deoxythymidine triphosphate  (Jena Bioscience)


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  • 93
    Name:
    dTTP Solution
    Description:

    Catalog Number:
    NU-1004-10ML
    Price:
    525.1
    Category:
    Molecular Biology
    Size:
    10 ml
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    Structured Review

    Jena Bioscience deoxythymidine triphosphate

    https://www.bioz.com/result/deoxythymidine triphosphate/product/Jena Bioscience
    Average 93 stars, based on 1 article reviews
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    Related Articles

    Isolation:

    Article Title: Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response
    Article Snippet: The BAC probes (T5H22, F5I10, available at www.arabidopsis.org) were kindly provided by T.Mandáková (CEITEC MU, Brno, CR). .. Approximately 1 µg of isolated BAC DNA (NucleoBond® Xtra Midi kit, Macherey-Nagel) were labeled by nick translation with biotin-dUTP or digoxigenin-dUTP (Jena Bioscience) using either commercial nick translation labelling kit (#07J00-001; Abbot - labelling was performed according to the manufactureŕs instructions using 5µl of the enzyme mix and 8h labelling time); or by home-made reaction mix , containing 5 µL of 10× NT buffer (0.5 M Tris-HCl, pH 7.5, 50 mM MgCl2, and 0.05% BSA), 10mM β-mercaptoethanol, 0.2mM each dATP, dCTP, dGTP and 40 µM dTTP (Jena Bioscience), 0.2 mM labelled-dUTP, 3 µL of DNase I (#10104159001, Roche, diluted to 8 µg/ml) and 5U of DNA polymerase I (#EP0041,Fermentas).The nick translation mixture was incubated at 15°C for ∼ 4h (or longer) to obtain a fragment length of ∼200 to 500 bp. .. The reaction was stopped by adding 1 µL of 0.5 M EDTA, pH 8.0, and incubation at 65°C for 10 min.

    BAC Assay:

    Article Title: Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response
    Article Snippet: The BAC probes (T5H22, F5I10, available at www.arabidopsis.org) were kindly provided by T.Mandáková (CEITEC MU, Brno, CR). .. Approximately 1 µg of isolated BAC DNA (NucleoBond® Xtra Midi kit, Macherey-Nagel) were labeled by nick translation with biotin-dUTP or digoxigenin-dUTP (Jena Bioscience) using either commercial nick translation labelling kit (#07J00-001; Abbot - labelling was performed according to the manufactureŕs instructions using 5µl of the enzyme mix and 8h labelling time); or by home-made reaction mix , containing 5 µL of 10× NT buffer (0.5 M Tris-HCl, pH 7.5, 50 mM MgCl2, and 0.05% BSA), 10mM β-mercaptoethanol, 0.2mM each dATP, dCTP, dGTP and 40 µM dTTP (Jena Bioscience), 0.2 mM labelled-dUTP, 3 µL of DNase I (#10104159001, Roche, diluted to 8 µg/ml) and 5U of DNA polymerase I (#EP0041,Fermentas).The nick translation mixture was incubated at 15°C for ∼ 4h (or longer) to obtain a fragment length of ∼200 to 500 bp. .. The reaction was stopped by adding 1 µL of 0.5 M EDTA, pH 8.0, and incubation at 65°C for 10 min.

    Labeling:

    Article Title: Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response
    Article Snippet: The BAC probes (T5H22, F5I10, available at www.arabidopsis.org) were kindly provided by T.Mandáková (CEITEC MU, Brno, CR). .. Approximately 1 µg of isolated BAC DNA (NucleoBond® Xtra Midi kit, Macherey-Nagel) were labeled by nick translation with biotin-dUTP or digoxigenin-dUTP (Jena Bioscience) using either commercial nick translation labelling kit (#07J00-001; Abbot - labelling was performed according to the manufactureŕs instructions using 5µl of the enzyme mix and 8h labelling time); or by home-made reaction mix , containing 5 µL of 10× NT buffer (0.5 M Tris-HCl, pH 7.5, 50 mM MgCl2, and 0.05% BSA), 10mM β-mercaptoethanol, 0.2mM each dATP, dCTP, dGTP and 40 µM dTTP (Jena Bioscience), 0.2 mM labelled-dUTP, 3 µL of DNase I (#10104159001, Roche, diluted to 8 µg/ml) and 5U of DNA polymerase I (#EP0041,Fermentas).The nick translation mixture was incubated at 15°C for ∼ 4h (or longer) to obtain a fragment length of ∼200 to 500 bp. .. The reaction was stopped by adding 1 µL of 0.5 M EDTA, pH 8.0, and incubation at 65°C for 10 min.

    Nick Translation:

    Article Title: Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response
    Article Snippet: The BAC probes (T5H22, F5I10, available at www.arabidopsis.org) were kindly provided by T.Mandáková (CEITEC MU, Brno, CR). .. Approximately 1 µg of isolated BAC DNA (NucleoBond® Xtra Midi kit, Macherey-Nagel) were labeled by nick translation with biotin-dUTP or digoxigenin-dUTP (Jena Bioscience) using either commercial nick translation labelling kit (#07J00-001; Abbot - labelling was performed according to the manufactureŕs instructions using 5µl of the enzyme mix and 8h labelling time); or by home-made reaction mix , containing 5 µL of 10× NT buffer (0.5 M Tris-HCl, pH 7.5, 50 mM MgCl2, and 0.05% BSA), 10mM β-mercaptoethanol, 0.2mM each dATP, dCTP, dGTP and 40 µM dTTP (Jena Bioscience), 0.2 mM labelled-dUTP, 3 µL of DNase I (#10104159001, Roche, diluted to 8 µg/ml) and 5U of DNA polymerase I (#EP0041,Fermentas).The nick translation mixture was incubated at 15°C for ∼ 4h (or longer) to obtain a fragment length of ∼200 to 500 bp. .. The reaction was stopped by adding 1 µL of 0.5 M EDTA, pH 8.0, and incubation at 65°C for 10 min.

    Incubation:

    Article Title: Low ribosomal RNA genes copy number provoke genomic instability and chromosomal segment duplication events that modify global gene expression and plant-pathogen response
    Article Snippet: The BAC probes (T5H22, F5I10, available at www.arabidopsis.org) were kindly provided by T.Mandáková (CEITEC MU, Brno, CR). .. Approximately 1 µg of isolated BAC DNA (NucleoBond® Xtra Midi kit, Macherey-Nagel) were labeled by nick translation with biotin-dUTP or digoxigenin-dUTP (Jena Bioscience) using either commercial nick translation labelling kit (#07J00-001; Abbot - labelling was performed according to the manufactureŕs instructions using 5µl of the enzyme mix and 8h labelling time); or by home-made reaction mix , containing 5 µL of 10× NT buffer (0.5 M Tris-HCl, pH 7.5, 50 mM MgCl2, and 0.05% BSA), 10mM β-mercaptoethanol, 0.2mM each dATP, dCTP, dGTP and 40 µM dTTP (Jena Bioscience), 0.2 mM labelled-dUTP, 3 µL of DNase I (#10104159001, Roche, diluted to 8 µg/ml) and 5U of DNA polymerase I (#EP0041,Fermentas).The nick translation mixture was incubated at 15°C for ∼ 4h (or longer) to obtain a fragment length of ∼200 to 500 bp. .. The reaction was stopped by adding 1 µL of 0.5 M EDTA, pH 8.0, and incubation at 65°C for 10 min.

    Concentration Assay:

    Article Title: Concerted bifunctionality of the dCTP deaminase-dUTPase from Methanocaldococcus jannaschii: a structural and pre-steady state kinetic analysis.
    Article Snippet: .. Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate. .. Two mutant dCTP deaminase-dUTPases from Methanocaldococcus jannaschii were crystallised and the crystal structures were solved: E145A in complex with the substrate analogue alpha,beta-imido-dUTP and E145Q in complex with diphosphate.

    Article Title: Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR
    Article Snippet: The titration experiments were performed with a PEAQ-ITC instrument (MicroCal, Northampton, MA, USA) with a cell volume of 200 μL and a 40-μL syringe. .. Lyophilized deoxyadenosine triphosphate, deoxycytidine trisphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate (Jena Bioscience, Germany) were dissolved in 1× PCR buffer (50 mM KCl, 10 mM Tris-HCl pH 8, and 4 mM MgCl2 ) to a stock concentration of 10 mM for each dNTP and then used at a concentration of 0.1 mM in the syringe. .. Protein concentration of Klenow fragment (product number EP0054, Thermo Scientific) was determined from the UV absorbance at 280 nm with a BioDrop μLITE instrument (BioDrop, Cambridge, UK) with a path length of 0.5 mm, a molar extinction coefficient of 55,450, and a molecular mass of 68 kDa.

    Polymerase Chain Reaction:

    Article Title: Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR
    Article Snippet: The titration experiments were performed with a PEAQ-ITC instrument (MicroCal, Northampton, MA, USA) with a cell volume of 200 μL and a 40-μL syringe. .. Lyophilized deoxyadenosine triphosphate, deoxycytidine trisphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate (Jena Bioscience, Germany) were dissolved in 1× PCR buffer (50 mM KCl, 10 mM Tris-HCl pH 8, and 4 mM MgCl2 ) to a stock concentration of 10 mM for each dNTP and then used at a concentration of 0.1 mM in the syringe. .. Protein concentration of Klenow fragment (product number EP0054, Thermo Scientific) was determined from the UV absorbance at 280 nm with a BioDrop μLITE instrument (BioDrop, Cambridge, UK) with a path length of 0.5 mm, a molar extinction coefficient of 55,450, and a molecular mass of 68 kDa.

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  • 92
    Jena Bioscience dupnpp
    Dimeric and trimeric S. aureus phagic Duts present completely different folding. ( A ) Cartoon representation of <t>ϕO11</t> dimeric Dut (protomers coloured in blue and yellow) and 80α trimeric Dut (PDB 3ZEZ; protomers coloured in blue, yellow and pink) showing the difference in folding between dimeric (all-alpha) and trimeric (all-beta) Duts. The <t>dUPNPP</t> molecules in the active centres are represented in stick and the Mg ions as spheres. For clarity only one dUPNPP molecule is shown in the trimeric structure. The structural motifs implicated in Stl recognition for trimeric Duts ( Maiques et al., 2016 ; Tormo-Más et al., 2013 ) are labelled and coloured in cyan, magenta and red for motif IV, V and VI, respectively. ( B ) Superimposition of dUPNPP molecules in the active centres of ϕO11 (green tones) and 80α (orange tones) shows that the bound nucleotide molecules acquire different conformations (stick representation), including the disposition of the Mg ions (sphere representation), and that the spatial arrangement of the structural elements conforming each active centre is essentially different. No structural element equivalent to the Stl binding motifs of 80α (coloured as in A) is observed in ϕO11.
    Dupnpp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Jena Bioscience stavudine triphosphate
    The effects of <t>d4T-TP,ddI-TP,ddA-TP,</t> ABC-TP and CBV-TP on SAMHD1 triphosphohydrolase activity. ( A ) Hydrolysis of 0.3 mM TTP, d4T-TP or ddI-TP in the absence (left) and presence (right) of 0.1 mM activator GTP. ( B ) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, d4T-TP or ddI-TP as activators. ( C ) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, d4T-TP or ddI-TP. Error bars are the standard error of the mean (SEM) of three independent measurements. ( D ) Concentration dependence of SAMHD1 ddI-TP hydrolysis in the presence of 0.2 mM GTP (saturating activator concentration). Nonlinear least squares fitting using a Hill equation gives the apparent binding constant K S = 226 ± 12 μM, catalytic constant k cat = 0.24 ± 0.04 s −1 and the Hill coefficient n = 1.7 ± 0.1 (Mean ± SEM). ( E ) ddI-TP allosteric activation of TTP hydrolysis. Rates were determined for 1 mM TTP at varying dd-ITP concentration. Nonlinear least squares fitting gives only lower estimates for the maximal rate and the ddI-TP concentration at half maximal activation of k max > 0.1 s −1 and K a > 300 μM. ( F ) Hydrolysis of 0.3 mM TTP, ABC-TP, CBV-TP and ddATP in the absence (left) and presence (right) of 0.1 mM activator GTP. ( G ) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, ABC-TP, CBV-TP or ddATP as activators. ( H ) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, ABC-TP, CBV-TP or ddATP. Error bars are the range of data from two independent measurements.
    Stavudine Triphosphate, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Jena Bioscience ϕdia73l duts against dupnpp
    Structures of ϕDI apo and ϕDI A73L dimeric <t>Duts</t> showed minimal conformational changes in the mutant, but a high impact on SaPI induction and Stl interaction. (A) Superimposition of three-dimensional structures of the ϕDI dimer in apo form (yellow tones), <t>dUPNPP</t> bound (magenta tones) and as A73L mutant (green tones) in cartoon representation. The nucleotide in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg as green spheres. (B) SaPIbov1 excision and replication following induction of the cloned ϕDI A73L dut gene. Strain JP6774 containing SaPIbov1 was complemented with plasmids expressing the 3xFLAG-tagged ϕDI and ϕDI A73L dimeric Dut or the empty pCN51 plasmid as a control. Samples were isolated at 3 hours after induction with 3μM CdCl 2 and Southern blots were performed using a probe for the SaPIbov1 integrase ( S4 Table ). The upper band is ‘bulk’ DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. (C) Native-PAGE experiment was performed to analyze the binding capacity of the ϕDI A73L Dut vs the ϕDI wild-type form. Proteins were mixed with Stl in equimolecular relationship (in monomers). A clear reduction in the band corresponding to the Stl-ϕDI Dut A73L complex with respect to the Stl-ϕDI Dut complex is observed. (D) Close-up view of the superimposed ϕDI-dUPNPP and ϕDI A73L active centers. Residues responsible for substrate coordination and binding are represented in sticks with carbon atoms colored according to the structure to which they correspond. The Leu73 residue at the bottom of the active center of the ϕDI A73L mutant that precludes the binding of the nucleotide is colored in yellow. The substrate dUPNPP in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg ions are represented as grey spheres.
    ϕdia73l Duts Against Dupnpp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Jena Bioscience deoxy thymidine triphosphate dttp
    Structures of ϕDI apo and ϕDI A73L dimeric <t>Duts</t> showed minimal conformational changes in the mutant, but a high impact on SaPI induction and Stl interaction. (A) Superimposition of three-dimensional structures of the ϕDI dimer in apo form (yellow tones), <t>dUPNPP</t> bound (magenta tones) and as A73L mutant (green tones) in cartoon representation. The nucleotide in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg as green spheres. (B) SaPIbov1 excision and replication following induction of the cloned ϕDI A73L dut gene. Strain JP6774 containing SaPIbov1 was complemented with plasmids expressing the 3xFLAG-tagged ϕDI and ϕDI A73L dimeric Dut or the empty pCN51 plasmid as a control. Samples were isolated at 3 hours after induction with 3μM CdCl 2 and Southern blots were performed using a probe for the SaPIbov1 integrase ( S4 Table ). The upper band is ‘bulk’ DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. (C) Native-PAGE experiment was performed to analyze the binding capacity of the ϕDI A73L Dut vs the ϕDI wild-type form. Proteins were mixed with Stl in equimolecular relationship (in monomers). A clear reduction in the band corresponding to the Stl-ϕDI Dut A73L complex with respect to the Stl-ϕDI Dut complex is observed. (D) Close-up view of the superimposed ϕDI-dUPNPP and ϕDI A73L active centers. Residues responsible for substrate coordination and binding are represented in sticks with carbon atoms colored according to the structure to which they correspond. The Leu73 residue at the bottom of the active center of the ϕDI A73L mutant that precludes the binding of the nucleotide is colored in yellow. The substrate dUPNPP in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg ions are represented as grey spheres.
    Deoxy Thymidine Triphosphate Dttp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Dimeric and trimeric S. aureus phagic Duts present completely different folding. ( A ) Cartoon representation of ϕO11 dimeric Dut (protomers coloured in blue and yellow) and 80α trimeric Dut (PDB 3ZEZ; protomers coloured in blue, yellow and pink) showing the difference in folding between dimeric (all-alpha) and trimeric (all-beta) Duts. The dUPNPP molecules in the active centres are represented in stick and the Mg ions as spheres. For clarity only one dUPNPP molecule is shown in the trimeric structure. The structural motifs implicated in Stl recognition for trimeric Duts ( Maiques et al., 2016 ; Tormo-Más et al., 2013 ) are labelled and coloured in cyan, magenta and red for motif IV, V and VI, respectively. ( B ) Superimposition of dUPNPP molecules in the active centres of ϕO11 (green tones) and 80α (orange tones) shows that the bound nucleotide molecules acquire different conformations (stick representation), including the disposition of the Mg ions (sphere representation), and that the spatial arrangement of the structural elements conforming each active centre is essentially different. No structural element equivalent to the Stl binding motifs of 80α (coloured as in A) is observed in ϕO11.

    Journal: eLife

    Article Title: Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer

    doi: 10.7554/eLife.26487

    Figure Lengend Snippet: Dimeric and trimeric S. aureus phagic Duts present completely different folding. ( A ) Cartoon representation of ϕO11 dimeric Dut (protomers coloured in blue and yellow) and 80α trimeric Dut (PDB 3ZEZ; protomers coloured in blue, yellow and pink) showing the difference in folding between dimeric (all-alpha) and trimeric (all-beta) Duts. The dUPNPP molecules in the active centres are represented in stick and the Mg ions as spheres. For clarity only one dUPNPP molecule is shown in the trimeric structure. The structural motifs implicated in Stl recognition for trimeric Duts ( Maiques et al., 2016 ; Tormo-Más et al., 2013 ) are labelled and coloured in cyan, magenta and red for motif IV, V and VI, respectively. ( B ) Superimposition of dUPNPP molecules in the active centres of ϕO11 (green tones) and 80α (orange tones) shows that the bound nucleotide molecules acquire different conformations (stick representation), including the disposition of the Mg ions (sphere representation), and that the spatial arrangement of the structural elements conforming each active centre is essentially different. No structural element equivalent to the Stl binding motifs of 80α (coloured as in A) is observed in ϕO11.

    Article Snippet: ϕO11 Dut crystallization and data collection ϕO11 Dut protein in complex with dUPNPP protein was crystallized using the sitting drop method in the Crystallogenesis facility of IBV. ϕO11 (at 10 mg/ml) was incubated with 0.5 mM dUPNPP (2-Deoxyuridine-5-[(α,β)-imido]triphosphate; Jena Biosciences) and 5 mM MgCl2 during 8 hr at 4°C and sitting drops were set up at 21°C.

    Techniques: Binding Assay

    The effects of d4T-TP,ddI-TP,ddA-TP, ABC-TP and CBV-TP on SAMHD1 triphosphohydrolase activity. ( A ) Hydrolysis of 0.3 mM TTP, d4T-TP or ddI-TP in the absence (left) and presence (right) of 0.1 mM activator GTP. ( B ) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, d4T-TP or ddI-TP as activators. ( C ) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, d4T-TP or ddI-TP. Error bars are the standard error of the mean (SEM) of three independent measurements. ( D ) Concentration dependence of SAMHD1 ddI-TP hydrolysis in the presence of 0.2 mM GTP (saturating activator concentration). Nonlinear least squares fitting using a Hill equation gives the apparent binding constant K S = 226 ± 12 μM, catalytic constant k cat = 0.24 ± 0.04 s −1 and the Hill coefficient n = 1.7 ± 0.1 (Mean ± SEM). ( E ) ddI-TP allosteric activation of TTP hydrolysis. Rates were determined for 1 mM TTP at varying dd-ITP concentration. Nonlinear least squares fitting gives only lower estimates for the maximal rate and the ddI-TP concentration at half maximal activation of k max > 0.1 s −1 and K a > 300 μM. ( F ) Hydrolysis of 0.3 mM TTP, ABC-TP, CBV-TP and ddATP in the absence (left) and presence (right) of 0.1 mM activator GTP. ( G ) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, ABC-TP, CBV-TP or ddATP as activators. ( H ) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, ABC-TP, CBV-TP or ddATP. Error bars are the range of data from two independent measurements.

    Journal: Scientific Reports

    Article Title: SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells

    doi: 10.1038/srep42824

    Figure Lengend Snippet: The effects of d4T-TP,ddI-TP,ddA-TP, ABC-TP and CBV-TP on SAMHD1 triphosphohydrolase activity. ( A ) Hydrolysis of 0.3 mM TTP, d4T-TP or ddI-TP in the absence (left) and presence (right) of 0.1 mM activator GTP. ( B ) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, d4T-TP or ddI-TP as activators. ( C ) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, d4T-TP or ddI-TP. Error bars are the standard error of the mean (SEM) of three independent measurements. ( D ) Concentration dependence of SAMHD1 ddI-TP hydrolysis in the presence of 0.2 mM GTP (saturating activator concentration). Nonlinear least squares fitting using a Hill equation gives the apparent binding constant K S = 226 ± 12 μM, catalytic constant k cat = 0.24 ± 0.04 s −1 and the Hill coefficient n = 1.7 ± 0.1 (Mean ± SEM). ( E ) ddI-TP allosteric activation of TTP hydrolysis. Rates were determined for 1 mM TTP at varying dd-ITP concentration. Nonlinear least squares fitting gives only lower estimates for the maximal rate and the ddI-TP concentration at half maximal activation of k max > 0.1 s −1 and K a > 300 μM. ( F ) Hydrolysis of 0.3 mM TTP, ABC-TP, CBV-TP and ddATP in the absence (left) and presence (right) of 0.1 mM activator GTP. ( G ) Activation of SAMHD1 TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP substrate upon addition of 0.1 mM GTP, ABC-TP, CBV-TP or ddATP as activators. ( H ) Inhibition of SAMHD1 GTP-activated TTP hydrolysis. Triphosphohydrolase activity was measured with 0.3 mM TTP and 0.1 mM GTP activator alone and with addition of 0.3 mM dApNHpp positive control, ABC-TP, CBV-TP or ddATP. Error bars are the range of data from two independent measurements.

    Article Snippet: Didanosine triphosphate (ddI-TP) and dideoxy ATP (ddATP) were from Trilink Biotechnologies, Stavudine triphosphate (d4T-TP) was from Jena biosciences and Chemcyte.

    Techniques: Activity Assay, Activation Assay, Inhibition, Positive Control, Concentration Assay, Binding Assay

    Anti-HIV-1 activities of nucleoside analogues in U937 cells expressing SAMHD1. Antiviral activities were determined in undifferentiated (PMA − ) and differentiated (PMA + ) U937 cells expressing SAMHD1 or the mutant HD206-7AA. Cells were cultured in the presence of increasing concentration of Aciclovir (ACV) ( A) , Ganciclovir (GCV) ( B ), Clofarabine (CFB) ( C ), Stavudine (d4T) (D) , Didanosine (ddI) (E) and Abacavir (ABC) (F) . Cells were incubated with HIV-1-GFP and the percentage of infected GFP + cells was measured by flow cytometry. PMA − cells expressing SAMHD1 (red filled circles), PMA + cells expressing the mutant HD206-7AA (blue open circles) and PMA + cells expressing SAMHD1 (blue filled circles) are shown. Infectivity data is plotted as the normalised percentage of GFP + cells against drug concentrations. Error bars represent the standard deviation from at least 2 independent experiments.

    Journal: Scientific Reports

    Article Title: SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells

    doi: 10.1038/srep42824

    Figure Lengend Snippet: Anti-HIV-1 activities of nucleoside analogues in U937 cells expressing SAMHD1. Antiviral activities were determined in undifferentiated (PMA − ) and differentiated (PMA + ) U937 cells expressing SAMHD1 or the mutant HD206-7AA. Cells were cultured in the presence of increasing concentration of Aciclovir (ACV) ( A) , Ganciclovir (GCV) ( B ), Clofarabine (CFB) ( C ), Stavudine (d4T) (D) , Didanosine (ddI) (E) and Abacavir (ABC) (F) . Cells were incubated with HIV-1-GFP and the percentage of infected GFP + cells was measured by flow cytometry. PMA − cells expressing SAMHD1 (red filled circles), PMA + cells expressing the mutant HD206-7AA (blue open circles) and PMA + cells expressing SAMHD1 (blue filled circles) are shown. Infectivity data is plotted as the normalised percentage of GFP + cells against drug concentrations. Error bars represent the standard deviation from at least 2 independent experiments.

    Article Snippet: Didanosine triphosphate (ddI-TP) and dideoxy ATP (ddATP) were from Trilink Biotechnologies, Stavudine triphosphate (d4T-TP) was from Jena biosciences and Chemcyte.

    Techniques: Expressing, Mutagenesis, Cell Culture, Concentration Assay, Incubation, Infection, Flow Cytometry, Cytometry, Standard Deviation

    Anti-HIV-1 activities of nucleoside analogues in differentiated THP-1 cells after Vpx knockdown of endogenous SAMHD1 expression. Antiviral activities were determined in differentiated (PMA + ) THP-1 cells expressing endogenous SAMHD1 and after transduction with Vpx. Cells were cultured in the presence of increasing concentrations of Aciclovir (ACV) ( A ), Ganciclovir (GCV) ( B ), Clofarabine (CFB) ( C ), Stavudine (d4T) ( D ), Didanosine (ddI) ( E ) and Abacavir (ABC) ( F ). Cells were incubated with HIV-1-GFP and the percentage of infected GFP + cells was measured by flow cytometry. Cells expressing SAMHD1 (Vpx − ) (blue filled circles), cells transduced with Vpx (Vpx + ) (red filled circles) are shown. Infectivity data is plotted as the normalised percentage of GFP + cells against drug concentrations. Error bars represent the standard deviation from at least 2 independent experiments.

    Journal: Scientific Reports

    Article Title: SAMHD1 enhances nucleoside-analogue efficacy against HIV-1 in myeloid cells

    doi: 10.1038/srep42824

    Figure Lengend Snippet: Anti-HIV-1 activities of nucleoside analogues in differentiated THP-1 cells after Vpx knockdown of endogenous SAMHD1 expression. Antiviral activities were determined in differentiated (PMA + ) THP-1 cells expressing endogenous SAMHD1 and after transduction with Vpx. Cells were cultured in the presence of increasing concentrations of Aciclovir (ACV) ( A ), Ganciclovir (GCV) ( B ), Clofarabine (CFB) ( C ), Stavudine (d4T) ( D ), Didanosine (ddI) ( E ) and Abacavir (ABC) ( F ). Cells were incubated with HIV-1-GFP and the percentage of infected GFP + cells was measured by flow cytometry. Cells expressing SAMHD1 (Vpx − ) (blue filled circles), cells transduced with Vpx (Vpx + ) (red filled circles) are shown. Infectivity data is plotted as the normalised percentage of GFP + cells against drug concentrations. Error bars represent the standard deviation from at least 2 independent experiments.

    Article Snippet: Didanosine triphosphate (ddI-TP) and dideoxy ATP (ddATP) were from Trilink Biotechnologies, Stavudine triphosphate (d4T-TP) was from Jena biosciences and Chemcyte.

    Techniques: Expressing, Transduction, Cell Culture, Incubation, Infection, Flow Cytometry, Cytometry, Standard Deviation

    Structures of ϕDI apo and ϕDI A73L dimeric Duts showed minimal conformational changes in the mutant, but a high impact on SaPI induction and Stl interaction. (A) Superimposition of three-dimensional structures of the ϕDI dimer in apo form (yellow tones), dUPNPP bound (magenta tones) and as A73L mutant (green tones) in cartoon representation. The nucleotide in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg as green spheres. (B) SaPIbov1 excision and replication following induction of the cloned ϕDI A73L dut gene. Strain JP6774 containing SaPIbov1 was complemented with plasmids expressing the 3xFLAG-tagged ϕDI and ϕDI A73L dimeric Dut or the empty pCN51 plasmid as a control. Samples were isolated at 3 hours after induction with 3μM CdCl 2 and Southern blots were performed using a probe for the SaPIbov1 integrase ( S4 Table ). The upper band is ‘bulk’ DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. (C) Native-PAGE experiment was performed to analyze the binding capacity of the ϕDI A73L Dut vs the ϕDI wild-type form. Proteins were mixed with Stl in equimolecular relationship (in monomers). A clear reduction in the band corresponding to the Stl-ϕDI Dut A73L complex with respect to the Stl-ϕDI Dut complex is observed. (D) Close-up view of the superimposed ϕDI-dUPNPP and ϕDI A73L active centers. Residues responsible for substrate coordination and binding are represented in sticks with carbon atoms colored according to the structure to which they correspond. The Leu73 residue at the bottom of the active center of the ϕDI A73L mutant that precludes the binding of the nucleotide is colored in yellow. The substrate dUPNPP in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg ions are represented as grey spheres.

    Journal: PLoS Pathogens

    Article Title: Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization

    doi: 10.1371/journal.ppat.1006581

    Figure Lengend Snippet: Structures of ϕDI apo and ϕDI A73L dimeric Duts showed minimal conformational changes in the mutant, but a high impact on SaPI induction and Stl interaction. (A) Superimposition of three-dimensional structures of the ϕDI dimer in apo form (yellow tones), dUPNPP bound (magenta tones) and as A73L mutant (green tones) in cartoon representation. The nucleotide in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg as green spheres. (B) SaPIbov1 excision and replication following induction of the cloned ϕDI A73L dut gene. Strain JP6774 containing SaPIbov1 was complemented with plasmids expressing the 3xFLAG-tagged ϕDI and ϕDI A73L dimeric Dut or the empty pCN51 plasmid as a control. Samples were isolated at 3 hours after induction with 3μM CdCl 2 and Southern blots were performed using a probe for the SaPIbov1 integrase ( S4 Table ). The upper band is ‘bulk’ DNA, including chromosomal, phage, and replicating SaPI. CCC indicates covalently closed circular SaPI DNA. In these experiments, as no helper phage was present, the excised and replicating SaPI DNA appears as part of the bulk DNA or as CCC molecules, rather than the linear monomers that are seen following helper phage-mediated induction and packaging. (C) Native-PAGE experiment was performed to analyze the binding capacity of the ϕDI A73L Dut vs the ϕDI wild-type form. Proteins were mixed with Stl in equimolecular relationship (in monomers). A clear reduction in the band corresponding to the Stl-ϕDI Dut A73L complex with respect to the Stl-ϕDI Dut complex is observed. (D) Close-up view of the superimposed ϕDI-dUPNPP and ϕDI A73L active centers. Residues responsible for substrate coordination and binding are represented in sticks with carbon atoms colored according to the structure to which they correspond. The Leu73 residue at the bottom of the active center of the ϕDI A73L mutant that precludes the binding of the nucleotide is colored in yellow. The substrate dUPNPP in the ϕDI-dUPNPP structure is represented in stick with carbon atoms in cyan and Mg ions are represented as grey spheres.

    Article Snippet: Isothermal Titration Microcalorimetry Isothermal Titration Microcalorimetry (ITC) was used to calculate the dissociation constant of ϕDI and ϕDIA73L Duts against dUPNPP (2-Deoxyuridine-5-[(α,β)-imido]triphosphate; Jena Biosciences), a nonhydrolyzable dUTP analog.

    Techniques: Mutagenesis, Clone Assay, Expressing, Plasmid Preparation, Isolation, Countercurrent Chromatography, Clear Native PAGE, Binding Assay

    The dUTP substrate but not the dUMP product reduces the Dut-Stl interaction. Native-Page gels were performed to analyze the effect of the substrate and product on the interaction of two inducing dimeric Duts, (A) ϕDI and (B) ϕO11, with Stl. Increasing concentrations (from 0 to 1000 μM) of dUPNPP (top gels) or dUMP (down gels) were added to equimolecular concentrations of each Dut with Stl, and the Dut-Stl complex formation was evaluated by Native-PAGE. Notice the reduction of the Stl-Dut complex band when the dUTP analogue but not the dUMP product was present. For ϕDI a new band (labeled with a red asterisk) corresponding to the complex between dUPNPP and the Dut can be observed when the Stl-Dut complex disappears. For each experiment a representative gel of 3 independents assays is shown.

    Journal: PLoS Pathogens

    Article Title: Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization

    doi: 10.1371/journal.ppat.1006581

    Figure Lengend Snippet: The dUTP substrate but not the dUMP product reduces the Dut-Stl interaction. Native-Page gels were performed to analyze the effect of the substrate and product on the interaction of two inducing dimeric Duts, (A) ϕDI and (B) ϕO11, with Stl. Increasing concentrations (from 0 to 1000 μM) of dUPNPP (top gels) or dUMP (down gels) were added to equimolecular concentrations of each Dut with Stl, and the Dut-Stl complex formation was evaluated by Native-PAGE. Notice the reduction of the Stl-Dut complex band when the dUTP analogue but not the dUMP product was present. For ϕDI a new band (labeled with a red asterisk) corresponding to the complex between dUPNPP and the Dut can be observed when the Stl-Dut complex disappears. For each experiment a representative gel of 3 independents assays is shown.

    Article Snippet: Isothermal Titration Microcalorimetry Isothermal Titration Microcalorimetry (ITC) was used to calculate the dissociation constant of ϕDI and ϕDIA73L Duts against dUPNPP (2-Deoxyuridine-5-[(α,β)-imido]triphosphate; Jena Biosciences), a nonhydrolyzable dUTP analog.

    Techniques: Clear Native PAGE, Labeling

    Structure of ϕDI dimeric Dut. (A) Cartoon representation of the ϕDI Dut dimer with protomers coloured in pink and orange, respectively (left). The secondary structural elements are numbered and labeled in order from the N to C terminus (the ‘ indicates elements from the second protomer). A molecule of dUPNPP (sticks coloured by atom type) and two Mg ions (green spheres) occupies the active center of each protomer. In an orthogonal view (right) the conserved and variable (motif VI) regions of the dimeric Duts from S . aureus phages are colored in pink and yellow tones, respectively. The structural elements exploited to dimerize (helices α2 and α6, and the latch) are highlighted in dark tones. Notice that the nucleotide binding sites and the motifs VI map in opposite faces of the dimer. (B) Sequence of the ϕDI Dut, with residues that interact with the substrate highlighted with red stars and residues that interact across the dimer interface in blue text. The Ala residue mutated in the ϕDI A73L is indicated in red. The locations of the five conserved motifs in the dimeric Duts are indicated. Structural elements are shown above the sequence coloured as (A) right panel. (C) Detailed view of the ϕDI Dut active center. The substrate dUPNPP is represented in stick with carbon atoms in cyan. The residues interacting with the nucleotide are shown in stick representation, with carbon atoms coloured according to the protomer to which they correspond and are labeled with a similar color text with the exception of A73 that is highlighted with red text. Nitrogen, oxygen, phosphorus atoms are coloured in dark blue, red and orange, respectively. The Mg ions are represented as green spheres.

    Journal: PLoS Pathogens

    Article Title: Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization

    doi: 10.1371/journal.ppat.1006581

    Figure Lengend Snippet: Structure of ϕDI dimeric Dut. (A) Cartoon representation of the ϕDI Dut dimer with protomers coloured in pink and orange, respectively (left). The secondary structural elements are numbered and labeled in order from the N to C terminus (the ‘ indicates elements from the second protomer). A molecule of dUPNPP (sticks coloured by atom type) and two Mg ions (green spheres) occupies the active center of each protomer. In an orthogonal view (right) the conserved and variable (motif VI) regions of the dimeric Duts from S . aureus phages are colored in pink and yellow tones, respectively. The structural elements exploited to dimerize (helices α2 and α6, and the latch) are highlighted in dark tones. Notice that the nucleotide binding sites and the motifs VI map in opposite faces of the dimer. (B) Sequence of the ϕDI Dut, with residues that interact with the substrate highlighted with red stars and residues that interact across the dimer interface in blue text. The Ala residue mutated in the ϕDI A73L is indicated in red. The locations of the five conserved motifs in the dimeric Duts are indicated. Structural elements are shown above the sequence coloured as (A) right panel. (C) Detailed view of the ϕDI Dut active center. The substrate dUPNPP is represented in stick with carbon atoms in cyan. The residues interacting with the nucleotide are shown in stick representation, with carbon atoms coloured according to the protomer to which they correspond and are labeled with a similar color text with the exception of A73 that is highlighted with red text. Nitrogen, oxygen, phosphorus atoms are coloured in dark blue, red and orange, respectively. The Mg ions are represented as green spheres.

    Article Snippet: Isothermal Titration Microcalorimetry Isothermal Titration Microcalorimetry (ITC) was used to calculate the dissociation constant of ϕDI and ϕDIA73L Duts against dUPNPP (2-Deoxyuridine-5-[(α,β)-imido]triphosphate; Jena Biosciences), a nonhydrolyzable dUTP analog.

    Techniques: Labeling, Binding Assay, Sequencing