obtained from Sigma-Aldrich (A-6642, St. Louis, MO).

    Techniques: Incubation, Acetylene Reduction Assay, Modification, Software

    Fig. 3. Comparison of native and recombinant telomerase. ( A ) Direct telomerase assay. Affinity-purified telomerase was incubated with (TTAGGG) 3 , dATP, dTTP and [α- 32 P]dGTP in presence of different concentrations of KCl. Telomerase products were separated on a sequencing gel. ( B ) Analysis of the native molecular mass of the telomerase RNP. Affinity-purified telomerase was fractionated by gel filtration on a Superose 6 column in running buffer containing 500 mM KCl and the activity of the fractions was determined using the TRAP assay. The void volume of the column and the elution volume of three marker proteins are indicated, together with the respective molecular masses and Stokes radii (in brackets).

    Journal: The EMBO Journal

    Article Title: Human telomerase contains two cooperating telomerase RNA molecules

    doi: 10.1093/emboj/20.13.3526

    Figure Lengend Snippet: Fig. 3. Comparison of native and recombinant telomerase. ( A ) Direct telomerase assay. Affinity-purified telomerase was incubated with (TTAGGG) 3 , dATP, dTTP and [α- 32 P]dGTP in presence of different concentrations of KCl. Telomerase products were separated on a sequencing gel. ( B ) Analysis of the native molecular mass of the telomerase RNP. Affinity-purified telomerase was fractionated by gel filtration on a Superose 6 column in running buffer containing 500 mM KCl and the activity of the fractions was determined using the TRAP assay. The void volume of the column and the elution volume of three marker proteins are indicated, together with the respective molecular masses and Stokes radii (in brackets).

    Article Snippet: Standard reaction conditions were 50 mM Tris–acetate pH 8.5, 50 mM KCl, 1 mM MgCl2 , 1 mM spermidine, 5 mM β-mercaptoethanol, 1 mM dATP, 1 mM dTTP, 2.5 µM dGTP, 15 µCi of [α-32 P]dGTP (3000 Ci/mmol; Amersham) and 2.5 µM (TTAGGG)3 .

    Techniques: Recombinant, Telomerase Assay, Affinity Purification, Incubation, Sequencing, Filtration, Activity Assay, TRAP Assay, Marker