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  • 99
    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: 99/100, based on 1202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 1512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dttp  (TaKaRa)
    99
    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: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore thymidine triphosphate dttp
    CDA deficiency leads to excess dCTP, inhibiting PARP-1 activity and leading to UFB formation. (A) Relative number of PAR foci in HeLa-Ctrl (CDA) (black bars) and HeLa-shCDA (gray bars) cell lines treated with 1 mM dC. Error bars represent means ± SD for five independent experiments ( > 1,200 cells per condition). (B) Mean number of UFBs per anaphase cell in HeLa-Ctrl (CDA) (black bars) and HeLa-shCDA (gray bars) cell lines left untreated or treated with 1 mM dC. Error bars represent means ± SD for three independent experiments ( > 95 anaphase cells per condition). (C) Percentage of centromeres left unreplicated in HeLa-Ctrl (CDA) (black bars) and in HeLa-shCDA (gray bars) prometaphase cells left untreated or treated with 1 mM dC. Error bars represent means ± SD for three independent experiments ( > 95 anaphase cells per condition). (D) In vitro analysis of PARP-1 activity in the presence of various amounts of dCTP. Error bars represent means ± SD from three independent experiments. Statistical significance was calculated with Student’s t -test. (E) In vitro analysis of PARP-1 activity in the presence of 1 mM 3-AB or 10 mM dCTP, <t>dUTP,</t> <t>dTTP,</t> dGTP, dATP. Error bars represent means ± SD from three independent experiments. (F) In vitro analysis of PARP-1 activity in the presence of 10 mM dCTP and various amounts of biotinylated NAD + (20; 50; 100 μM). Error bars represent means ± SD from three independent experiments. Statistical significance was calculated with Student’s t -test. (G) (1) CDA deficiency leads to intracellular dCTP accumulation, decreasing basal PARP-1 activity. (2) The decrease in basal PARP-1 activity leads to an increase in unreplicated DNA sequences (preferentially at centromeres and CFS sites). (3) During late G2/early mitosis, some of this unreplicated DNA is probably processed by MUS81-EME1 and ERCC1 nucleases as previously suggested [ 36 , 12 ], leading to DNA synthesis during metaphase (EdU foci) and preventing supernumerary UFB formation. (4) Some of the excess unreplicated DNA sequences are not processed by the nucleases, leading to supernumerary UFB formation.
    Thymidine Triphosphate Dttp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer h 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.
    H Deoxythymidine Triphosphate Dttp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    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: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    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: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Promega dttp
    The detection of polyA RNA in permeabilized HeLa cells. The cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100 ( A–H ) or fixed and permeabilized in methanol/acetone ( I ). Cy3-labelled secondary antibodies were used in all of the experiments. (A) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, <t>dTTP,</t> <t>dATP,</t> dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (B) The immunolocalization of biotin in the cells without incubation in RM. (C) The immunolocalization of biotin after the incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP and dGTP (without oligo dT15). (D) The detection of polyA RNA after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated BrdU. (E) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP and dGTP (without oligo dT15). (F) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dA15. (G) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and random hexanucleotide. (H) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (I) The detection of polyA RNA after the incubation of the methanol/acetone-fixed cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of BrdU. The images of the biotin immunolocalization were acquired at 2000 ms (A–C, H), whereas the images of the BrdU immunolocalization at 70 ms (D–G, I). Bar: 20 µm.
    Dttp, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 307 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    ICN Pharmaceuticals thymidine 5 triphosphate
    The detection of polyA RNA in permeabilized HeLa cells. The cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100 ( A–H ) or fixed and permeabilized in methanol/acetone ( I ). Cy3-labelled secondary antibodies were used in all of the experiments. (A) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, <t>dTTP,</t> <t>dATP,</t> dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (B) The immunolocalization of biotin in the cells without incubation in RM. (C) The immunolocalization of biotin after the incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP and dGTP (without oligo dT15). (D) The detection of polyA RNA after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated BrdU. (E) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP and dGTP (without oligo dT15). (F) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dA15. (G) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and random hexanucleotide. (H) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (I) The detection of polyA RNA after the incubation of the methanol/acetone-fixed cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of BrdU. The images of the biotin immunolocalization were acquired at 2000 ms (A–C, H), whereas the images of the BrdU immunolocalization at 70 ms (D–G, I). Bar: 20 µm.
    Thymidine 5 Triphosphate, supplied by ICN Pharmaceuticals, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare p dttp
    The detection of polyA RNA in permeabilized HeLa cells. The cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100 ( A–H ) or fixed and permeabilized in methanol/acetone ( I ). Cy3-labelled secondary antibodies were used in all of the experiments. (A) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, <t>dTTP,</t> <t>dATP,</t> dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (B) The immunolocalization of biotin in the cells without incubation in RM. (C) The immunolocalization of biotin after the incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP and dGTP (without oligo dT15). (D) The detection of polyA RNA after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated BrdU. (E) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP and dGTP (without oligo dT15). (F) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dA15. (G) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and random hexanucleotide. (H) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (I) The detection of polyA RNA after the incubation of the methanol/acetone-fixed cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of BrdU. The images of the biotin immunolocalization were acquired at 2000 ms (A–C, H), whereas the images of the BrdU immunolocalization at 70 ms (D–G, I). Bar: 20 µm.
    P Dttp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    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: 88/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Moravek Biochemicals 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 Moravek Biochemicals, used in various techniques. Bioz Stars score: 88/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    GE Healthcare α 32 p dttp
    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and <t>dTTP,</t> in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
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    GE Healthcare 32p dttp
    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and <t>dTTP,</t> in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
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    DuPont de Nemours h dttp
    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and <t>dTTP,</t> in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
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    Kaneka Corp mastermix dttp
    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and <t>dTTP,</t> in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
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    Kaneka Corp i dttp
    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and <t>dTTP,</t> in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
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    PerkinElmer methyl 3 h thymidine triphosphate
    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and <t>dTTP,</t> in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
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    Kaneka Corp dttp blue
    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and <t>dTTP,</t> in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
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    Thermo Fisher dttp nt
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Millipore thymidine
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Thermo Fisher γ methylene dttp
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    GE Healthcare h labeled thymidine 5 triphosphate
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Kaneka Corp sybr assay dttp
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Glen Research α thio dttp
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Biotech AB dttp
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Brookhaven Instruments dttp
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Meridian Life Science dttp
    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single <t>dGTP,</t> dCTP or <t>dTTP</t> incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value
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    Image Search Results


    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

    (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:

    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

    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

    CDA deficiency leads to excess dCTP, inhibiting PARP-1 activity and leading to UFB formation. (A) Relative number of PAR foci in HeLa-Ctrl (CDA) (black bars) and HeLa-shCDA (gray bars) cell lines treated with 1 mM dC. Error bars represent means ± SD for five independent experiments ( > 1,200 cells per condition). (B) Mean number of UFBs per anaphase cell in HeLa-Ctrl (CDA) (black bars) and HeLa-shCDA (gray bars) cell lines left untreated or treated with 1 mM dC. Error bars represent means ± SD for three independent experiments ( > 95 anaphase cells per condition). (C) Percentage of centromeres left unreplicated in HeLa-Ctrl (CDA) (black bars) and in HeLa-shCDA (gray bars) prometaphase cells left untreated or treated with 1 mM dC. Error bars represent means ± SD for three independent experiments ( > 95 anaphase cells per condition). (D) In vitro analysis of PARP-1 activity in the presence of various amounts of dCTP. Error bars represent means ± SD from three independent experiments. Statistical significance was calculated with Student’s t -test. (E) In vitro analysis of PARP-1 activity in the presence of 1 mM 3-AB or 10 mM dCTP, dUTP, dTTP, dGTP, dATP. Error bars represent means ± SD from three independent experiments. (F) In vitro analysis of PARP-1 activity in the presence of 10 mM dCTP and various amounts of biotinylated NAD + (20; 50; 100 μM). Error bars represent means ± SD from three independent experiments. Statistical significance was calculated with Student’s t -test. (G) (1) CDA deficiency leads to intracellular dCTP accumulation, decreasing basal PARP-1 activity. (2) The decrease in basal PARP-1 activity leads to an increase in unreplicated DNA sequences (preferentially at centromeres and CFS sites). (3) During late G2/early mitosis, some of this unreplicated DNA is probably processed by MUS81-EME1 and ERCC1 nucleases as previously suggested [ 36 , 12 ], leading to DNA synthesis during metaphase (EdU foci) and preventing supernumerary UFB formation. (4) Some of the excess unreplicated DNA sequences are not processed by the nucleases, leading to supernumerary UFB formation.

    Journal: PLoS Genetics

    Article Title: Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA

    doi: 10.1371/journal.pgen.1005384

    Figure Lengend Snippet: CDA deficiency leads to excess dCTP, inhibiting PARP-1 activity and leading to UFB formation. (A) Relative number of PAR foci in HeLa-Ctrl (CDA) (black bars) and HeLa-shCDA (gray bars) cell lines treated with 1 mM dC. Error bars represent means ± SD for five independent experiments ( > 1,200 cells per condition). (B) Mean number of UFBs per anaphase cell in HeLa-Ctrl (CDA) (black bars) and HeLa-shCDA (gray bars) cell lines left untreated or treated with 1 mM dC. Error bars represent means ± SD for three independent experiments ( > 95 anaphase cells per condition). (C) Percentage of centromeres left unreplicated in HeLa-Ctrl (CDA) (black bars) and in HeLa-shCDA (gray bars) prometaphase cells left untreated or treated with 1 mM dC. Error bars represent means ± SD for three independent experiments ( > 95 anaphase cells per condition). (D) In vitro analysis of PARP-1 activity in the presence of various amounts of dCTP. Error bars represent means ± SD from three independent experiments. Statistical significance was calculated with Student’s t -test. (E) In vitro analysis of PARP-1 activity in the presence of 1 mM 3-AB or 10 mM dCTP, dUTP, dTTP, dGTP, dATP. Error bars represent means ± SD from three independent experiments. (F) In vitro analysis of PARP-1 activity in the presence of 10 mM dCTP and various amounts of biotinylated NAD + (20; 50; 100 μM). Error bars represent means ± SD from three independent experiments. Statistical significance was calculated with Student’s t -test. (G) (1) CDA deficiency leads to intracellular dCTP accumulation, decreasing basal PARP-1 activity. (2) The decrease in basal PARP-1 activity leads to an increase in unreplicated DNA sequences (preferentially at centromeres and CFS sites). (3) During late G2/early mitosis, some of this unreplicated DNA is probably processed by MUS81-EME1 and ERCC1 nucleases as previously suggested [ 36 , 12 ], leading to DNA synthesis during metaphase (EdU foci) and preventing supernumerary UFB formation. (4) Some of the excess unreplicated DNA sequences are not processed by the nucleases, leading to supernumerary UFB formation.

    Article Snippet: Deoxyuridine (dU), deoxycytidine (dC), deoxyuridine triphosphate (dUTP), deoxycytidine triphosphate (dCTP), deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP) and thymidine triphosphate (dTTP) were provided by Sigma Aldrich (D5412; D0779; D4001, D4635, D6500, D4010 and T0251 respectively); tetrahydrouridine (THU) was provided by Calbiochem (584222); camptothecin (CPT) was provided by Sigma Aldrich (C9911) and olaparib was provided by SelleckChem (S1060).

    Techniques: Activity Assay, In Vitro, DNA Synthesis

    (A) Time-dependent release of TMR-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). (B) Fluorescence spectra corresponding to the release of TMR-D upon exposing the microcapsules loaded with TMR-D to various concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 25, (e) 50 and (f) 75 mM. Inset: derived calibration curve corresponding to the fluorescence intensities of the released TMR-D at λ em = 582 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of TMR-D upon the treatment of the ATP-responsive microcapsules with different nucleotide triphosphates for a fixed time interval of 30 min: (a) 25 mM ATP, (b) 25 mM TTP, (c) 25 mM CTP, (d) 25 mM GTP, and (e) untreated.

    Journal: Chemical Science

    Article Title: pH- and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules and ligand-induced release of loads from DNA–acrylamide hydrogel microcapsules †Electronic supplementary information (ESI) available: Nucleic acid sequences; characterization of nucleic acid-modified copolymers; SEM images, confocal images of microcapsules; calibration curves of TMR-D, TR-D, and DOX-D; synthesis of DOX-D; CD spectra of pH-responsive hydrogel; characterization of hydrogel membranes. See DOI: 10.1039/c6sc04770jClick here for additional data file.

    doi: 10.1039/c6sc04770j

    Figure Lengend Snippet: (A) Time-dependent release of TMR-D from the ATP-responsive hydrogel microcapsules treated with 50 mM ATP (a) and without added ATP (b). (B) Fluorescence spectra corresponding to the release of TMR-D upon exposing the microcapsules loaded with TMR-D to various concentrations of ATP for a fixed time interval of 30 min: (a) 0, (b) 5, (c) 10, (d) 25, (e) 50 and (f) 75 mM. Inset: derived calibration curve corresponding to the fluorescence intensities of the released TMR-D at λ em = 582 nm upon treatment with different concentrations of ATP. (C) Fluorescence spectra corresponding to the release of TMR-D upon the treatment of the ATP-responsive microcapsules with different nucleotide triphosphates for a fixed time interval of 30 min: (a) 25 mM ATP, (b) 25 mM TTP, (c) 25 mM CTP, (d) 25 mM GTP, and (e) untreated.

    Article Snippet: Reagents and materials Magnesium chloride, sodium chloride, doxorubicin hydrochloride (DOX), 4-carboxyphenylboronic acid, dextran (MW = 40 kDa), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES base), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES acid), N -(3-dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride (EDC), N -hydroxysulfosuccinimide sodium salt (sulfo-NHS), poly(acrylic acid) (MW = 30, 450, and 1250 kDa), 2-(N -morpholino)ethanesulfonic acid (MES), ammonium persulfate (APS), N ,N ,N ′,N ′-tetramethylethylenediamine (TEMED), acrylamide solution (40%), poly(allylamine hydrochloride) (PAH, MW = 58 kDa), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and thymidine triphosphate (TTP) were purchased from Sigma-Aldrich.

    Techniques: Fluorescence, Derivative Assay

    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

    8-oxodGTP is a telomerase chain terminator. ( a ) Telomerase catalytic cycle. Blue indicates newly added nucleotides and grey indicates the telomerase template. Numbers represents steps in the cycle. ( b ) Telomerase reactions were conducted using (TTAGGG) 3 primer (3R) with high dNTPs (500 μM dGTP, 500 μM dATP, and 2.9 μM dTTP) (lanes 2 – 4) or cellular dNTPs (37 μM dTTP, 24 μM dATP, 29 μM dCTP, 5.2 μM dGTP) (lanes 6 – 8) and 0.3 μM 32 P-α-dTTP. dGTP and 8-oxodGTP were mixed at 1:0, 1:1, and 0:1 ratios for a total of 500 μM or 5.2 μM unlabeled guanine nucleotide. Products were separated on denaturing gels. The LC was a 32 P end-labeled 18-mer oligonucleotide. Numbers on the left show number of added repeats, and letters on right indicate template base. ( c ) Total products were normalized to the LC and used to calculate processivity and relative activity. Bars represent the mean ± sd from three independent experiments. * p

    Journal: Nature structural & molecular biology

    Article Title: Oxidative guanine base damage regulates human telomerase activity

    doi: 10.1038/nsmb.3319

    Figure Lengend Snippet: 8-oxodGTP is a telomerase chain terminator. ( a ) Telomerase catalytic cycle. Blue indicates newly added nucleotides and grey indicates the telomerase template. Numbers represents steps in the cycle. ( b ) Telomerase reactions were conducted using (TTAGGG) 3 primer (3R) with high dNTPs (500 μM dGTP, 500 μM dATP, and 2.9 μM dTTP) (lanes 2 – 4) or cellular dNTPs (37 μM dTTP, 24 μM dATP, 29 μM dCTP, 5.2 μM dGTP) (lanes 6 – 8) and 0.3 μM 32 P-α-dTTP. dGTP and 8-oxodGTP were mixed at 1:0, 1:1, and 0:1 ratios for a total of 500 μM or 5.2 μM unlabeled guanine nucleotide. Products were separated on denaturing gels. The LC was a 32 P end-labeled 18-mer oligonucleotide. Numbers on the left show number of added repeats, and letters on right indicate template base. ( c ) Total products were normalized to the LC and used to calculate processivity and relative activity. Bars represent the mean ± sd from three independent experiments. * p

    Article Snippet: Briefly, reactions (20 μl) contained 1x human telomerase buffer, 1 μM oligonucleotide substrate, 0.3 μM of 3,000 Ci/mmol 32 P-α-dGTP or 32 P-α-dTTP (Perkin Elmer) and dNTP (Invitrogen) mix as indicated in the figure legend.

    Techniques: Labeling, 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

    The detection of polyA RNA in permeabilized HeLa cells. The cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100 ( A–H ) or fixed and permeabilized in methanol/acetone ( I ). Cy3-labelled secondary antibodies were used in all of the experiments. (A) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (B) The immunolocalization of biotin in the cells without incubation in RM. (C) The immunolocalization of biotin after the incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP and dGTP (without oligo dT15). (D) The detection of polyA RNA after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated BrdU. (E) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP and dGTP (without oligo dT15). (F) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dA15. (G) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and random hexanucleotide. (H) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (I) The detection of polyA RNA after the incubation of the methanol/acetone-fixed cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of BrdU. The images of the biotin immunolocalization were acquired at 2000 ms (A–C, H), whereas the images of the BrdU immunolocalization at 70 ms (D–G, I). Bar: 20 µm.

    Journal: Nucleic Acids Research

    Article Title: In situ reverse transcription: the magic of strength and anonymity

    doi: 10.1093/nar/gkq619

    Figure Lengend Snippet: The detection of polyA RNA in permeabilized HeLa cells. The cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100 ( A–H ) or fixed and permeabilized in methanol/acetone ( I ). Cy3-labelled secondary antibodies were used in all of the experiments. (A) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (B) The immunolocalization of biotin in the cells without incubation in RM. (C) The immunolocalization of biotin after the incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP and dGTP (without oligo dT15). (D) The detection of polyA RNA after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated BrdU. (E) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP and dGTP (without oligo dT15). (F) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dA15. (G) The immunolocalization of BrdU after the incubation of the cells in RM containing BrdUTP, dATP, dCTP, dGTP and random hexanucleotide. (H) The detection of polyA RNA after the incubation of the cells in RM containing biotin-dUTP, BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of the incorporated biotin. (I) The detection of polyA RNA after the incubation of the methanol/acetone-fixed cells in RM containing BrdUTP, dATP, dCTP, dGTP and oligo dT15 by means of an immunolocalization of BrdU. The images of the biotin immunolocalization were acquired at 2000 ms (A–C, H), whereas the images of the BrdU immunolocalization at 70 ms (D–G, I). Bar: 20 µm.

    Article Snippet: The following components were used in the RM during the control experiments with DNA polymerase I, E. coli (Fermentas): 1 × DNA polymerase I, E. coli buffer (Fermentas, 10x polymerase I buffer: 500 mM Tris–HCl, 100 mM MgCl2 , 10 mM DTT), 0.2 U/µl DNA polymerase I, E. coli , 0.25 mM dATP, dGTP, dCTP and dTTP (Promega), 0.25 mM BrdUTP and 0.05 mM biotin-dUTP.

    Techniques: Incubation, Mass Spectrometry

    The detection of polyA RNA by means of Alexa-dUTP in permeabilized HeLa cells. The cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100. ( A ) The detection of polyA RNA after an incubation of the cells in RM containing AMV reverse transcriptase, Alexa-dUTP, BrdUTP, dATP, dCTP, dGTP and oligo dT15. ( B ) The detection of polyA RNA after an incubation of the cells in RM containing AMV reverse transcriptase, Alexa-dUTP, dTTP, dATP, dCTP, dGTP and oligo dT15. All of the images were acquired at 2000 ms. Bar: 10 µm.

    Journal: Nucleic Acids Research

    Article Title: In situ reverse transcription: the magic of strength and anonymity

    doi: 10.1093/nar/gkq619

    Figure Lengend Snippet: The detection of polyA RNA by means of Alexa-dUTP in permeabilized HeLa cells. The cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100. ( A ) The detection of polyA RNA after an incubation of the cells in RM containing AMV reverse transcriptase, Alexa-dUTP, BrdUTP, dATP, dCTP, dGTP and oligo dT15. ( B ) The detection of polyA RNA after an incubation of the cells in RM containing AMV reverse transcriptase, Alexa-dUTP, dTTP, dATP, dCTP, dGTP and oligo dT15. All of the images were acquired at 2000 ms. Bar: 10 µm.

    Article Snippet: The following components were used in the RM during the control experiments with DNA polymerase I, E. coli (Fermentas): 1 × DNA polymerase I, E. coli buffer (Fermentas, 10x polymerase I buffer: 500 mM Tris–HCl, 100 mM MgCl2 , 10 mM DTT), 0.2 U/µl DNA polymerase I, E. coli , 0.25 mM dATP, dGTP, dCTP and dTTP (Promega), 0.25 mM BrdUTP and 0.05 mM biotin-dUTP.

    Techniques: Incubation, Mass Spectrometry

    The comparison of DNA polymerase I and AMV reverse transcriptase activity in situ . The HeLa cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100. Cy3-labelled secondary antibodies were used in all of the experiments. ( A ) The immunolocalization of biotin after a 1-h incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP and dGTP and AMV reverse transcriptase. ( B ) The immunolocalization of biotin after a 10-min incubation of the cells in RM containing DNA polymerase I, biotin-dUTP, dTTP, dATP, dCTP and dGTP. ( C ) The immunolocalization of biotin after a 10-min pre-incubation of the cells in RM containing DNA polymerase I, dTTP, dATP, dCTP and dGTP and a subsequent incubation in RM containing AMV reverse transcriptase, biotin-dUTP, dTTP, dATP, dCTP and dGTP. ( D ) The immunolocalization of BrdU after an incubation of the cells in RM containing AMV reverse transcriptase, BrdUTP, dATP, dCTP and dGTP. ( E ) The immunolocalization of BrdU after a 10-min incubation of the cells in RM containing DNA polymerase I, BrdUTP, dATP, dCTP and dGTP. ( F ) The immunolocalization of BrdU after a 10-min pre-incubation of the cells in RM containing DNA polymerase I, dTTP, dATP, dCTP and dGTP and a subsequent incubation in RM containing AMV reverse transcriptase, BrdUTP, dATP, dCTP and dGTP. All of the images were acquired at 2000 ms. Bar: 20 µm.

    Journal: Nucleic Acids Research

    Article Title: In situ reverse transcription: the magic of strength and anonymity

    doi: 10.1093/nar/gkq619

    Figure Lengend Snippet: The comparison of DNA polymerase I and AMV reverse transcriptase activity in situ . The HeLa cells were fixed with formaldehyde and permeabilized with 0.2% Triton X-100. Cy3-labelled secondary antibodies were used in all of the experiments. ( A ) The immunolocalization of biotin after a 1-h incubation of the cells in RM containing biotin-dUTP, dTTP, dATP, dCTP and dGTP and AMV reverse transcriptase. ( B ) The immunolocalization of biotin after a 10-min incubation of the cells in RM containing DNA polymerase I, biotin-dUTP, dTTP, dATP, dCTP and dGTP. ( C ) The immunolocalization of biotin after a 10-min pre-incubation of the cells in RM containing DNA polymerase I, dTTP, dATP, dCTP and dGTP and a subsequent incubation in RM containing AMV reverse transcriptase, biotin-dUTP, dTTP, dATP, dCTP and dGTP. ( D ) The immunolocalization of BrdU after an incubation of the cells in RM containing AMV reverse transcriptase, BrdUTP, dATP, dCTP and dGTP. ( E ) The immunolocalization of BrdU after a 10-min incubation of the cells in RM containing DNA polymerase I, BrdUTP, dATP, dCTP and dGTP. ( F ) The immunolocalization of BrdU after a 10-min pre-incubation of the cells in RM containing DNA polymerase I, dTTP, dATP, dCTP and dGTP and a subsequent incubation in RM containing AMV reverse transcriptase, BrdUTP, dATP, dCTP and dGTP. All of the images were acquired at 2000 ms. Bar: 20 µm.

    Article Snippet: The following components were used in the RM during the control experiments with DNA polymerase I, E. coli (Fermentas): 1 × DNA polymerase I, E. coli buffer (Fermentas, 10x polymerase I buffer: 500 mM Tris–HCl, 100 mM MgCl2 , 10 mM DTT), 0.2 U/µl DNA polymerase I, E. coli , 0.25 mM dATP, dGTP, dCTP and dTTP (Promega), 0.25 mM BrdUTP and 0.05 mM biotin-dUTP.

    Techniques: Activity Assay, In Situ, Incubation, Mass Spectrometry

    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

    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.

    Journal: The EMBO Journal

    Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

    doi: 10.1093/emboj/19.7.1731

    Figure Lengend Snippet: Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.

    Article Snippet: [γ–32 P]ATP (3000 Ci/mmol), [α–32 P]dATP (3000 Ci/mmol) and [α–32 P]dTTP (3000 Ci/mmol) were obtained from Amersham International Plc.

    Techniques: Inhibition, Incubation, Concentration Assay

    Fig. 4. Pol μ has terminal transferase activity, but requires a template–primer structure for optimal efficiency. ( A ) Terminal transferase activity associated with human Pol μ. The assay was carried out as described in Materials and methods, using 3.2 nM 5′–labelled single-stranded 19mer (P19) as substrate, 1 mM MnCl 2 as a source of activating metal ions, 80 μM each individual deoxynucleotide, and either TdT (2.5 U/41 ng) or Pol μ (20 ng). A control reaction in the absence of enzyme ( C ) was also carried out. After incubation for 30 min at 30°C, extension of the 5′–labelled oligonucleotide was analysed by 8 M urea–20% PAGE and autoradiography. ( B ) Template-dependent polymerization catalysed by Pol μ. Polymerization efficiency was assayed comparatively on either poly(dA) (○), oligo(dT) (□) or a poly(dA)/oligo(dT) hybrid (•) to provide a homopolymeric template (dA)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dTTP, Pol μ (20 ng) and 0.5 μM each DNA substrate. After incubation for the indicated times at 37°C, dTMP incorporation was quantitated as described in Materials and methods.

    Journal: The EMBO Journal

    Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

    doi: 10.1093/emboj/19.7.1731

    Figure Lengend Snippet: Fig. 4. Pol μ has terminal transferase activity, but requires a template–primer structure for optimal efficiency. ( A ) Terminal transferase activity associated with human Pol μ. The assay was carried out as described in Materials and methods, using 3.2 nM 5′–labelled single-stranded 19mer (P19) as substrate, 1 mM MnCl 2 as a source of activating metal ions, 80 μM each individual deoxynucleotide, and either TdT (2.5 U/41 ng) or Pol μ (20 ng). A control reaction in the absence of enzyme ( C ) was also carried out. After incubation for 30 min at 30°C, extension of the 5′–labelled oligonucleotide was analysed by 8 M urea–20% PAGE and autoradiography. ( B ) Template-dependent polymerization catalysed by Pol μ. Polymerization efficiency was assayed comparatively on either poly(dA) (○), oligo(dT) (□) or a poly(dA)/oligo(dT) hybrid (•) to provide a homopolymeric template (dA)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dTTP, Pol μ (20 ng) and 0.5 μM each DNA substrate. After incubation for the indicated times at 37°C, dTMP incorporation was quantitated as described in Materials and methods.

    Article Snippet: [γ–32 P]ATP (3000 Ci/mmol), [α–32 P]dATP (3000 Ci/mmol) and [α–32 P]dTTP (3000 Ci/mmol) were obtained from Amersham International Plc.

    Techniques: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value

    Journal: PLoS ONE

    Article Title: A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

    doi: 10.1371/journal.pone.0020208

    Figure Lengend Snippet: Multiple incorrect nt incorporations at the K65 position with subtype B RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype B template. The full-length product contains a single dGTP, dCTP or dTTP incorporation opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype B RT on the subtype C template. Dislocation is observed with the subtype C template as two distinct nt incorporations at the P+1nt and P+2nt positions. (B) Graphical representation of DNA synthesis with subtype B RT on the subtype B and C templates. The values indicated with an asterisk have a p -value

    Article Snippet: Experiments were performed as described above with the addition of 10 µM of each of the dGTP, dCTP and dTTP nt (Invitrogen, Carlsbad, CA, USA) for the primer-independent reactions versus the addition of 10 µM of each of the four dNTPs (Invitrogen, Carlsbad, CA, USA) for the primer-dependent reactions.

    Techniques: DNA Synthesis

    Multiple incorrect nt incorporations at the K65 position with subtype C RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product is observed as a single dGTP, dCTP or dTTP incorporation occurred opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.

    Journal: PLoS ONE

    Article Title: A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

    doi: 10.1371/journal.pone.0020208

    Figure Lengend Snippet: Multiple incorrect nt incorporations at the K65 position with subtype C RT on subtype B and C templates. (A) Lanes 1 through 10 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype B template. The full-length product is observed as a single dGTP, dCTP or dTTP incorporation occurred opposite the T in the template strand at the P+1nt position. Lanes 11 through 20 depict (+)dsDNA synthesis from the (−)ssDNA intermediate with subtype C RT on the subtype C template. Dislocation is only present with the subtype C template. (B) Graphical representation of DNA synthesis with subtype C RT on the subtype B and C templates. The same trend is observed regardless of origin of the RT enzyme used. (C) Depiction of the primer, template and nt used in the reaction. The homopolymeric regions are underlined and the base responsible for the K65R mutation is indicated in bold.

    Article Snippet: Experiments were performed as described above with the addition of 10 µM of each of the dGTP, dCTP and dTTP nt (Invitrogen, Carlsbad, CA, USA) for the primer-independent reactions versus the addition of 10 µM of each of the four dNTPs (Invitrogen, Carlsbad, CA, USA) for the primer-dependent reactions.

    Techniques: DNA Synthesis, Mutagenesis