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  • 93
    Thermo Fisher deoxythymidine triphosphate dttp
    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, <t>dGTP,</t> <t>dTTP)</t> mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.
    Deoxythymidine Triphosphate Dttp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore deoxythymidine triphosphate dttp
    Depletion of uridine is responsible for AICAR-induced apoptosis in LKB1-null cells A. H460, H157 and Lkb1 −/− MEF cells were seeded in 96-well plates, and treated with the indicated concentration of uridine, cytidine, adenosine, <t>thymidine,</t> <t>dTTP</t> alone, or their respective combination with 1 mM AICAR. Plates were subjected to SRB assay 48 hrs after treatment. Reactions were carried out in quadruplicates, and the error bars represent one standard deviation. B. Lkb1 −/− MEF and H460 cells were seeded in 6-well plates and treated with 1 mM AICAR alone, 5 mM uridine alone or their combination. Lysates were collected 24 hrs after treatment for immunoblot analysis of total caspase-3. C. For Annexin-V and 7AAD analysis of apoptosis, Lkb1 −/− MEF and H460 cells were seeded in 6-well plates, and treated with 1 mM AICAR alone, 5 mM uridine alone, or their combination. Both floating and attached cells were collected 24 hrs after treatment and subjected to flow analysis. Percentage of Annexin-V positive cells was reported. D. Isogenic H157-pBabe and H157-LKB1-WT cells were treated with 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected and analyzed by immunoblot with indicated antibodies. LKB1 was depleted in H1299 cells by siRNA and treated with 0, 0.1, or 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected for immunblot of LKB1 and total caspase-3. E. H460 cells were treated with 0.25 mM phenformin, 25 μM leflunomide, or their combination for 10 days in a colony formation assay.
    Deoxythymidine Triphosphate Dttp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher dttp
    Proliferating cells from the DPZ contribute to the growth of embryonic articular cartilage. (A-E) <t>EdU</t> pulse-chase experiments were carried out in pregnant mice 13.5 and 15.5 dpc. (A) Schematic of the experimental design. (B,D) Mouse hindlimbs 2 h post EdU injection. (C,E) Mouse hindlimbs 24 h post EdU injection. Red asterisks show EdU-incorporated cells of DPZ and white asterisks indicate DPZ cells devoid of EdU. White arrowheads denote interzone cells devoid of EdU and red arrowheads highlight EdU-incorporated cells in the interzone. For D,E, corresponding Col2a 1 and Gdf5 . (F) Schematic showing the experimental design for <t>EdU-dTTP</t> competition in mouse embryos. (G) Simultaneous injection of EdU and excess dTTP abolished any selective uptake of EdU. (H) Many EdU + cells are detected in the interzone when excess dTTP is administered 2 h post EdU injection. White arrowhead indicates interzone. MT, metatarsus; PH, phalangeal element. (I) Schematic of EdU/BrdU double pulse-chase experiment. (J-M) J is merged image of K-M. Many cells in the interzone of MTP incorporated EdU (J,L, white arrowheads), but did not incorporate BrdU. However, cells flanking the interzone displayed BrdU immunoreactivity (J,K, asterisks). (M) DAPI-stained section. For J-M, corresponding Col2a1 and Gdf5 . (N-R) Genetic lineage tracing using Col2a1-CreER T ; Rosa mT/mG . (N) Schematic for the experimental design. (O) At 15.5 dpc, Col2a1 is not expressed at the interzone of the MTP joint (black asterisk). (P,Q) Adjacent sections from 18.5 dpc Col2a1-CreER T2 ; Rosa mT/mG MTP joint. (P) Col2a1 . (R) Higher magnification view of the region marked in Q shows multiple EdU + and GFP + . Dashed lines represent the margins of the skeletal elements in the developing digits.
    Dttp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dttp  (TaKaRa)
    93
    TaKaRa dttp
    Comparative analysis of metal ion activation for pol β of Danio <t>rerio</t> and rat . Dependence on the Mg 2+ (A) or Mn 2+ (B) concentration of the pol β activity of Danio rerio (closed lozenge) and rat (open circle) were assayed in 0-30 mM Mg 2+ and 0-5 mM Mn 2+ . Comparison of the activity of D. rerio and rat pol β by Mg 2+ and Mn 2+ was performed at 1 mM MgCl 2 and at 0.5 mM MnCl 2 (C). The activity measured by the amount of incorporated <t>dTTP.</t> Each activity was normalized by the activity of rat pol β in the presence of Mg 2+ .
    Dttp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare deoxythymidine triphosphate dttp
    Assessment of the ratio of SN to LP BER after repair incubation. ( A ) Schematic diagram of uracil-containing plasmid, ‘pPAL1’, is shown. A uracil residue (U) was placed in a central position of the Nco I/ Sac I fragment. Three strategic restriction enzyme sites, Nco I, Kpn I and Sac I, were designed around the lesion such that SN and LP BER could be analyzed in the same repair reaction mixture. After completion of the BER reaction in the presence of <t>[α-</t> 32 P]dCTP, repair products were restricted with either Nco I and Sac I or Nco I, Sac I and Kpn I, which generated 47-, 25- and 22-mer DNA fragments representing total, LP and SN BER products, respectively. To calculate SN and LP BER products, counts in the 25-mer fragment were subtracted from the total counts in 22-mer fragment, because for every incorporation of dCMP at the second position, next to uracil, one dCMP will be incorporated first at the lesion site. ( B ) pPAL1 (20 nM) was incubated with 10 μg of MEF extract in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl 2 , 20 mM NaCl and 1 mM DTT. The reaction was conducted at 37°C for 30 min in the presence of 20 μM each of dATP, dGTP, <t>dTTP</t> and 2.3 μM [α- 32 P]dCTP. A 16-mer radiolabeled DNA fragment was added in each reaction mixture as an internal control prior to phenol/chloroform extraction and ethanol precipitation. The reaction products were analyzed by 15% denaturing PAGE. The combinations of restriction enzymes used are shown at top of the PhosphorImager panel. The description of each radiolabeled band is indicated on the right-hand side of the image. ( C ) Quantification of total, SN and LP BER is shown in a bar graph. Band intensity of each radiolabeled DNA fragment, 47-, 25- and 22-mer, was measured in terms of arbitrary PhosphorImager units and plotted as total BER, SN BER and LP BER. The experiments were repeated three times and a PhosphorImage of a representative experiment is shown.
    Deoxythymidine Triphosphate Dttp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer dttp
    Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using <t>[α-</t> 32 P]ATP, -GTP, -CTP, -UTP, or <t>-dTTP.</t> Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.
    Dttp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 271 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche deoxythymidine triphosphate dttp
    Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using <t>[α-</t> 32 P]ATP, -GTP, -CTP, -UTP, or <t>-dTTP.</t> Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.
    Deoxythymidine Triphosphate Dttp, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega dttp
    Non-templated addition reactions of various starter molecule conformations. Assays of NTA to 5’- 32 P-labeled (*) single-stranded (ss) DNA (34 nt R2R DNA; left), a 34-bp RNA/DNA duplex (R2 RNA/R2R DNA; center), and a 34-bp DNA/DNA duplex (R2 DNA/R2R DNA; right). Reactions were done with 500 nM GsI-IIC RT and 50 nM substrate in reaction medium containing 200 mM NaCl and 4 mM dNTPs (an equimolar mix of 1 mM <t>dATP,</t> dCTP, dGTP, and <t>dTTP)</t> with time points ranging from 10 to 7,200 s. The reaction products were analyzed as in Fig. S4 .
    Dttp, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ICN Pharmaceuticals thymidine 5 triphosphate
    Non-templated addition reactions of various starter molecule conformations. Assays of NTA to 5’- 32 P-labeled (*) single-stranded (ss) DNA (34 nt R2R DNA; left), a 34-bp RNA/DNA duplex (R2 RNA/R2R DNA; center), and a 34-bp DNA/DNA duplex (R2 DNA/R2R DNA; right). Reactions were done with 500 nM GsI-IIC RT and 50 nM substrate in reaction medium containing 200 mM NaCl and 4 mM dNTPs (an equimolar mix of 1 mM <t>dATP,</t> dCTP, dGTP, and <t>dTTP)</t> with time points ranging from 10 to 7,200 s. The reaction products were analyzed as in Fig. S4 .
    Thymidine 5 Triphosphate, supplied by ICN Pharmaceuticals, 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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    89
    GE Healthcare p dttp
    Non-templated addition reactions of various starter molecule conformations. Assays of NTA to 5’- 32 P-labeled (*) single-stranded (ss) DNA (34 nt R2R DNA; left), a 34-bp RNA/DNA duplex (R2 RNA/R2R DNA; center), and a 34-bp DNA/DNA duplex (R2 DNA/R2R DNA; right). Reactions were done with 500 nM GsI-IIC RT and 50 nM substrate in reaction medium containing 200 mM NaCl and 4 mM dNTPs (an equimolar mix of 1 mM <t>dATP,</t> dCTP, dGTP, and <t>dTTP)</t> with time points ranging from 10 to 7,200 s. The reaction products were analyzed as in Fig. S4 .
    P Dttp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare h dttp
    Differential Sensitivity of the Pol D/N752 Virus and Protein Variants to Aphidicolin and Structural Modeling of the Pol Region of Interest (A) Cells were infected with the Pol D752 revertant (•) or N752 mutant (○) virus, treated with aphidicolin, incubated 3 d, then lysed; final virus yield was titrated on new cells. (B) DNA was also extracted after the lysing step and qPCR performed to quantify normalized viral genome copies. (C) The DNA polymerase activity of Pol D752 and Pol N752 proteins, in the absence and in the presence of <t>pORF18</t> (Pol accessory subunit), was analyzed by measuring the incorporation of [ 3 <t>H]dTTP</t> into a poly(dA)-oligo(dT) template. (▪) Pol D752; (□) Pol N752; (•) Pol D752 + pORF18; (○) Pol N752 + pORF18. (D) The effect of aphidicolin on polymerase activity of Pol D752 (•) and of Pol N752 (○) was assayed by measuring the incorporation of [ 3 H]dTTP into a poly(dA)-oligo(dT) template in the presence of pORF18. Graphs show the average of three experiments with standard deviations (error bars). Asterisk * indicates p
    H Dttp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore dttp
    (A) Structures of 2' deoxynucleoside triphosphates used or referred to in this study are <t>dATP,</t> dCTP, dGTP, <t>dTTP,</t> Ind-TP, 5-FITP, 5-AITP, 5-NITP, 5-PhITP, 5-CE-ITP, 5-CH-ITP, and 5-NapITP. For convenience, dR is used to represent the 2'-deoxyribose 5'-triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes T or the presence of a tetrahydrofuran moiety that mimics an abasic site.
    Dttp, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Jena Bioscience thymidine 5 triphosphate
    (A) Structures of 2' deoxynucleoside triphosphates used or referred to in this study are <t>dATP,</t> dCTP, dGTP, <t>dTTP,</t> Ind-TP, 5-FITP, 5-AITP, 5-NITP, 5-PhITP, 5-CE-ITP, 5-CH-ITP, and 5-NapITP. For convenience, dR is used to represent the 2'-deoxyribose 5'-triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes T or the presence of a tetrahydrofuran moiety that mimics an abasic site.
    Thymidine 5 Triphosphate, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 85/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Article Snippet: Termination of purified DPE activity with dideoxyCTP DPE reactions were prepared as described above with a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM dideoxyCTP (ddCTP) (Affymetrix cat# 77332).

    Techniques: Purification, Polymerase Chain Reaction, Construct

    Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

    Article Snippet: Termination of purified DPE activity with dideoxyCTP DPE reactions were prepared as described above with a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM dideoxyCTP (ddCTP) (Affymetrix cat# 77332).

    Techniques: Polymerase Chain Reaction, Lysis, Derivative Assay, Activity Assay, Real-time Polymerase Chain Reaction, Generated

    Depletion of uridine is responsible for AICAR-induced apoptosis in LKB1-null cells A. H460, H157 and Lkb1 −/− MEF cells were seeded in 96-well plates, and treated with the indicated concentration of uridine, cytidine, adenosine, thymidine, dTTP alone, or their respective combination with 1 mM AICAR. Plates were subjected to SRB assay 48 hrs after treatment. Reactions were carried out in quadruplicates, and the error bars represent one standard deviation. B. Lkb1 −/− MEF and H460 cells were seeded in 6-well plates and treated with 1 mM AICAR alone, 5 mM uridine alone or their combination. Lysates were collected 24 hrs after treatment for immunoblot analysis of total caspase-3. C. For Annexin-V and 7AAD analysis of apoptosis, Lkb1 −/− MEF and H460 cells were seeded in 6-well plates, and treated with 1 mM AICAR alone, 5 mM uridine alone, or their combination. Both floating and attached cells were collected 24 hrs after treatment and subjected to flow analysis. Percentage of Annexin-V positive cells was reported. D. Isogenic H157-pBabe and H157-LKB1-WT cells were treated with 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected and analyzed by immunoblot with indicated antibodies. LKB1 was depleted in H1299 cells by siRNA and treated with 0, 0.1, or 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected for immunblot of LKB1 and total caspase-3. E. H460 cells were treated with 0.25 mM phenformin, 25 μM leflunomide, or their combination for 10 days in a colony formation assay.

    Journal: Oncotarget

    Article Title: LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    doi: 10.18632/oncotarget.6224

    Figure Lengend Snippet: Depletion of uridine is responsible for AICAR-induced apoptosis in LKB1-null cells A. H460, H157 and Lkb1 −/− MEF cells were seeded in 96-well plates, and treated with the indicated concentration of uridine, cytidine, adenosine, thymidine, dTTP alone, or their respective combination with 1 mM AICAR. Plates were subjected to SRB assay 48 hrs after treatment. Reactions were carried out in quadruplicates, and the error bars represent one standard deviation. B. Lkb1 −/− MEF and H460 cells were seeded in 6-well plates and treated with 1 mM AICAR alone, 5 mM uridine alone or their combination. Lysates were collected 24 hrs after treatment for immunoblot analysis of total caspase-3. C. For Annexin-V and 7AAD analysis of apoptosis, Lkb1 −/− MEF and H460 cells were seeded in 6-well plates, and treated with 1 mM AICAR alone, 5 mM uridine alone, or their combination. Both floating and attached cells were collected 24 hrs after treatment and subjected to flow analysis. Percentage of Annexin-V positive cells was reported. D. Isogenic H157-pBabe and H157-LKB1-WT cells were treated with 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected and analyzed by immunoblot with indicated antibodies. LKB1 was depleted in H1299 cells by siRNA and treated with 0, 0.1, or 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected for immunblot of LKB1 and total caspase-3. E. H460 cells were treated with 0.25 mM phenformin, 25 μM leflunomide, or their combination for 10 days in a colony formation assay.

    Article Snippet: Uridine, cytidine, adenosine, thymidine, deoxythymidine triphosphate (dTTP), uracil, phenformin and 5-fluorouridine were purchased from Sigma-Aldrich Co. LLC, (St Louis, MO).

    Techniques: Concentration Assay, Sulforhodamine B Assay, Standard Deviation, Flow Cytometry, Colony Assay

    Proliferating cells from the DPZ contribute to the growth of embryonic articular cartilage. (A-E) EdU pulse-chase experiments were carried out in pregnant mice 13.5 and 15.5 dpc. (A) Schematic of the experimental design. (B,D) Mouse hindlimbs 2 h post EdU injection. (C,E) Mouse hindlimbs 24 h post EdU injection. Red asterisks show EdU-incorporated cells of DPZ and white asterisks indicate DPZ cells devoid of EdU. White arrowheads denote interzone cells devoid of EdU and red arrowheads highlight EdU-incorporated cells in the interzone. For D,E, corresponding Col2a 1 and Gdf5 . (F) Schematic showing the experimental design for EdU-dTTP competition in mouse embryos. (G) Simultaneous injection of EdU and excess dTTP abolished any selective uptake of EdU. (H) Many EdU + cells are detected in the interzone when excess dTTP is administered 2 h post EdU injection. White arrowhead indicates interzone. MT, metatarsus; PH, phalangeal element. (I) Schematic of EdU/BrdU double pulse-chase experiment. (J-M) J is merged image of K-M. Many cells in the interzone of MTP incorporated EdU (J,L, white arrowheads), but did not incorporate BrdU. However, cells flanking the interzone displayed BrdU immunoreactivity (J,K, asterisks). (M) DAPI-stained section. For J-M, corresponding Col2a1 and Gdf5 . (N-R) Genetic lineage tracing using Col2a1-CreER T ; Rosa mT/mG . (N) Schematic for the experimental design. (O) At 15.5 dpc, Col2a1 is not expressed at the interzone of the MTP joint (black asterisk). (P,Q) Adjacent sections from 18.5 dpc Col2a1-CreER T2 ; Rosa mT/mG MTP joint. (P) Col2a1 . (R) Higher magnification view of the region marked in Q shows multiple EdU + and GFP + . Dashed lines represent the margins of the skeletal elements in the developing digits.

    Journal: Development (Cambridge, England)

    Article Title: Precise spatial restriction of BMP signaling is essential for articular cartilage differentiation

    doi: 10.1242/dev.110940

    Figure Lengend Snippet: Proliferating cells from the DPZ contribute to the growth of embryonic articular cartilage. (A-E) EdU pulse-chase experiments were carried out in pregnant mice 13.5 and 15.5 dpc. (A) Schematic of the experimental design. (B,D) Mouse hindlimbs 2 h post EdU injection. (C,E) Mouse hindlimbs 24 h post EdU injection. Red asterisks show EdU-incorporated cells of DPZ and white asterisks indicate DPZ cells devoid of EdU. White arrowheads denote interzone cells devoid of EdU and red arrowheads highlight EdU-incorporated cells in the interzone. For D,E, corresponding Col2a 1 and Gdf5 . (F) Schematic showing the experimental design for EdU-dTTP competition in mouse embryos. (G) Simultaneous injection of EdU and excess dTTP abolished any selective uptake of EdU. (H) Many EdU + cells are detected in the interzone when excess dTTP is administered 2 h post EdU injection. White arrowhead indicates interzone. MT, metatarsus; PH, phalangeal element. (I) Schematic of EdU/BrdU double pulse-chase experiment. (J-M) J is merged image of K-M. Many cells in the interzone of MTP incorporated EdU (J,L, white arrowheads), but did not incorporate BrdU. However, cells flanking the interzone displayed BrdU immunoreactivity (J,K, asterisks). (M) DAPI-stained section. For J-M, corresponding Col2a1 and Gdf5 . (N-R) Genetic lineage tracing using Col2a1-CreER T ; Rosa mT/mG . (N) Schematic for the experimental design. (O) At 15.5 dpc, Col2a1 is not expressed at the interzone of the MTP joint (black asterisk). (P,Q) Adjacent sections from 18.5 dpc Col2a1-CreER T2 ; Rosa mT/mG MTP joint. (P) Col2a1 . (R) Higher magnification view of the region marked in Q shows multiple EdU + and GFP + . Dashed lines represent the margins of the skeletal elements in the developing digits.

    Article Snippet: In the EdU competition assay, five times molar excess of dTTP (Thermo Fisher Scientific) was used.

    Techniques: Pulse Chase, Mouse Assay, Injection, Staining

    Comparative analysis of metal ion activation for pol β of Danio rerio and rat . Dependence on the Mg 2+ (A) or Mn 2+ (B) concentration of the pol β activity of Danio rerio (closed lozenge) and rat (open circle) were assayed in 0-30 mM Mg 2+ and 0-5 mM Mn 2+ . Comparison of the activity of D. rerio and rat pol β by Mg 2+ and Mn 2+ was performed at 1 mM MgCl 2 and at 0.5 mM MnCl 2 (C). The activity measured by the amount of incorporated dTTP. Each activity was normalized by the activity of rat pol β in the presence of Mg 2+ .

    Journal: Microbial Cell Factories

    Article Title: Characterization of DNA polymerase ? from Danio rerio by overexpression in E. coli using the in vivo/in vitro compatible pIVEX plasmid

    doi: 10.1186/1475-2859-10-84

    Figure Lengend Snippet: Comparative analysis of metal ion activation for pol β of Danio rerio and rat . Dependence on the Mg 2+ (A) or Mn 2+ (B) concentration of the pol β activity of Danio rerio (closed lozenge) and rat (open circle) were assayed in 0-30 mM Mg 2+ and 0-5 mM Mn 2+ . Comparison of the activity of D. rerio and rat pol β by Mg 2+ and Mn 2+ was performed at 1 mM MgCl 2 and at 0.5 mM MnCl 2 (C). The activity measured by the amount of incorporated dTTP. Each activity was normalized by the activity of rat pol β in the presence of Mg 2+ .

    Article Snippet: Kinetic assays of Danio rerio pol β Kinetic Incorporation of dTTP (Takara) by pol β of Danio rerio or rat was performed in the same reaction buffer and temperature (30°C) that was used for the respective DNA polymerization assay in a total volume of 10 μl containing 0.5 μM of the primer-template complex.

    Techniques: Activation Assay, Concentration Assay, Activity Assay

    Assessment of the ratio of SN to LP BER after repair incubation. ( A ) Schematic diagram of uracil-containing plasmid, ‘pPAL1’, is shown. A uracil residue (U) was placed in a central position of the Nco I/ Sac I fragment. Three strategic restriction enzyme sites, Nco I, Kpn I and Sac I, were designed around the lesion such that SN and LP BER could be analyzed in the same repair reaction mixture. After completion of the BER reaction in the presence of [α- 32 P]dCTP, repair products were restricted with either Nco I and Sac I or Nco I, Sac I and Kpn I, which generated 47-, 25- and 22-mer DNA fragments representing total, LP and SN BER products, respectively. To calculate SN and LP BER products, counts in the 25-mer fragment were subtracted from the total counts in 22-mer fragment, because for every incorporation of dCMP at the second position, next to uracil, one dCMP will be incorporated first at the lesion site. ( B ) pPAL1 (20 nM) was incubated with 10 μg of MEF extract in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl 2 , 20 mM NaCl and 1 mM DTT. The reaction was conducted at 37°C for 30 min in the presence of 20 μM each of dATP, dGTP, dTTP and 2.3 μM [α- 32 P]dCTP. A 16-mer radiolabeled DNA fragment was added in each reaction mixture as an internal control prior to phenol/chloroform extraction and ethanol precipitation. The reaction products were analyzed by 15% denaturing PAGE. The combinations of restriction enzymes used are shown at top of the PhosphorImager panel. The description of each radiolabeled band is indicated on the right-hand side of the image. ( C ) Quantification of total, SN and LP BER is shown in a bar graph. Band intensity of each radiolabeled DNA fragment, 47-, 25- and 22-mer, was measured in terms of arbitrary PhosphorImager units and plotted as total BER, SN BER and LP BER. The experiments were repeated three times and a PhosphorImage of a representative experiment is shown.

    Journal: Nucleic Acids Research

    Article Title: Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity

    doi: 10.1093/nar/gkm639

    Figure Lengend Snippet: Assessment of the ratio of SN to LP BER after repair incubation. ( A ) Schematic diagram of uracil-containing plasmid, ‘pPAL1’, is shown. A uracil residue (U) was placed in a central position of the Nco I/ Sac I fragment. Three strategic restriction enzyme sites, Nco I, Kpn I and Sac I, were designed around the lesion such that SN and LP BER could be analyzed in the same repair reaction mixture. After completion of the BER reaction in the presence of [α- 32 P]dCTP, repair products were restricted with either Nco I and Sac I or Nco I, Sac I and Kpn I, which generated 47-, 25- and 22-mer DNA fragments representing total, LP and SN BER products, respectively. To calculate SN and LP BER products, counts in the 25-mer fragment were subtracted from the total counts in 22-mer fragment, because for every incorporation of dCMP at the second position, next to uracil, one dCMP will be incorporated first at the lesion site. ( B ) pPAL1 (20 nM) was incubated with 10 μg of MEF extract in 50 mM Tris–HCl, pH 7.5, 5 mM MgCl 2 , 20 mM NaCl and 1 mM DTT. The reaction was conducted at 37°C for 30 min in the presence of 20 μM each of dATP, dGTP, dTTP and 2.3 μM [α- 32 P]dCTP. A 16-mer radiolabeled DNA fragment was added in each reaction mixture as an internal control prior to phenol/chloroform extraction and ethanol precipitation. The reaction products were analyzed by 15% denaturing PAGE. The combinations of restriction enzymes used are shown at top of the PhosphorImager panel. The description of each radiolabeled band is indicated on the right-hand side of the image. ( C ) Quantification of total, SN and LP BER is shown in a bar graph. Band intensity of each radiolabeled DNA fragment, 47-, 25- and 22-mer, was measured in terms of arbitrary PhosphorImager units and plotted as total BER, SN BER and LP BER. The experiments were repeated three times and a PhosphorImage of a representative experiment is shown.

    Article Snippet: The [α-32 P]dCTP and dTTP (3000 Ci/mmol) were from GE HealthCare (Piscataway, NJ, USA).

    Techniques: Incubation, Plasmid Preparation, Generated, Ethanol Precipitation, Polyacrylamide Gel Electrophoresis

    Incorporation of labeled dCMP and dTMP into the repair patch produced during LP BER of the THF-containing plasmid. The repair reaction was performed with THF-containing plasmid, pUN2 and MEF extract. The reaction conditions and product analyses were as described under ‘Materials and Methods section’. ( A ) BER reaction was performed in a10 μl reaction mixture that contained 20 nM pUN2, 10 μg MEF extract and either 32 P-dCTP (lanes 1and 2) or 32 P-dTTP (lanes 3 and 4). Incubation was for 30 min at 37°C. The reaction products were analyzed as in Figure 3 . The restriction enzymes used are shown at the top of the PhosphorImager panel. The description of each radiolabeled band is indicated on both sides of the image. ( B ) Quantification of total BER (41-mer), SN BER (25-mer) and LP BER (16-mer) is shown in a bar graph. A small portion of each reaction was spotted on the gel filter for calculations of incorporation of 32 P-dCMP or 32 P-dTMP in DNA. The band intensity of each radiolabeled DNA fragment, 41-, 25- and 16-mer, was measured in terms of arbitrary PhosphorImager units and then converted into relative dCMP or dTMP incorporation. The experiments were repeated three times, and the PhosphorImage of a representative experiment is shown. ( C ) The positions of dCMP (filled circle) or dTMP (cross) incorporation in the 41-, 25- and 16-mer fragments, and the restriction sites are indicated.

    Journal: Nucleic Acids Research

    Article Title: Comparative assessment of plasmid and oligonucleotide DNA substrates in measurement of in vitro base excision repair activity

    doi: 10.1093/nar/gkm639

    Figure Lengend Snippet: Incorporation of labeled dCMP and dTMP into the repair patch produced during LP BER of the THF-containing plasmid. The repair reaction was performed with THF-containing plasmid, pUN2 and MEF extract. The reaction conditions and product analyses were as described under ‘Materials and Methods section’. ( A ) BER reaction was performed in a10 μl reaction mixture that contained 20 nM pUN2, 10 μg MEF extract and either 32 P-dCTP (lanes 1and 2) or 32 P-dTTP (lanes 3 and 4). Incubation was for 30 min at 37°C. The reaction products were analyzed as in Figure 3 . The restriction enzymes used are shown at the top of the PhosphorImager panel. The description of each radiolabeled band is indicated on both sides of the image. ( B ) Quantification of total BER (41-mer), SN BER (25-mer) and LP BER (16-mer) is shown in a bar graph. A small portion of each reaction was spotted on the gel filter for calculations of incorporation of 32 P-dCMP or 32 P-dTMP in DNA. The band intensity of each radiolabeled DNA fragment, 41-, 25- and 16-mer, was measured in terms of arbitrary PhosphorImager units and then converted into relative dCMP or dTMP incorporation. The experiments were repeated three times, and the PhosphorImage of a representative experiment is shown. ( C ) The positions of dCMP (filled circle) or dTMP (cross) incorporation in the 41-, 25- and 16-mer fragments, and the restriction sites are indicated.

    Article Snippet: The [α-32 P]dCTP and dTTP (3000 Ci/mmol) were from GE HealthCare (Piscataway, NJ, USA).

    Techniques: Labeling, Produced, Plasmid Preparation, Incubation

    Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *

    doi: 10.1074/jbc.M111.227603

    Figure Lengend Snippet: Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Article Snippet: For nucleotide specificity assays, in vitro reactions were conducted as above with α-32 P-labeled ATP, GTP, CTP, UTP, or dTTP (PerkinElmer Life Sciences) containing 1 mm of each respective cold dNTP.

    Techniques: Purification, Incubation, In Vitro, SDS Page, Autoradiography, Staining, Sequencing, Mutagenesis, Modification

    RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

    doi: 10.1128/MCB.24.6.2423-2443.2004

    Figure Lengend Snippet: RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences.

    Techniques: Plasmid Preparation, Sequencing, Construct, Polymerase Chain Reaction, Amplification, Labeling, Footprinting, DNA Sequencing, Marker, Autoradiography

    Non-templated addition reactions of various starter molecule conformations. Assays of NTA to 5’- 32 P-labeled (*) single-stranded (ss) DNA (34 nt R2R DNA; left), a 34-bp RNA/DNA duplex (R2 RNA/R2R DNA; center), and a 34-bp DNA/DNA duplex (R2 DNA/R2R DNA; right). Reactions were done with 500 nM GsI-IIC RT and 50 nM substrate in reaction medium containing 200 mM NaCl and 4 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP) with time points ranging from 10 to 7,200 s. The reaction products were analyzed as in Fig. S4 .

    Journal: bioRxiv

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

    doi: 10.1101/792986

    Figure Lengend Snippet: Non-templated addition reactions of various starter molecule conformations. Assays of NTA to 5’- 32 P-labeled (*) single-stranded (ss) DNA (34 nt R2R DNA; left), a 34-bp RNA/DNA duplex (R2 RNA/R2R DNA; center), and a 34-bp DNA/DNA duplex (R2 DNA/R2R DNA; right). Reactions were done with 500 nM GsI-IIC RT and 50 nM substrate in reaction medium containing 200 mM NaCl and 4 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP) with time points ranging from 10 to 7,200 s. The reaction products were analyzed as in Fig. S4 .

    Article Snippet: Unless specified otherwise, reactions were done with 500 nM GsI-IIC RT, 50 nM R2 RNA/R2R DNA starter duplex, and 100 nM acceptor oligonucleotide in 25 μl of medium containing 200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.5, 5 mM fresh DTT, and an equimolar mix of 1 mM each dATP, dCTP, dGTP, and dTTP (Promega) to give 4 mM total dNTP concentration.

    Techniques: Labeling

    Non-templated nucleotide addition activity of GsI-IIC RT using a mixture of all four dNTPs. Reactions included 500 nM GsI-II RT and 50 nM of a blunt-end starter duplex with 5’- 32 P-labeled (*) DNA primer in reaction medium containing 200 mM NaCl and varying dNTP concentrations (0.04, 0.4, 1, and 4 mM, where 4 mM is an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP). Aliquots were stopped after times ranging from 10 to 7,200 s, and the products were analyzed by electrophoresis in a denaturing polyacrylamide gel, which was dried and scanned with a phosphorimager. Each product band was quantified individually and summed to estimate the rate of step 1. Product bands 2 and 3 were summed to estimate the rate of step 2, and product band 3 was used to estimate the rate of step 3. The data were plotted and fit by a single-exponential function to calculate the k obs and amplitude parameters for each reaction. A , Representative gel showing the labeled DNA primer (P) and bands resulting from NTA of 1, 2, and 3 nucleotides to the 3’ end of the DNA. B , A plot of k obs values as a function of dNTP concentration fit by a hyperbolic function to calculate k add , the catalytic rate at saturating substrate concentration; K 1/2 , the substrate concentration at half maximum k add ; and k add /K 1/2 for each NTA step (values summarized in tables to the right of the plots). The individual parameter values k add and K 1/2 were not well defined for steps two and three because saturation was not reached at 4 mM dNTP, and they are therefore indicated as N.D. (not determined). Although the progress curve of the second and third NTA products ( Fig. S4 ) would be expected to include kinetic lags in principle, the rate constants for NTA are progressively lower with repeated additions, such that the data for these additions are adequately described by simple exponential functions without lag phases. All reactions were performed at least twice, and some time points were collected three times. Data were averaged for each time point, and these averages were fit by a single-exponential function to obtain the k obs values.

    Journal: bioRxiv

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

    doi: 10.1101/792986

    Figure Lengend Snippet: Non-templated nucleotide addition activity of GsI-IIC RT using a mixture of all four dNTPs. Reactions included 500 nM GsI-II RT and 50 nM of a blunt-end starter duplex with 5’- 32 P-labeled (*) DNA primer in reaction medium containing 200 mM NaCl and varying dNTP concentrations (0.04, 0.4, 1, and 4 mM, where 4 mM is an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP). Aliquots were stopped after times ranging from 10 to 7,200 s, and the products were analyzed by electrophoresis in a denaturing polyacrylamide gel, which was dried and scanned with a phosphorimager. Each product band was quantified individually and summed to estimate the rate of step 1. Product bands 2 and 3 were summed to estimate the rate of step 2, and product band 3 was used to estimate the rate of step 3. The data were plotted and fit by a single-exponential function to calculate the k obs and amplitude parameters for each reaction. A , Representative gel showing the labeled DNA primer (P) and bands resulting from NTA of 1, 2, and 3 nucleotides to the 3’ end of the DNA. B , A plot of k obs values as a function of dNTP concentration fit by a hyperbolic function to calculate k add , the catalytic rate at saturating substrate concentration; K 1/2 , the substrate concentration at half maximum k add ; and k add /K 1/2 for each NTA step (values summarized in tables to the right of the plots). The individual parameter values k add and K 1/2 were not well defined for steps two and three because saturation was not reached at 4 mM dNTP, and they are therefore indicated as N.D. (not determined). Although the progress curve of the second and third NTA products ( Fig. S4 ) would be expected to include kinetic lags in principle, the rate constants for NTA are progressively lower with repeated additions, such that the data for these additions are adequately described by simple exponential functions without lag phases. All reactions were performed at least twice, and some time points were collected three times. Data were averaged for each time point, and these averages were fit by a single-exponential function to obtain the k obs values.

    Article Snippet: Unless specified otherwise, reactions were done with 500 nM GsI-IIC RT, 50 nM R2 RNA/R2R DNA starter duplex, and 100 nM acceptor oligonucleotide in 25 μl of medium containing 200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.5, 5 mM fresh DTT, and an equimolar mix of 1 mM each dATP, dCTP, dGTP, and dTTP (Promega) to give 4 mM total dNTP concentration.

    Techniques: Activity Assay, Labeling, Electrophoresis, Concentration Assay

    Non-templated nucleotide addition reactions to blunt-end and 1-nt 3’ overhang starter duplexes using individual dNTPs. A , Assays of non-templated nucleotide addition to the 32 P-labeled blunt-end RNA/DNA duplex (left) and 1-nt 3’-overhang RNA/DNA duplex (right) using 0.04, 0.4, 1, and 4 mM dATP, dTTP, dCTP and dGTP individually. Reactions included 500 nM GsI-IIC RT and 50 nM of starter duplex in reaction medium containing 200 mM NaCl and were stopped at times ranging from 10 to 7,200 s. The reaction products were analyzed as in Fig. S4 . The values obtained from these experiments were used for global fitting analysis in Fig. S6 and for the summary of second-order rate constants in Fig. 5 .

    Journal: bioRxiv

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

    doi: 10.1101/792986

    Figure Lengend Snippet: Non-templated nucleotide addition reactions to blunt-end and 1-nt 3’ overhang starter duplexes using individual dNTPs. A , Assays of non-templated nucleotide addition to the 32 P-labeled blunt-end RNA/DNA duplex (left) and 1-nt 3’-overhang RNA/DNA duplex (right) using 0.04, 0.4, 1, and 4 mM dATP, dTTP, dCTP and dGTP individually. Reactions included 500 nM GsI-IIC RT and 50 nM of starter duplex in reaction medium containing 200 mM NaCl and were stopped at times ranging from 10 to 7,200 s. The reaction products were analyzed as in Fig. S4 . The values obtained from these experiments were used for global fitting analysis in Fig. S6 and for the summary of second-order rate constants in Fig. 5 .

    Article Snippet: Unless specified otherwise, reactions were done with 500 nM GsI-IIC RT, 50 nM R2 RNA/R2R DNA starter duplex, and 100 nM acceptor oligonucleotide in 25 μl of medium containing 200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.5, 5 mM fresh DTT, and an equimolar mix of 1 mM each dATP, dCTP, dGTP, and dTTP (Promega) to give 4 mM total dNTP concentration.

    Techniques: Labeling

    Overview of template-switching experiments and determination of saturating enzyme concentrations. A , Outline of the experiments. GsI-IIC RT was pre-bound to a starter duplex (magenta) consisting of a 34-nt RNA oligonucleotide containing an Illumina Read 2 (R2) sequence annealed to a complementary 35-nt DNA primer (R2R) leaving a 1-nt, 3’-DNA overhang (N) ( Table S1 ). The 3’-over-hang nucleotide (N) base pairs with the 3’ nucleotide (N’) of an acceptor RNA (black) for template switching, leading to the synthesis of a full-length cDNA copy of the acceptor RNA with the R2R adapter linked to its 5’ end. The cDNAs were incubated with NaOH to degrade RNA and neutralized with equimolar HCl prior to further analysis. For the biochemical experiments (left branch), the R2R DNA primer in the starter duplex was 5’- 32 P-labeled (*), and the cDNA products were analyzed by electrophoresis in a denaturing polyacrylamide gel, which was dried and quantified with a phosphorimager. For RNA-seq experiments (right branch), the cDNAs were cleaned up by using a MinElute column (Qiagen; not shown) prior to ligating a 5’-adenylated R1R adapter to the 3’ end of the cDNA using the thermostable 5’ app DNA/RNA ligase (New England BioLabs). After another MinElute clean-up, Illumina RNA-seq capture sites (P5 and P7) and indexes were added by PCR, and he resulting libraries were cleaned up by using AMPure XP beads (Beckman Coulter) prior to sequencing on an Illumina NextSeq 500. B , Determination of saturating enzyme concentrations. Template-switching reactions included various concentrations of GsI-IIC RT as indicated, 50 nM RNA template/DNA primer starter duplex (5’- 32 P-labeled on DNA primer indicated by *), 100 nM of a 50-nt RNA acceptor template, and 4 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP) in reaction medium containing 200 mM NaCl at 60 °C. Aliquots were quenched at times ranging from 6 to 1,800 s, and the products were analyzed by denaturing PAGE, as described in Experimental Procedures. The numbers to the left of the gel indicate size markers (a 5’ 32 P-labeled ssDNA ladder; ss20 DNA Ladder, Simplex Sciences) run in a parallel lane, and the labels to the right of the gel indicate the products resulting from the initial template switch (1x) and subsequent end-to-end template switches from the 5’ end of one acceptor to the 3’ end of another (2x, 3x, etc .). The plot at the right shows time courses for the production of template-switching products ( i.e. , products > 2 nt larger than the primer), with each data set fit by a single-exponential function and the error bars indicating the standard deviations for three experiments. The inset table indicates the k obs and amplitude parameters obtained from the fit of an exponential function to the average values from three independent determinations, along with standard errors obtained from the fit (see Experimental Procedures).

    Journal: bioRxiv

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

    doi: 10.1101/792986

    Figure Lengend Snippet: Overview of template-switching experiments and determination of saturating enzyme concentrations. A , Outline of the experiments. GsI-IIC RT was pre-bound to a starter duplex (magenta) consisting of a 34-nt RNA oligonucleotide containing an Illumina Read 2 (R2) sequence annealed to a complementary 35-nt DNA primer (R2R) leaving a 1-nt, 3’-DNA overhang (N) ( Table S1 ). The 3’-over-hang nucleotide (N) base pairs with the 3’ nucleotide (N’) of an acceptor RNA (black) for template switching, leading to the synthesis of a full-length cDNA copy of the acceptor RNA with the R2R adapter linked to its 5’ end. The cDNAs were incubated with NaOH to degrade RNA and neutralized with equimolar HCl prior to further analysis. For the biochemical experiments (left branch), the R2R DNA primer in the starter duplex was 5’- 32 P-labeled (*), and the cDNA products were analyzed by electrophoresis in a denaturing polyacrylamide gel, which was dried and quantified with a phosphorimager. For RNA-seq experiments (right branch), the cDNAs were cleaned up by using a MinElute column (Qiagen; not shown) prior to ligating a 5’-adenylated R1R adapter to the 3’ end of the cDNA using the thermostable 5’ app DNA/RNA ligase (New England BioLabs). After another MinElute clean-up, Illumina RNA-seq capture sites (P5 and P7) and indexes were added by PCR, and he resulting libraries were cleaned up by using AMPure XP beads (Beckman Coulter) prior to sequencing on an Illumina NextSeq 500. B , Determination of saturating enzyme concentrations. Template-switching reactions included various concentrations of GsI-IIC RT as indicated, 50 nM RNA template/DNA primer starter duplex (5’- 32 P-labeled on DNA primer indicated by *), 100 nM of a 50-nt RNA acceptor template, and 4 mM dNTPs (an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP) in reaction medium containing 200 mM NaCl at 60 °C. Aliquots were quenched at times ranging from 6 to 1,800 s, and the products were analyzed by denaturing PAGE, as described in Experimental Procedures. The numbers to the left of the gel indicate size markers (a 5’ 32 P-labeled ssDNA ladder; ss20 DNA Ladder, Simplex Sciences) run in a parallel lane, and the labels to the right of the gel indicate the products resulting from the initial template switch (1x) and subsequent end-to-end template switches from the 5’ end of one acceptor to the 3’ end of another (2x, 3x, etc .). The plot at the right shows time courses for the production of template-switching products ( i.e. , products > 2 nt larger than the primer), with each data set fit by a single-exponential function and the error bars indicating the standard deviations for three experiments. The inset table indicates the k obs and amplitude parameters obtained from the fit of an exponential function to the average values from three independent determinations, along with standard errors obtained from the fit (see Experimental Procedures).

    Article Snippet: Unless specified otherwise, reactions were done with 500 nM GsI-IIC RT, 50 nM R2 RNA/R2R DNA starter duplex, and 100 nM acceptor oligonucleotide in 25 μl of medium containing 200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.5, 5 mM fresh DTT, and an equimolar mix of 1 mM each dATP, dCTP, dGTP, and dTTP (Promega) to give 4 mM total dNTP concentration.

    Techniques: Sequencing, Incubation, Labeling, Electrophoresis, RNA Sequencing Assay, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis

    Template-switching reactions from blunt-end and 1-nt 3’-overhang starter duplexes with varying concentrations of dNTPs. A and B , Template-switching reactions with 50 nM blunt-end (A) or 1-nt overhang (B) starter duplexes with a 32 P-labeled (*) DNA primer and 100 nM 50-nt acceptor RNA were done with 500 nM GsI-IIC RNA RT and 0.04, 0.4, 1 or 4 mM dNTPs (where 4 mM dNTPs = 1 mM each of dATP, dCTP, dGTP, and dTTP). The reactions were stopped after times ranging from 6 to 900 s, and the products were analyzed by denaturing PAGE and quantified from a phosphorimager scan of the dried gel. The data were fit by a single exponential function to obtain the observed rates and amplitudes, which are summarized in the associated tables. C and D , The same template-switching reactions as in A and B but adding 0.04, 0.4, 1, or 4 mM dGTP, the nucleotide complementary to the last 2 nucleotides (CC) of the acceptor RNA, so that the template-switching products for the (C) blunt-end duplex is 2 nt longer than the DNA primer, and that for the (D) 1-nt overhang duplex is only 1 nt longer than the DNA primer.

    Journal: bioRxiv

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

    doi: 10.1101/792986

    Figure Lengend Snippet: Template-switching reactions from blunt-end and 1-nt 3’-overhang starter duplexes with varying concentrations of dNTPs. A and B , Template-switching reactions with 50 nM blunt-end (A) or 1-nt overhang (B) starter duplexes with a 32 P-labeled (*) DNA primer and 100 nM 50-nt acceptor RNA were done with 500 nM GsI-IIC RNA RT and 0.04, 0.4, 1 or 4 mM dNTPs (where 4 mM dNTPs = 1 mM each of dATP, dCTP, dGTP, and dTTP). The reactions were stopped after times ranging from 6 to 900 s, and the products were analyzed by denaturing PAGE and quantified from a phosphorimager scan of the dried gel. The data were fit by a single exponential function to obtain the observed rates and amplitudes, which are summarized in the associated tables. C and D , The same template-switching reactions as in A and B but adding 0.04, 0.4, 1, or 4 mM dGTP, the nucleotide complementary to the last 2 nucleotides (CC) of the acceptor RNA, so that the template-switching products for the (C) blunt-end duplex is 2 nt longer than the DNA primer, and that for the (D) 1-nt overhang duplex is only 1 nt longer than the DNA primer.

    Article Snippet: Unless specified otherwise, reactions were done with 500 nM GsI-IIC RT, 50 nM R2 RNA/R2R DNA starter duplex, and 100 nM acceptor oligonucleotide in 25 μl of medium containing 200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.5, 5 mM fresh DTT, and an equimolar mix of 1 mM each dATP, dCTP, dGTP, and dTTP (Promega) to give 4 mM total dNTP concentration.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis

    Non-templated nucleotide addition activity to blunt-end and 1-nt 3’ overhang starter duplexes using mixed dNTPs. A , Reactions included 500 nM GsI-II RT and 50 nM of a blunt-end starter duplex with 5’- 32 P-labeled (*) DNA primer in a solution containing 200 mM NaCl and varying dNTP concentrations (0.04, 0.4, 1, and 4 mM, where 4 mM is an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP). Aliquots were quenched at times from 10 s to 7,200 s, and the products were analyzed by electrophoresis in a denaturing polyacrylamide gel, which was dried and scanned with a phosphorimager (gels and calculated kinetic parameters shown in Fig. 4 ). B , Reactions included 500 nM GsI-II RT and 50 nM of a 1-nt G overhang starter duplex and 200 mM NaCl. Reaction aliquots were stopped at times from 10 to 7,200 s, and the products were analyzed as in A. The observed rates for the first NTA from this template were used to calculate the kinetic parameters shown below the plot.

    Journal: bioRxiv

    Article Title: Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

    doi: 10.1101/792986

    Figure Lengend Snippet: Non-templated nucleotide addition activity to blunt-end and 1-nt 3’ overhang starter duplexes using mixed dNTPs. A , Reactions included 500 nM GsI-II RT and 50 nM of a blunt-end starter duplex with 5’- 32 P-labeled (*) DNA primer in a solution containing 200 mM NaCl and varying dNTP concentrations (0.04, 0.4, 1, and 4 mM, where 4 mM is an equimolar mix of 1 mM dATP, dCTP, dGTP, and dTTP). Aliquots were quenched at times from 10 s to 7,200 s, and the products were analyzed by electrophoresis in a denaturing polyacrylamide gel, which was dried and scanned with a phosphorimager (gels and calculated kinetic parameters shown in Fig. 4 ). B , Reactions included 500 nM GsI-II RT and 50 nM of a 1-nt G overhang starter duplex and 200 mM NaCl. Reaction aliquots were stopped at times from 10 to 7,200 s, and the products were analyzed as in A. The observed rates for the first NTA from this template were used to calculate the kinetic parameters shown below the plot.

    Article Snippet: Unless specified otherwise, reactions were done with 500 nM GsI-IIC RT, 50 nM R2 RNA/R2R DNA starter duplex, and 100 nM acceptor oligonucleotide in 25 μl of medium containing 200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl pH 7.5, 5 mM fresh DTT, and an equimolar mix of 1 mM each dATP, dCTP, dGTP, and dTTP (Promega) to give 4 mM total dNTP concentration.

    Techniques: Activity Assay, Labeling, Electrophoresis

    Differential Sensitivity of the Pol D/N752 Virus and Protein Variants to Aphidicolin and Structural Modeling of the Pol Region of Interest (A) Cells were infected with the Pol D752 revertant (•) or N752 mutant (○) virus, treated with aphidicolin, incubated 3 d, then lysed; final virus yield was titrated on new cells. (B) DNA was also extracted after the lysing step and qPCR performed to quantify normalized viral genome copies. (C) The DNA polymerase activity of Pol D752 and Pol N752 proteins, in the absence and in the presence of pORF18 (Pol accessory subunit), was analyzed by measuring the incorporation of [ 3 H]dTTP into a poly(dA)-oligo(dT) template. (▪) Pol D752; (□) Pol N752; (•) Pol D752 + pORF18; (○) Pol N752 + pORF18. (D) The effect of aphidicolin on polymerase activity of Pol D752 (•) and of Pol N752 (○) was assayed by measuring the incorporation of [ 3 H]dTTP into a poly(dA)-oligo(dT) template in the presence of pORF18. Graphs show the average of three experiments with standard deviations (error bars). Asterisk * indicates p

    Journal: PLoS Pathogens

    Article Title: A Point Mutation in a Herpesvirus Polymerase Determines Neuropathogenicity

    doi: 10.1371/journal.ppat.0030160

    Figure Lengend Snippet: Differential Sensitivity of the Pol D/N752 Virus and Protein Variants to Aphidicolin and Structural Modeling of the Pol Region of Interest (A) Cells were infected with the Pol D752 revertant (•) or N752 mutant (○) virus, treated with aphidicolin, incubated 3 d, then lysed; final virus yield was titrated on new cells. (B) DNA was also extracted after the lysing step and qPCR performed to quantify normalized viral genome copies. (C) The DNA polymerase activity of Pol D752 and Pol N752 proteins, in the absence and in the presence of pORF18 (Pol accessory subunit), was analyzed by measuring the incorporation of [ 3 H]dTTP into a poly(dA)-oligo(dT) template. (▪) Pol D752; (□) Pol N752; (•) Pol D752 + pORF18; (○) Pol N752 + pORF18. (D) The effect of aphidicolin on polymerase activity of Pol D752 (•) and of Pol N752 (○) was assayed by measuring the incorporation of [ 3 H]dTTP into a poly(dA)-oligo(dT) template in the presence of pORF18. Graphs show the average of three experiments with standard deviations (error bars). Asterisk * indicates p

    Article Snippet: Basal DNA polymerase activity of Pol D752 and of Pol N752 and stimulation of their activity by pORF18 were assayed by measuring the incorporation of [3 H]dTTP (Amersham Bioscience-GE Healthcare, Milan, Italy) into a poly(dA)-oligo(dT) template (Amersham Bioscience-GE Healthcare) as previously reported [ ], using 12 μl of in vitro transcribed-translated Pol D752 or Pol N752 in the absence or in the presence of 600 fmol of purified baculovirus-expressed pORF18 in a 60-μl reaction volume.

    Techniques: Infection, Mutagenesis, Incubation, Real-time Polymerase Chain Reaction, Activity Assay

    (A) Structures of 2' deoxynucleoside triphosphates used or referred to in this study are dATP, dCTP, dGTP, dTTP, Ind-TP, 5-FITP, 5-AITP, 5-NITP, 5-PhITP, 5-CE-ITP, 5-CH-ITP, and 5-NapITP. For convenience, dR is used to represent the 2'-deoxyribose 5'-triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes T or the presence of a tetrahydrofuran moiety that mimics an abasic site.

    Journal: Biochemistry

    Article Title: The Mechanism and Dynamics of Translesion DNA Synthesis Catalyzed by the Escherichia coli Klenow fragment

    doi: 10.1021/bi800324r

    Figure Lengend Snippet: (A) Structures of 2' deoxynucleoside triphosphates used or referred to in this study are dATP, dCTP, dGTP, dTTP, Ind-TP, 5-FITP, 5-AITP, 5-NITP, 5-PhITP, 5-CE-ITP, 5-CH-ITP, and 5-NapITP. For convenience, dR is used to represent the 2'-deoxyribose 5'-triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes T or the presence of a tetrahydrofuran moiety that mimics an abasic site.

    Article Snippet: Oligonucleotides, including those containing a tetrahydrofuran moiety mimicking an abasic site, were synthesized by Operon Technologies (Alameda, CA). dATP, dCTP, dGTP, and dTTP were obtained from Sigma in greater than 99% purity.

    Techniques: