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

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

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

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

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

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

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

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

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: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

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

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: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

Article Snippet: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

Techniques: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

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: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

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

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: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

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

Journal: PLoS ONE

Article Title: The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5?-Triphosphate towards Influenza A Virus Polymerase

doi: 10.1371/journal.pone.0068347

Figure Lengend Snippet: Inhibition of recombinant IAVpol using a 50-mer RNA template. (A) Principle of the reaction. Recombinant IAVpol (PA/PB1/PB2) was incubated in the presence of a 50-mer RNA template sequence derived from the 3′-end of the PA gene of the NanChang strain [12] . The 15-nt 5′vRNA oligo that is partially complementary to the 3′vRNA is needed as promoter for the enzyme. The 5′-pApG dinucleotide primer is extended and allows for multiple incorporation events of α- 33 P-GMP used as tracer (star). (B) Representative curves of inhibition potency of 3′dGTP and T-705 RTP against IAVpol RNA synthesis activity. IC 50 s were determined by adding increasing concentrations of each inhibitor, and quantitative analysis of the amount of remaining full length RNA product is expressed as % inhibition (see Materials and Methods ). Each experiment was conducted at least twice to calculate the average value and standard deviation. (C) The inhibition percentage was measured in the presence of a saturating concentration of T-705 RTP (100 µM), and either low (3 µM) or high (300 µM) concentration of one of the two purines GTP or ATP. (D) The same experiment as (C) instead with either low (3 µM) or high (300 µM) of pyrimidine UTP or CTP.

Article Snippet: 3′dGTP was purchased from Trilink (San Diego, CA), T-705 RTP and the nucleoprotein inhibitor were synthesized at Alios BioPharma.

Techniques: Inhibition, Recombinant, Incubation, Sequencing, Derivative Assay, Activity Assay, Standard Deviation, Concentration Assay

Journal: PLoS ONE

Article Title: The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5?-Triphosphate towards Influenza A Virus Polymerase

doi: 10.1371/journal.pone.0068347

Figure Lengend Snippet: GMP and T-705 RMP incorporation opposite C on template. (A) A 14-mer RNA template sequence (t14-1) was designed to favor the extension of the 5′-pApG dinucleotide primer (AG) to a 9-mer in the presence of CTP and UTP, or a 14-mer full-length product (FL) in the presence of the additional GTP that can be specifically incorporated at position +10. (B) Example of a polyacrylamide gel electrophoresis result showing the products of primer extension. In addition to 25 µM CTP and UTP, 100 µM of GTP (lane 4), T-705 RTP (lane 5), ATP (lane 6), or 3′dGTP (lane 7) were added to the enzymatic reaction. The 9-mer and 14-mer product sequences were chemically synthesized, radiolabeled, and used as molecular size markers during the gel migration (lane 1 and 2). (C) Natural GTP was added to the primer extension reaction at increasing concentrations up to 10 µM in the presence of 25 µM CTP and UTP. (D) Quantitative analysis of GMP incorporation, based on the extension of the 9-mer RNA product obtained in Fig. 3C. The percentage of the extended products from 9-mer was plotted against GTP concentration and the data was fitted to a hyperbolic equation (see Materials and Methods ) to derive the K app for GTP incorporation. The inset shows the same plot on semi-log scale. Each experiment was conducted at least twice to calculate the average value and standard deviation (see Table 2 ).

Article Snippet: 3′dGTP was purchased from Trilink (San Diego, CA), T-705 RTP and the nucleoprotein inhibitor were synthesized at Alios BioPharma.

Techniques: Sequencing, Polyacrylamide Gel Electrophoresis, Synthesized, Migration, Concentration Assay, Standard Deviation

Journal: PLoS ONE

Article Title: The Ambiguous Base-Pairing and High Substrate Efficiency of T-705 (Favipiravir) Ribofuranosyl 5?-Triphosphate towards Influenza A Virus Polymerase

doi: 10.1371/journal.pone.0068347

Figure Lengend Snippet: Inhibition of the influenza virus RNP complex by 3′dGTP and T-705 RTP. (A) Chemical structures of the obligate chain terminator 3′dGTP and the base-modified T-705 ribofuranosyl 5′-triphosphate (T-705 RTP). (B) Polyacrylamide gel electrophoresis (6%) showing the decrease in radiolabeled viral RNA product from the enzymatic reaction in the presence of increasing concentrations of 3′dGTP. Concentrations of inhibitor are as follows: lane 1 (0), lane 2 (0.023), lane 3 (0.069), lane 4 (0.21), lane 5 (0.62), lane 6 (1.9), lane 7 (5.6), lane 8 (16.7), lane 9 (50), and lane 10 (150 µM). (C) same as (B), with the same concentration range of T-705 RTP as inhibitor.

Article Snippet: 3′dGTP was purchased from Trilink (San Diego, CA), T-705 RTP and the nucleoprotein inhibitor were synthesized at Alios BioPharma.

Techniques: Inhibition, Modification, Polyacrylamide Gel Electrophoresis, Concentration Assay

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