Structured Review

Millipore datp
OxsA sequentially hydrolyzes inorganic phosphate molecules from OXT and deoxyribonucleotide molecules. HPLC analysis of OxsA hydrolysis products when incubated with <t>dATP,</t> <t>dADP,</t> or dAMP. Each red trace corresponds to a deoxyribonucleotide standard. The
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Images

1) Product Images from "An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis"

Article Title: An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis

Journal:

doi: 10.1073/pnas.1613610113

OxsA sequentially hydrolyzes inorganic phosphate molecules from OXT and deoxyribonucleotide molecules. HPLC analysis of OxsA hydrolysis products when incubated with dATP, dADP, or dAMP. Each red trace corresponds to a deoxyribonucleotide standard. The
Figure Legend Snippet: OxsA sequentially hydrolyzes inorganic phosphate molecules from OXT and deoxyribonucleotide molecules. HPLC analysis of OxsA hydrolysis products when incubated with dATP, dADP, or dAMP. Each red trace corresponds to a deoxyribonucleotide standard. The

Techniques Used: High Performance Liquid Chromatography, Incubation

2) Product Images from "The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation"

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation

Journal:

doi: 10.1128/MCB.01883-06

E295K has no polymerase activity. (A) An in vitro primer extension assay under steady-state conditions. Typically, 5 nM pol β and 300 nM DNA substrate were incubated with 10 mM MgCl 2 and 50 μM each of dATP, dCTP, dGTP, and dTTP at 37°C
Figure Legend Snippet: E295K has no polymerase activity. (A) An in vitro primer extension assay under steady-state conditions. Typically, 5 nM pol β and 300 nM DNA substrate were incubated with 10 mM MgCl 2 and 50 μM each of dATP, dCTP, dGTP, and dTTP at 37°C

Techniques Used: Activity Assay, In Vitro, Primer Extension Assay, Incubation

3) Product Images from "Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity"

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp201

The Pol β polymorphism R137Q is defective in polymerase activity. ( A ) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP- 32 P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. ( B ) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. ( C ) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32 P as shown in figure. ( D ) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.
Figure Legend Snippet: The Pol β polymorphism R137Q is defective in polymerase activity. ( A ) Top panel, schematic of the biotin-labeled 1-nt gapped DNA substrate (Pol-GAP); bottom panel, polymerase activity assay in which Pol-GAP was incubated with 50 μM each of dATP, dGTP, dTTP and 8 μM dCTP- 32 P and varying amounts of purified Pol β protein (WT and R137Q). WT and R137Q DNA polymerization products were separated by denaturing gel electrophoresis and visualized with a phosphorimager. ( B ) DNA polymerization products were pulled down by Sepharose–avidin beads. After washing, the amount of radio-nucleotide incorporated into the products was determined by liquid scintillation counting. ( C ) Gel shift assay of DNA-binding affinity of R137Q and WT Pol β. In this assay, Pol-GAP was labeled by 32 P as shown in figure. ( D ) ELISA-based isotherm adsorption assay of DNA-binding affinity of R137Q and WT Pol β. The DNA substrate was the same as that used in (A). WT, filled squares; R137, filled circles.

Techniques Used: Activity Assay, Labeling, Incubation, Purification, Nucleic Acid Electrophoresis, Avidin-Biotin Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Adsorption

4) Product Images from "Modified nucleoside triphosphates exist in mammals †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc05472f"

Article Title: Modified nucleoside triphosphates exist in mammals †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc05472f

Journal: Chemical Science

doi: 10.1039/c7sc05472f

Extracted ion chromatograms of the 34 NTPs without labeling (A) or with 8-DMQ labeling (B) followed by LC-ESI-MS/MS analysis. 1, ATP; 2, dATP; 3, GTP; 4, dGTP; 5, CTP; 6, dCTP; 7, UTP; 8, TTP; 9, N 6 -meATP; 10, 7-meGTP; 11, ITP; 12, XTP; 13, N 1 -meATP; 14, 2- S -UTP; 15, 5-hmCTP; 16, 5-meCTP; 17, 5-medCTP; 18, 5-hmdCTP; 19, 2′- O -meATP; 20, 2′- O -me-GTP; 21, 5-cadCTP; 22, YTP; 23, 2′- O -me-ITP; 24, 5-meUTP; 25, 2′- O -meCTP; 26, 5-caseUTP; 27, N 6 -medATP; 28, N 4 -medCTP; 29, 2′- O -meYTP; 30, 2′- O -meUTP; 31, 5-fodCTP; 32, 5-foCTP; 33, O 6 -meGTP; and 34, N 1 -meGTP.
Figure Legend Snippet: Extracted ion chromatograms of the 34 NTPs without labeling (A) or with 8-DMQ labeling (B) followed by LC-ESI-MS/MS analysis. 1, ATP; 2, dATP; 3, GTP; 4, dGTP; 5, CTP; 6, dCTP; 7, UTP; 8, TTP; 9, N 6 -meATP; 10, 7-meGTP; 11, ITP; 12, XTP; 13, N 1 -meATP; 14, 2- S -UTP; 15, 5-hmCTP; 16, 5-meCTP; 17, 5-medCTP; 18, 5-hmdCTP; 19, 2′- O -meATP; 20, 2′- O -me-GTP; 21, 5-cadCTP; 22, YTP; 23, 2′- O -me-ITP; 24, 5-meUTP; 25, 2′- O -meCTP; 26, 5-caseUTP; 27, N 6 -medATP; 28, N 4 -medCTP; 29, 2′- O -meYTP; 30, 2′- O -meUTP; 31, 5-fodCTP; 32, 5-foCTP; 33, O 6 -meGTP; and 34, N 1 -meGTP.

Techniques Used: Labeling, Liquid Chromatography, Mass Spectrometry

5) Product Images from "Aberrant localization of apoptosis protease activating factor-1 in lipid raft sub-domains of diffuse large B cell lymphomas"

Article Title: Aberrant localization of apoptosis protease activating factor-1 in lipid raft sub-domains of diffuse large B cell lymphomas

Journal: Oncotarget

doi: 10.18632/oncotarget.13336

Disruption of Apaf-1 from S-100 pellet increases caspase-3 activity a. , c. S-100 cytosol was prepared from primary BL cells and Raji cells after incubation with MCD (200μM), MPO (5μM), MPO-Zn (200μM), LY30 (25μM) or NaN 3 (5μg/ml) for 1hr. Caspase-3 enzyme activity was measured using DEVD-AFC in the treated and untreated S-100 cytosol of B-cell lymphoma in the presence and absence of 1mM dATP and 4μM cytochrome C. b. , d. , f. Apaf-1 expression was determined in S-100 cytosol of treated and untreated BL cells by western blotting. Membranes were probed with anti β-actin for loading control of cytosolic fractions. e. S-100 cytosolic fraction was purified from Raji cells pre-treated with MPO (5μM) for 1hr followed by treated with etoposide (5μM) and daunorubicin (0.4μg/ml). Caspase 3 enzyme activity was then measured in the presence and absence of 1mM dATP and 4μM cytochrome c.
Figure Legend Snippet: Disruption of Apaf-1 from S-100 pellet increases caspase-3 activity a. , c. S-100 cytosol was prepared from primary BL cells and Raji cells after incubation with MCD (200μM), MPO (5μM), MPO-Zn (200μM), LY30 (25μM) or NaN 3 (5μg/ml) for 1hr. Caspase-3 enzyme activity was measured using DEVD-AFC in the treated and untreated S-100 cytosol of B-cell lymphoma in the presence and absence of 1mM dATP and 4μM cytochrome C. b. , d. , f. Apaf-1 expression was determined in S-100 cytosol of treated and untreated BL cells by western blotting. Membranes were probed with anti β-actin for loading control of cytosolic fractions. e. S-100 cytosolic fraction was purified from Raji cells pre-treated with MPO (5μM) for 1hr followed by treated with etoposide (5μM) and daunorubicin (0.4μg/ml). Caspase 3 enzyme activity was then measured in the presence and absence of 1mM dATP and 4μM cytochrome c.

Techniques Used: Activity Assay, Incubation, Expressing, Western Blot, Purification

6) Product Images from "Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction"

Article Title: Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1546

RecA-mediated HR in the presence of heterologous ends. ( A ) Scheme of the lds substrate with heterology at the 3'- and 5'-end (in red) with the css (+) substrate and the expected product ( prd ). ( B ) Circular 3199 nt ssDNA (10 μM) and 4374 bp Nco I-linearized dsDNA substrate (20 μM in nt) were pre-incubated with SsbA (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, then the preformed SsbA·ssDNA complex was incubated or not with RecO (0.2 μM) or DprA (0.3 μM) for 5 min at 37°C. Finally RecA (1.2 μM) was added, and the reaction was incubated for 30 or 60 min at 37ºC and products separated by 0.8% AGE. In lane 17, the cds DNA substrate was treated with DNase I to generate nc DNA. The positions of the bands corresponding to css, lds, prd and jm are indicated. The symbols + and – denote the presence or absence of the indicated protein(s). Amounts of recombination intermediates and products are indicated, and expressed as percentage of total substrate added. The results are the average of more than three independent experiments (the results given are within a 5% standard error).
Figure Legend Snippet: RecA-mediated HR in the presence of heterologous ends. ( A ) Scheme of the lds substrate with heterology at the 3'- and 5'-end (in red) with the css (+) substrate and the expected product ( prd ). ( B ) Circular 3199 nt ssDNA (10 μM) and 4374 bp Nco I-linearized dsDNA substrate (20 μM in nt) were pre-incubated with SsbA (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, then the preformed SsbA·ssDNA complex was incubated or not with RecO (0.2 μM) or DprA (0.3 μM) for 5 min at 37°C. Finally RecA (1.2 μM) was added, and the reaction was incubated for 30 or 60 min at 37ºC and products separated by 0.8% AGE. In lane 17, the cds DNA substrate was treated with DNase I to generate nc DNA. The positions of the bands corresponding to css, lds, prd and jm are indicated. The symbols + and – denote the presence or absence of the indicated protein(s). Amounts of recombination intermediates and products are indicated, and expressed as percentage of total substrate added. The results are the average of more than three independent experiments (the results given are within a 5% standard error).

Techniques Used: Incubation

SsbA and RecO or SsbA and DprA contribute to RecA-mediated bidirectional recombination in the presence of different nucleotide cofactors. ( A and B ) Scheme of the thr ee-strand exchange reaction between circular 3199 nt ssDNA ( css , + strand in black) and the 4374 bp lds substrate with homology restricted to the 5′-end (in black, A) or the 3′-end (in black, B) of the (-) strand. The expected prd final products of RecA-mediated DNA strand exchange are illustrated. The relevant restriction sites are indicated. The relative lengths of homology and heterology (denoted in red) are indicated. ( C and D ) Circular ssDNA (10 μM) and 4374 bp Pst I- or Eco RI-linearised dsDNA substrate (20 μM in nt) were pre-incubated with SsbA (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, then the preformed SsbA·ssDNA complex was incubated with RecO (C, 0.2 μM) or DprA (D, 0.3 μM) for 5 min at 37°C. Finally RecA (1.2 μM) was added, and the reaction was incubated for 30 or 60 min at 37ºC and products separated by 0.8% AGE. (C) In lane 14, the supercoiled DNA substrate ( cds ) was treated with DNase I to generate nicked DNA ( nc ) that co-migrate with the prd . (D) In lane 14, SsbA was omitted and the reaction incubated by 60 min. The positions of the bands corresponding to css, lds, prd and jm are indicated. Amounts of recombination intermediates and products are indicated, and expressed as percentage of total substrate added. The results are the average value obtained from more than three independent experiments (the results given are within a 5% standard error).
Figure Legend Snippet: SsbA and RecO or SsbA and DprA contribute to RecA-mediated bidirectional recombination in the presence of different nucleotide cofactors. ( A and B ) Scheme of the thr ee-strand exchange reaction between circular 3199 nt ssDNA ( css , + strand in black) and the 4374 bp lds substrate with homology restricted to the 5′-end (in black, A) or the 3′-end (in black, B) of the (-) strand. The expected prd final products of RecA-mediated DNA strand exchange are illustrated. The relevant restriction sites are indicated. The relative lengths of homology and heterology (denoted in red) are indicated. ( C and D ) Circular ssDNA (10 μM) and 4374 bp Pst I- or Eco RI-linearised dsDNA substrate (20 μM in nt) were pre-incubated with SsbA (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, then the preformed SsbA·ssDNA complex was incubated with RecO (C, 0.2 μM) or DprA (D, 0.3 μM) for 5 min at 37°C. Finally RecA (1.2 μM) was added, and the reaction was incubated for 30 or 60 min at 37ºC and products separated by 0.8% AGE. (C) In lane 14, the supercoiled DNA substrate ( cds ) was treated with DNase I to generate nicked DNA ( nc ) that co-migrate with the prd . (D) In lane 14, SsbA was omitted and the reaction incubated by 60 min. The positions of the bands corresponding to css, lds, prd and jm are indicated. Amounts of recombination intermediates and products are indicated, and expressed as percentage of total substrate added. The results are the average value obtained from more than three independent experiments (the results given are within a 5% standard error).

Techniques Used: Incubation

7) Product Images from "Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair"

Article Title: Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv545

Effect of RecO on RecA nucleation and polymerization. Circular pGEM3 Zf (+) ssDNA (10 μM in nt) was pre-incubated with the indicated concentration of RecO for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. Then RecA (0.8 μM) was added and the (d)ATPase activity measured for 25 min. All reactions were repeated three or more times with similar results. The amount of ATP or dATP hydrolyzed was calculated as described in ‘Materials and Methods’ section.
Figure Legend Snippet: Effect of RecO on RecA nucleation and polymerization. Circular pGEM3 Zf (+) ssDNA (10 μM in nt) was pre-incubated with the indicated concentration of RecO for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. Then RecA (0.8 μM) was added and the (d)ATPase activity measured for 25 min. All reactions were repeated three or more times with similar results. The amount of ATP or dATP hydrolyzed was calculated as described in ‘Materials and Methods’ section.

Techniques Used: Incubation, Concentration Assay, Activity Assay

RecO facilitates RecA assembly on SsbA- or SsbB-coated ssDNA. ( A ) Circular pGEM3 Zf (+) ssDNA (10 μM in nt) was pre-incubated with SsbA (0.3 μM) or SsbA and RecO for 5 min at 37°C in buffer A containing 5 mM ATP. Then RecA (0.8 μM) was added and the ATPase activity measured for 25 min. ( B ) Circular ssDNA was pre-incubated with SsbB (0.3 μM) (or both SsbA [0.3 μM] and SsbB [0.3 μM]) and when indicated with RecO (0.1 and 0.2 μM) for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. Then RecA (0.8 μM) was added and the (d)ATPase activity measured for 25 min. ( C ) The circular ssDNA was pre-incubated with a fixed amount of SsbB (0.6 μM), SsbA (0.3 μM) plus SsbB (0.3 μM) or a fixed amount of SsbB (0.3 μM) and increasing SsbA (0.03 to 0.3 μM) concentrations for 5 min at 37°C in buffer A containing 5 mM ATP. Then RecO (0.1 μM) was added to the preformed SSB·ssDNA complexes and incubated for 5 min at 37°C. Finally RecA (0.8 μM) was added and the ATPase activity measured for 25 min. All reactions were repeated three or more times with similar results.
Figure Legend Snippet: RecO facilitates RecA assembly on SsbA- or SsbB-coated ssDNA. ( A ) Circular pGEM3 Zf (+) ssDNA (10 μM in nt) was pre-incubated with SsbA (0.3 μM) or SsbA and RecO for 5 min at 37°C in buffer A containing 5 mM ATP. Then RecA (0.8 μM) was added and the ATPase activity measured for 25 min. ( B ) Circular ssDNA was pre-incubated with SsbB (0.3 μM) (or both SsbA [0.3 μM] and SsbB [0.3 μM]) and when indicated with RecO (0.1 and 0.2 μM) for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. Then RecA (0.8 μM) was added and the (d)ATPase activity measured for 25 min. ( C ) The circular ssDNA was pre-incubated with a fixed amount of SsbB (0.6 μM), SsbA (0.3 μM) plus SsbB (0.3 μM) or a fixed amount of SsbB (0.3 μM) and increasing SsbA (0.03 to 0.3 μM) concentrations for 5 min at 37°C in buffer A containing 5 mM ATP. Then RecO (0.1 μM) was added to the preformed SSB·ssDNA complexes and incubated for 5 min at 37°C. Finally RecA (0.8 μM) was added and the ATPase activity measured for 25 min. All reactions were repeated three or more times with similar results.

Techniques Used: Incubation, Activity Assay

SsbA and RecO contribute to RecA-promoted DNA strand exchange. ( A ) Circular pGEM3 Zf (+) ssDNA (10 μM in nt) and homologous linear dsDNA (20 μM in nt) were pre-incubated with increasing concentrations of SsbA or SsbB (0.15 and 0.3 μM) or RecO (0.2 and 0.4 μM) for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. In the indicated reactions, RecO (0.2 and 0.4 μM) was added to the preformed SsbA·ssDNA or SsbB·ssDNA (SSB 0.3 μM) complexes and incubated for 5 min at 37°C. Finally, RecA (1.2 μM) was added and the reaction was incubated for 60 min at 37°C. ( B ) The circular ssDNA and homologous linear dsDNA were pre-incubated with SsbA or SsbB (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, and RecO (0.2 μM) was added to the preformed SsbA·ssDNA or SsbB·ssDNA complexes and incubated for 10 min at 37°C. Finally, RecA (1.2 μM) was added and the reaction was incubated for 60 min at 37°C. The products of the reactions were deproteinized, separated on a 0.8% AGE with ethidium bromide and quantified as described in ‘Materials and Methods’ section. The positions of the bands corresponding to css, cds, lds, jm, nc and ATPγS-generated prd products are indicated. Symbols + and − denote the presence or absence, respectively, of the indicated protein. In A (lane 14) and in B (lane 17) nicked circular pGEM3 Zf (+) plasmid DNA was added as a mobility control ( C ). The amounts of jm s and products ( nc and prd ) are indicated and expressed as the percentage of total substrate added. The results are the average value obtained from more than three independent experiments (the results given stand within a 5% standard error).
Figure Legend Snippet: SsbA and RecO contribute to RecA-promoted DNA strand exchange. ( A ) Circular pGEM3 Zf (+) ssDNA (10 μM in nt) and homologous linear dsDNA (20 μM in nt) were pre-incubated with increasing concentrations of SsbA or SsbB (0.15 and 0.3 μM) or RecO (0.2 and 0.4 μM) for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. In the indicated reactions, RecO (0.2 and 0.4 μM) was added to the preformed SsbA·ssDNA or SsbB·ssDNA (SSB 0.3 μM) complexes and incubated for 5 min at 37°C. Finally, RecA (1.2 μM) was added and the reaction was incubated for 60 min at 37°C. ( B ) The circular ssDNA and homologous linear dsDNA were pre-incubated with SsbA or SsbB (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, and RecO (0.2 μM) was added to the preformed SsbA·ssDNA or SsbB·ssDNA complexes and incubated for 10 min at 37°C. Finally, RecA (1.2 μM) was added and the reaction was incubated for 60 min at 37°C. The products of the reactions were deproteinized, separated on a 0.8% AGE with ethidium bromide and quantified as described in ‘Materials and Methods’ section. The positions of the bands corresponding to css, cds, lds, jm, nc and ATPγS-generated prd products are indicated. Symbols + and − denote the presence or absence, respectively, of the indicated protein. In A (lane 14) and in B (lane 17) nicked circular pGEM3 Zf (+) plasmid DNA was added as a mobility control ( C ). The amounts of jm s and products ( nc and prd ) are indicated and expressed as the percentage of total substrate added. The results are the average value obtained from more than three independent experiments (the results given stand within a 5% standard error).

Techniques Used: Incubation, Generated, Plasmid Preparation

8) Product Images from "Contractility and kinetics of human fetal and human adult skeletal muscle"

Article Title: Contractility and kinetics of human fetal and human adult skeletal muscle

Journal:

doi: 10.1113/jphysiol.2013.252650

In vitro motility unregulated F-actin speeds for increasing [ATP] or [dATP]
Figure Legend Snippet: In vitro motility unregulated F-actin speeds for increasing [ATP] or [dATP]

Techniques Used: In Vitro

9) Product Images from "Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination"

Article Title: Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination

Journal:

doi: 10.1074/jbc.M114.577924

RecA preferentially hydrolyzes dATP. A , circular 3,199-nt ssDNA (10 μ m in nt) was incubated with RecA (0.8 μ m ) in buffer B, A, or C (pH 6.5, 7.5, or 8.5) containing 5 m m ATP or dATP. Then the ssDNA-dependent (d)ATPase activity was measured
Figure Legend Snippet: RecA preferentially hydrolyzes dATP. A , circular 3,199-nt ssDNA (10 μ m in nt) was incubated with RecA (0.8 μ m ) in buffer B, A, or C (pH 6.5, 7.5, or 8.5) containing 5 m m ATP or dATP. Then the ssDNA-dependent (d)ATPase activity was measured

Techniques Used: Incubation, Activity Assay

Limiting dATP concentrations facilitate RecA·ATP NPF formation and DNA strand exchange. A , circular 3,199-nt ssDNA (10 μ m in nt) was incubated with RecA (0.8 μ m ) in buffer A containing a total of 5 m m of dATP/ATP at different ratios
Figure Legend Snippet: Limiting dATP concentrations facilitate RecA·ATP NPF formation and DNA strand exchange. A , circular 3,199-nt ssDNA (10 μ m in nt) was incubated with RecA (0.8 μ m ) in buffer A containing a total of 5 m m of dATP/ATP at different ratios

Techniques Used: Incubation

10) Product Images from "Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells"

Article Title: Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells

Journal:

doi: 10.3858/emm.2008.40.1.19

Effects of the P2 receptor agonist on the release of IL-10 from microglial cells. Microglial cells (3 × 10 4 cells/well) were treated with 2-meSATP, 2-meSADP, α,β-meATP, BzATP, UTP, UDP, and dATP at the indicated concentrations.
Figure Legend Snippet: Effects of the P2 receptor agonist on the release of IL-10 from microglial cells. Microglial cells (3 × 10 4 cells/well) were treated with 2-meSATP, 2-meSADP, α,β-meATP, BzATP, UTP, UDP, and dATP at the indicated concentrations.

Techniques Used:

11) Product Images from "ATP-Mediated Activation of RNA Polymerase II Transcription Complexes"

Article Title: ATP-Mediated Activation of RNA Polymerase II Transcription Complexes

Journal: Gene Expression

doi:

Time course of activated complex formation. (A) Preinitiation complexes were assembled on pMBPT3 template in HeLa nuclear extract (HNE) and then activated at time 0 by the addition of dATP (100 μM). Sarkosyl (0.02%) was added along with the dATP. At time I, activated complexes were allowed to transcribe by adding [α-32 P]CTP, GTP, and 10-fold molar excess ATPγS (1 mM) and incubating for 10 min. (B) Quantitation of transcriptional activity from activated complexes (closed circles) and correction for (d)ATP-dependent decay (open circles) (see text for details of the correction). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution. Points represent the average of at least three independent experiments, and error bars represent SEM.
Figure Legend Snippet: Time course of activated complex formation. (A) Preinitiation complexes were assembled on pMBPT3 template in HeLa nuclear extract (HNE) and then activated at time 0 by the addition of dATP (100 μM). Sarkosyl (0.02%) was added along with the dATP. At time I, activated complexes were allowed to transcribe by adding [α-32 P]CTP, GTP, and 10-fold molar excess ATPγS (1 mM) and incubating for 10 min. (B) Quantitation of transcriptional activity from activated complexes (closed circles) and correction for (d)ATP-dependent decay (open circles) (see text for details of the correction). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution. Points represent the average of at least three independent experiments, and error bars represent SEM.

Techniques Used: Quantitation Assay, Activity Assay

Detection of activated pol II transcription complexes in vitro. (A) ATP-dependent activation of pol II preinitiation complexes (see text for details). (B) Requirement for dATP pretreatment to generate activated complexes. Transcription complexes were assembled on the pMBPT3 template in HeLa nuclear extract (HNE) followed by the addition of dATP (20 μM) and Sarkosyl (0.02%) at time 0. The dATP allows activation of preassembled complexes, whereas Sarkosyl prevents the assembly of any additional complexes and limits transcription to a single round. After a 2-min dATP pretreatment, [α-32 P]CTP, GTP, and ATPγS (200 μM) were added, and the incubations allowed to continue for an additional 10 min (lane 1). Control reactions received no dATP (lane 3) or received dATP at the same time as the other nucleotides (lane 2). (C) Quantitation of transcription activity from complexes that received no dATP (stippled bar), dATP at the same time as the other nucleotides (open bar), or a 2-min dATP pretreatment (closed bar). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution.
Figure Legend Snippet: Detection of activated pol II transcription complexes in vitro. (A) ATP-dependent activation of pol II preinitiation complexes (see text for details). (B) Requirement for dATP pretreatment to generate activated complexes. Transcription complexes were assembled on the pMBPT3 template in HeLa nuclear extract (HNE) followed by the addition of dATP (20 μM) and Sarkosyl (0.02%) at time 0. The dATP allows activation of preassembled complexes, whereas Sarkosyl prevents the assembly of any additional complexes and limits transcription to a single round. After a 2-min dATP pretreatment, [α-32 P]CTP, GTP, and ATPγS (200 μM) were added, and the incubations allowed to continue for an additional 10 min (lane 1). Control reactions received no dATP (lane 3) or received dATP at the same time as the other nucleotides (lane 2). (C) Quantitation of transcription activity from complexes that received no dATP (stippled bar), dATP at the same time as the other nucleotides (open bar), or a 2-min dATP pretreatment (closed bar). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution.

Techniques Used: In Vitro, Activation Assay, Quantitation Assay, Activity Assay

Stability of total transcription complexes. (A) Preinitiation complexes were assembled on pMBPT3 template in HeLa nuclear extract (HNE) and then activated at time 0 by the addition of dATP (20 μM). Sarkosyl (0.02%) was added along with the dATP. At time t , ATP, [α-32 P]CTP, and GTP were added and the reactions incubated an additional 10 min, thus allowing all functional transcription complexes (both activated and unactivated) to generate RNA products. (B) Quantitation of transcriptional activity from total complexes treated with dATP as described in (A) (closed circles) or from complexes that were not treated with dATP (closed squares). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution. Points represent the average of at least three independent experiments, and error bars represent SEM.
Figure Legend Snippet: Stability of total transcription complexes. (A) Preinitiation complexes were assembled on pMBPT3 template in HeLa nuclear extract (HNE) and then activated at time 0 by the addition of dATP (20 μM). Sarkosyl (0.02%) was added along with the dATP. At time t , ATP, [α-32 P]CTP, and GTP were added and the reactions incubated an additional 10 min, thus allowing all functional transcription complexes (both activated and unactivated) to generate RNA products. (B) Quantitation of transcriptional activity from total complexes treated with dATP as described in (A) (closed circles) or from complexes that were not treated with dATP (closed squares). Quantitation was made relative to a recovery control (RC) RNA added with the stop solution. Points represent the average of at least three independent experiments, and error bars represent SEM.

Techniques Used: Incubation, Functional Assay, Quantitation Assay, Activity Assay

12) Product Images from "Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences"

Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.00237

RecA⋅ATP-mediated recombination of a short heterologous (77-bp, ∼54% sequence divergence) substrate ( lds het ) in the presence of MutSL. (A) Scheme of the reaction between css hom and the heterologous lds het (black square) substrates. The 77-bp heterologous segment was restricted to an internal region toward the 3′ end. The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, (MutS, MutL or MutSL) and the lds het substrate, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS or MutL. C denotes the DNA substrates control. The percentage of jm intermediates and nc products are shown beneath the gel. Results shown as mean ± 5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP. The lds het substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration were then added and incubated (60 min, 37°C). (C) The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, the [γ 32 P]- lds hom (lanes 2–5) or [γ 32 P]- lds het lanes 7–10 substrate, in the presence or absence of increasing MutS concentrations, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS.
Figure Legend Snippet: RecA⋅ATP-mediated recombination of a short heterologous (77-bp, ∼54% sequence divergence) substrate ( lds het ) in the presence of MutSL. (A) Scheme of the reaction between css hom and the heterologous lds het (black square) substrates. The 77-bp heterologous segment was restricted to an internal region toward the 3′ end. The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, (MutS, MutL or MutSL) and the lds het substrate, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS or MutL. C denotes the DNA substrates control. The percentage of jm intermediates and nc products are shown beneath the gel. Results shown as mean ± 5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP. The lds het substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration were then added and incubated (60 min, 37°C). (C) The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A with 5 mM ATP, followed by RecA, the [γ 32 P]- lds hom (lanes 2–5) or [γ 32 P]- lds het lanes 7–10 substrate, in the presence or absence of increasing MutS concentrations, and incubated (60 min, 37°C). Band positions correspond to substrates as in Figure 2 . The minus (–) symbol denotes absence of MutS.

Techniques Used: Sequencing, Incubation, Concentration Assay

A short homeologous (77-bp segment, ∼16% sequence divergence) region on an otherwise homologous substrate ( lds mis ) delays RecA⋅ATP-mediated DNA strand exchange. The scheme shows the three-strand exchange reaction between css hom (+ strand) and the homeologous lds mis (filled circle) or lds hom ( open circle) substrates. The 77-bp homeologous/homologous segment was restricted to an internal region toward the 3′ end. (A) The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, followed by 3276-bp kpn I-linearized lds mis (lanes 1–7) or lds hom DNA (lanes 8–14) and RecA, and the reaction was incubated for various times (min) at 37°C. The reaction was separated by 0.8% agarose gel electrophoresis. Band positions correspond to substrates ( lds and css ), intermediates ( jm ) and product ( nc ). C denotes the DNA substrates control. The amount of recombination intermediates ( jm ) and products ( nc ) are expressed as a percentage of total substrate added. MutSL affects RecA⋅ATP rather than RecA⋅dATP-mediated DNA strand exchange. (B,C) The 3,277-nt homologous circular ssDNA ( css hom ) was pre-incubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP or dATP. Then, RecA and increasing MutS and the 3,276-bp lds mis (B) or lds hom (C) DNA (20 μM in nt) substrate were added. The reaction was incubated (60 min, 37°C) and separated by 0.8% agarose gel electrophoresis. The amount of recombination products is expressed as a percentage of total substrate added. Quantification of intermediate/products beneath the gel shown as mean ± SEM of ≥3 independent experiments.
Figure Legend Snippet: A short homeologous (77-bp segment, ∼16% sequence divergence) region on an otherwise homologous substrate ( lds mis ) delays RecA⋅ATP-mediated DNA strand exchange. The scheme shows the three-strand exchange reaction between css hom (+ strand) and the homeologous lds mis (filled circle) or lds hom ( open circle) substrates. The 77-bp homeologous/homologous segment was restricted to an internal region toward the 3′ end. (A) The 3276-nt css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, followed by 3276-bp kpn I-linearized lds mis (lanes 1–7) or lds hom DNA (lanes 8–14) and RecA, and the reaction was incubated for various times (min) at 37°C. The reaction was separated by 0.8% agarose gel electrophoresis. Band positions correspond to substrates ( lds and css ), intermediates ( jm ) and product ( nc ). C denotes the DNA substrates control. The amount of recombination intermediates ( jm ) and products ( nc ) are expressed as a percentage of total substrate added. MutSL affects RecA⋅ATP rather than RecA⋅dATP-mediated DNA strand exchange. (B,C) The 3,277-nt homologous circular ssDNA ( css hom ) was pre-incubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP or dATP. Then, RecA and increasing MutS and the 3,276-bp lds mis (B) or lds hom (C) DNA (20 μM in nt) substrate were added. The reaction was incubated (60 min, 37°C) and separated by 0.8% agarose gel electrophoresis. The amount of recombination products is expressed as a percentage of total substrate added. Quantification of intermediate/products beneath the gel shown as mean ± SEM of ≥3 independent experiments.

Techniques Used: Sequencing, Incubation, Agarose Gel Electrophoresis

RecA⋅ATP-mediated strand exchange with short internal heterology and at the 5′-end in the presence of MutSL. (A) Scheme of the reaction between css hom and the lds het–ins substrate with internal region toward the 3′ end and at the 5′-end (black and gray squares, respectively). The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, after which RecA, (MutS, MutL or MutSL) and the lds het–ins substrate were added and incubated (60 min, 37°C). The percentage of jm intermediates and nc products are shown beneath the gel. Lane 1, the css and lds substrates (termed C). Results shown as the mean ±5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP, followed by the 3353-bp lds het–ins substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration, and incubated (60 min, 37°C). The reaction was separated by gel electrophoresis and quantified. The left-hand side bar denotes the fraction of jm intermediates (gray bar) and the right-hand side scale denotes the fraction of unreactive lds het–ins substrate (black bars). The minus (–) symbol indicates lack of MutS or MutL.
Figure Legend Snippet: RecA⋅ATP-mediated strand exchange with short internal heterology and at the 5′-end in the presence of MutSL. (A) Scheme of the reaction between css hom and the lds het–ins substrate with internal region toward the 3′ end and at the 5′-end (black and gray squares, respectively). The css hom DNA (10 μM in nt) was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM ATP, after which RecA, (MutS, MutL or MutSL) and the lds het–ins substrate were added and incubated (60 min, 37°C). The percentage of jm intermediates and nc products are shown beneath the gel. Lane 1, the css and lds substrates (termed C). Results shown as the mean ±5% SEM of ≥3 independent experiments. (B) Experiments with dATP and increasing RecA concentrations. The 3276-nt css hom DNA was preincubated with SsbA and RecO (5 min, 37°C) in buffer A containing 5 mM dATP, followed by the 3353-bp lds het–ins substrate, increasing RecA concentrations and, where indicated, a fixed MutS concentration, and incubated (60 min, 37°C). The reaction was separated by gel electrophoresis and quantified. The left-hand side bar denotes the fraction of jm intermediates (gray bar) and the right-hand side scale denotes the fraction of unreactive lds het–ins substrate (black bars). The minus (–) symbol indicates lack of MutS or MutL.

Techniques Used: Incubation, Concentration Assay, Nucleic Acid Electrophoresis

13) Product Images from "Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli"

Article Title: Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli

Journal: eLife

doi: 10.7554/eLife.07141

Molecular basis for communication between substrate and effector binding sites in E. coli class Ia RNR. Structures are shown as sticks on the left with 2D representation of hydrogen-bonding interactions on the right for ( A ) CDP/dATP, ( B ) UDP/dATP, ( C ) ADP/dGTP, and ( D ) GDP/TTP. Atoms are colored as in Figure 7 . Hydrogen-bonding interactions are shown in black dashed lines with distances given in Å. In the 2D representation, each residue is colored a different color, residues 295–297 are shown as a black line, and hydrogen bonds are indicated with black dashed lines. DOI: http://dx.doi.org/10.7554/eLife.07141.014
Figure Legend Snippet: Molecular basis for communication between substrate and effector binding sites in E. coli class Ia RNR. Structures are shown as sticks on the left with 2D representation of hydrogen-bonding interactions on the right for ( A ) CDP/dATP, ( B ) UDP/dATP, ( C ) ADP/dGTP, and ( D ) GDP/TTP. Atoms are colored as in Figure 7 . Hydrogen-bonding interactions are shown in black dashed lines with distances given in Å. In the 2D representation, each residue is colored a different color, residues 295–297 are shown as a black line, and hydrogen bonds are indicated with black dashed lines. DOI: http://dx.doi.org/10.7554/eLife.07141.014

Techniques Used: Binding Assay, IA

Specific activity for wild-type and mutant forms of E. coli RNR in the presence of different substrate/effector pairs. Wild-type is shown in black, Gln294Ala in grey, and Arg298Ala in white. Activity was measured by a coupled assay that follows nicotinamide adenine dinucleotide phosphate (NAPDH ) consumption (see Materials and methods) for the following substrate and effector concentrations: 1 mM CDP and 3 mM ATP (far left), 1 mM CDP and 175 μM dATP (second to left), 1 mM ADP and 120 μM dGTP, 1 mM GDP and 250 μM TTP, and 1 mM CDP/UDP and 1 μM dATP (far right). Since dATP at high concentrations (175 μM) inhibits the enzyme, the sets of bars at the far left represent control experiments to show activity levels under active (CDP/ATP) and inactive (CDP/dATP) conditions. DOI: http://dx.doi.org/10.7554/eLife.07141.015
Figure Legend Snippet: Specific activity for wild-type and mutant forms of E. coli RNR in the presence of different substrate/effector pairs. Wild-type is shown in black, Gln294Ala in grey, and Arg298Ala in white. Activity was measured by a coupled assay that follows nicotinamide adenine dinucleotide phosphate (NAPDH ) consumption (see Materials and methods) for the following substrate and effector concentrations: 1 mM CDP and 3 mM ATP (far left), 1 mM CDP and 175 μM dATP (second to left), 1 mM ADP and 120 μM dGTP, 1 mM GDP and 250 μM TTP, and 1 mM CDP/UDP and 1 μM dATP (far right). Since dATP at high concentrations (175 μM) inhibits the enzyme, the sets of bars at the far left represent control experiments to show activity levels under active (CDP/ATP) and inactive (CDP/dATP) conditions. DOI: http://dx.doi.org/10.7554/eLife.07141.015

Techniques Used: Activity Assay, Mutagenesis

Composite omit electron density confirms that loop 2 is ordered in our E. coli class Ia RNR structures. Protein is shown as grey ribbons with substrate, loop 2, and specificity effector in sticks and labeled. Carbon is colored in yellow, oxygen in red, nitrogen in blue, phosphorus in orange. 2 F o − F c composite omit density is shown contoured at +1.0 σ (green mesh). ( A ) α 4 β 4 -CDP/dATP structure. ( B ) α 4 β 4 -UDP/dATP structure. ( C ) α 4 β 4 -ADP/dGTP structure. (D) α 4 β 4 -GDP/TTP structure. DOI: http://dx.doi.org/10.7554/eLife.07141.005
Figure Legend Snippet: Composite omit electron density confirms that loop 2 is ordered in our E. coli class Ia RNR structures. Protein is shown as grey ribbons with substrate, loop 2, and specificity effector in sticks and labeled. Carbon is colored in yellow, oxygen in red, nitrogen in blue, phosphorus in orange. 2 F o − F c composite omit density is shown contoured at +1.0 σ (green mesh). ( A ) α 4 β 4 -CDP/dATP structure. ( B ) α 4 β 4 -UDP/dATP structure. ( C ) α 4 β 4 -ADP/dGTP structure. (D) α 4 β 4 -GDP/TTP structure. DOI: http://dx.doi.org/10.7554/eLife.07141.005

Techniques Used: IA, Labeling

Conformations of E. coli class Ia RNR loop 2 in the presence and absence of substrate-effector pairs. Structures are shown in wall-eyed stereo as sticks for ( A ) CDP/dATP, ( B ) UDP/dATP, ( C ) ADP/dGTP, and ( D ) GDP/TTP. ( E ) α 2 with no substrates or effectors bound (PDB ID: 3R1R). Substrates are shown with carbons in yellow, effectors are shown with carbons in cyan, and loop 2 is shown with carbons in grey. Other atoms colored as in previous figures. Hydrogen-bonding interactions are shown in black dashed lines with distances given in Å. Only interactions between protein residues are shown. DOI: http://dx.doi.org/10.7554/eLife.07141.013
Figure Legend Snippet: Conformations of E. coli class Ia RNR loop 2 in the presence and absence of substrate-effector pairs. Structures are shown in wall-eyed stereo as sticks for ( A ) CDP/dATP, ( B ) UDP/dATP, ( C ) ADP/dGTP, and ( D ) GDP/TTP. ( E ) α 2 with no substrates or effectors bound (PDB ID: 3R1R). Substrates are shown with carbons in yellow, effectors are shown with carbons in cyan, and loop 2 is shown with carbons in grey. Other atoms colored as in previous figures. Hydrogen-bonding interactions are shown in black dashed lines with distances given in Å. Only interactions between protein residues are shown. DOI: http://dx.doi.org/10.7554/eLife.07141.013

Techniques Used: IA

Cα difference distance matrix plot reveals movements that occur concurrent with substrate binding for E. coli class Ia RNR. ( A ) Superposition of α from our CDP/dATP structure (N-terminus and one half of the active site barrel in purple and the other half barrel in blue) and α from a substrate-free E. coli RNR structure (PDB ID: 3R1R, ( Eriksson et al., 1997 )) (N-terminus and one half of active site barrel in pink and the other half barrel in red). The two chains were aligned by the active site finger loop, which is colored green. CDP is shown as sticks. ( B ) Distances in chain A of the CDP/dATP structure (this work) were subtracted from chain A of the substrate-free α 2 structure (PDB ID: 3R1R) for residues 4–737. Scale is shown on the top and is ±3.0 Å (positive values in blue indicate a shorter distance in the CDP/dATP structure and negative values in red indicate a longer distance). Regions that move in a concerted fashion are indicated with colored lines and residue ranges are listed to the left of the plot. ( C ) One α chain is shown in ribbons with residue ranges from ( B ) colored. Region 1 (blue), the N-terminal 225 residues, contracts towards region 2 (yellow). Region 3 (red) includes loop 2 residues and moves towards the active site (in region 4). A flexible loop, region 5 (green), undergoes a large motion towards regions 2 and 4 whereas region 4 undergoes little movement with respect to the rest of the structure. DOI: http://dx.doi.org/10.7554/eLife.07141.009
Figure Legend Snippet: Cα difference distance matrix plot reveals movements that occur concurrent with substrate binding for E. coli class Ia RNR. ( A ) Superposition of α from our CDP/dATP structure (N-terminus and one half of the active site barrel in purple and the other half barrel in blue) and α from a substrate-free E. coli RNR structure (PDB ID: 3R1R, ( Eriksson et al., 1997 )) (N-terminus and one half of active site barrel in pink and the other half barrel in red). The two chains were aligned by the active site finger loop, which is colored green. CDP is shown as sticks. ( B ) Distances in chain A of the CDP/dATP structure (this work) were subtracted from chain A of the substrate-free α 2 structure (PDB ID: 3R1R) for residues 4–737. Scale is shown on the top and is ±3.0 Å (positive values in blue indicate a shorter distance in the CDP/dATP structure and negative values in red indicate a longer distance). Regions that move in a concerted fashion are indicated with colored lines and residue ranges are listed to the left of the plot. ( C ) One α chain is shown in ribbons with residue ranges from ( B ) colored. Region 1 (blue), the N-terminal 225 residues, contracts towards region 2 (yellow). Region 3 (red) includes loop 2 residues and moves towards the active site (in region 4). A flexible loop, region 5 (green), undergoes a large motion towards regions 2 and 4 whereas region 4 undergoes little movement with respect to the rest of the structure. DOI: http://dx.doi.org/10.7554/eLife.07141.009

Techniques Used: Binding Assay, IA

Snapshots of higher and lower affinity substrate-bound states of RNR. ( A ) Cartoon of a high-affinity complex for CDP/UDP bound to RNR. ( B ) Packing of active site in E. coli class Ia RNR CDP/dATP structure (this work). ( C ) Packing of active site for UDP/dATP structure (this work). ( D ) Cartoon of a lower-affinity complex in which positioning of Gln into the active site holds loop 2 away such that Arg cannot reach the substrate diphosphate. ( E ) Packing of active site of ADP-bound S. cerevisiae RNR structure (PDB ID: 2CVX). With Gln288 (Gln294 in E. coli ) in the active site, Arg293 (Arg298 in E. coli ) does not reach the diphosphate of substrate. ( F ) Same structure as in ( E ), but Gln is not shown. Shape complementary suggests that a tighter complex could form than the one that is visualized in this crystal structure. ( G ) Cartoon of a high-affinity complex for ADP/GDP bound to RNR. ( H ) Packing of active site in GDP structure of class II RNR from T. maritima (PDB ID: IXJE) is similar to that of the E. coli class Ia RNR with ADP/dGTP bound (shown in panel I) and the E. coli GDP/TTP structure that is shown in Figure 4C . ( I ) Packing of active site in E. coli class Ia RNR with ADP/dGTP bound (this work). DOI: http://dx.doi.org/10.7554/eLife.07141.017
Figure Legend Snippet: Snapshots of higher and lower affinity substrate-bound states of RNR. ( A ) Cartoon of a high-affinity complex for CDP/UDP bound to RNR. ( B ) Packing of active site in E. coli class Ia RNR CDP/dATP structure (this work). ( C ) Packing of active site for UDP/dATP structure (this work). ( D ) Cartoon of a lower-affinity complex in which positioning of Gln into the active site holds loop 2 away such that Arg cannot reach the substrate diphosphate. ( E ) Packing of active site of ADP-bound S. cerevisiae RNR structure (PDB ID: 2CVX). With Gln288 (Gln294 in E. coli ) in the active site, Arg293 (Arg298 in E. coli ) does not reach the diphosphate of substrate. ( F ) Same structure as in ( E ), but Gln is not shown. Shape complementary suggests that a tighter complex could form than the one that is visualized in this crystal structure. ( G ) Cartoon of a high-affinity complex for ADP/GDP bound to RNR. ( H ) Packing of active site in GDP structure of class II RNR from T. maritima (PDB ID: IXJE) is similar to that of the E. coli class Ia RNR with ADP/dGTP bound (shown in panel I) and the E. coli GDP/TTP structure that is shown in Figure 4C . ( I ) Packing of active site in E. coli class Ia RNR with ADP/dGTP bound (this work). DOI: http://dx.doi.org/10.7554/eLife.07141.017

Techniques Used: IA

Interactions that anchor specificity effectors in E. coli class Ia RNR involve residues outside of loop 2. Interactions are shown for ( A ) CDP/dATP, ( B ) UDP/dATP, ( C ) ADP/dGTP, and ( D ) GDP/TTP. The dNTP effector carbons are colored cyan and the protein carbons are colored grey. Magnesium ions are colored grey and waters in red. The side chain of Leu234 is omitted for clarity. Hydrogen-bonding interactions are shown with black dashed lines. Figures are displayed in wall-eyed stereo. DOI: http://dx.doi.org/10.7554/eLife.07141.012
Figure Legend Snippet: Interactions that anchor specificity effectors in E. coli class Ia RNR involve residues outside of loop 2. Interactions are shown for ( A ) CDP/dATP, ( B ) UDP/dATP, ( C ) ADP/dGTP, and ( D ) GDP/TTP. The dNTP effector carbons are colored cyan and the protein carbons are colored grey. Magnesium ions are colored grey and waters in red. The side chain of Leu234 is omitted for clarity. Hydrogen-bonding interactions are shown with black dashed lines. Figures are displayed in wall-eyed stereo. DOI: http://dx.doi.org/10.7554/eLife.07141.012

Techniques Used: IA

14) Product Images from "Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1"

Article Title: Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1

Journal:

doi: 10.1073/pnas.1218528110

Structure of the hOAS1●dsRNA●dATP ternary complex. The complex is shown in two orientations related by 90° rotation. Protein is shown as ribbons; dsRNA (purple) and dATP (light blue) are shown as molecular surfaces. Two Mg 2+ ions
Figure Legend Snippet: Structure of the hOAS1●dsRNA●dATP ternary complex. The complex is shown in two orientations related by 90° rotation. Protein is shown as ribbons; dsRNA (purple) and dATP (light blue) are shown as molecular surfaces. Two Mg 2+ ions

Techniques Used:

Related Articles

Clone Assay:

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: To create the pMAD_lic vector we oligomerized the primers pMAD_lic (EcoRI) and pMAD_lic (BamHI) listed in Supplementary Table , and cloned the double DNA strand dimer into pMAD using EcoRI and BamHI restriction enzymes. .. The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C.

Amplification:

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C. .. To anneal the insert into the pMAD_lic vector, the mix of vector and insert was incubated for 5 min at 22°C and then, EDTA (6.25 mM) was added and a further incubation of 5 min at 22°C was applied.

Autoradiography:

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β. .. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer.

Construct:

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: A LIC-modified pMAD vector was constructed in order to enable efficient directional cloning without restriction enzyme digestion or ligation reactions ( ). .. The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C.

Electrophoresis:

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation
Article Snippet: Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma). .. Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma).

Primer Extension Assay:

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation
Article Snippet: Paragraph title: In vitro primer extension assay. ... Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma).

Incubation:

Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
Article Snippet: For the nicking reaction, 900 ng of lambda phage DNA (New England Biolabs) was digested with 30 units of Nt.BspQI nicking enzyme (New England Biolabs) for 2 h at 50 °C in the presence of 3 μL of 10× buffer 3.1 (New England Biolabs) and ultrapure water to a total volume of 30 μL. .. Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL. .. Following labeling, DNA was repaired for 30 min at 45 °C with 12 units of Taq DNA ligase (New England Biolabs) in the presence of 1.5 μL of 10× thermopol buffer, 1 mM NAD+ (New England Biolabs), and ultrapure water to a total reaction volume of 60 μL.

Article Title: Aberrant localization of apoptosis protease activating factor-1 in lipid raft sub-domains of diffuse large B cell lymphomas
Article Snippet: The S-100 pellet was dissolved in 1X RIPA lysis buffer with protease and phosphatase inhibitors and stored at -800 C or used immediately. .. For cell free caspase-3 activation, S-100 cytosol (120μg) from cells treated with MPO (5μM), MPO-Zn (200μM), LY30 (25μM), NaN3 (5μg/ml), etoposide (5μΜ) or daundorubicin (0.5μl/μl) were incubated in the presence or absence of 1mM dATP and 4μM cytochrome C (Sigma, St Louis, MO) for 30min at 370 C. Caspase-3 activity was then measured by using fluorogenic substrate DEVD-AFC as described previously [ ]. .. S-100 cytosol and pellet fractions or lipid raft fractions were denatured in loading buffer and resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membranes.

Article Title: Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing
Article Snippet: Subsequently, each sample underwent column cleaning and was eluted in 34 μl EB followed by A-base addition using an A-addition master mix and incubated at 37 °C for 30 minutes. .. The A-addition master mix consisted of 5 μl 10× NEBuffer 2 (New England Biolabs, Ipswich, MA, USA), 10 μl dATP (1 mM; Sigma Aldrich, MO, USA), and 3 μl Klenow [3′- > 5′ exo-] (Thermo Scientific).

Diffusion-based Assay:

Article Title: Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1
Article Snippet: The crystallization mixture contained 15 mg/mL hOAS1, 550 μM dsRNA, 2 mM dATP (Sigma), 10 mM Hepes (pH 7.4), 350 mM NaCl, and 5 mM MgCl2 . .. The crystallization mixture contained 15 mg/mL hOAS1, 550 μM dsRNA, 2 mM dATP (Sigma), 10 mM Hepes (pH 7.4), 350 mM NaCl, and 5 mM MgCl2 .

Activity Assay:

Article Title: Aberrant localization of apoptosis protease activating factor-1 in lipid raft sub-domains of diffuse large B cell lymphomas
Article Snippet: The S-100 pellet was dissolved in 1X RIPA lysis buffer with protease and phosphatase inhibitors and stored at -800 C or used immediately. .. For cell free caspase-3 activation, S-100 cytosol (120μg) from cells treated with MPO (5μM), MPO-Zn (200μM), LY30 (25μM), NaN3 (5μg/ml), etoposide (5μΜ) or daundorubicin (0.5μl/μl) were incubated in the presence or absence of 1mM dATP and 4μM cytochrome C (Sigma, St Louis, MO) for 30min at 370 C. Caspase-3 activity was then measured by using fluorogenic substrate DEVD-AFC as described previously [ ]. .. S-100 cytosol and pellet fractions or lipid raft fractions were denatured in loading buffer and resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membranes.

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: WT and R137Q genes were inserted into the pET28b vector and expressed in Escherichia coli strain BL21. .. The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β. .. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer.

Article Title: An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis
Article Snippet: A fresh aliquot of protein was removed from the freezer each day that experiments were performed, and each new aliquot was assayed with OXT-P and dAMP to ensure a consistent level of activity was observed between experiments performed on different days. .. Standard compounds, dATP (≥97% purity), dADP (≥95% purity), and dAMP (98–100% purity) were purchased from Sigma Aldrich.

BIA-KA:

Article Title: Modified nucleoside triphosphates exist in mammals †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc05472f
Article Snippet: The stable isotopic ATP and dATP (13 C10 ,15 N5 -ATP and 13 C10 ,15 N5 -dATP) were purchased from Sigma-Aldrich (Beijing, China). .. The stable isotopic ATP and dATP (13 C10 ,15 N5 -ATP and 13 C10 ,15 N5 -dATP) were purchased from Sigma-Aldrich (Beijing, China).

Crystallization Assay:

Article Title: Structural basis for cytosolic double-stranded RNA surveillance by human oligoadenylate synthetase 1
Article Snippet: dsRNA was reconstituted from cRNA strands purchased from Dharmacon. .. The crystallization mixture contained 15 mg/mL hOAS1, 550 μM dsRNA, 2 mM dATP (Sigma), 10 mM Hepes (pH 7.4), 350 mM NaCl, and 5 mM MgCl2 . .. The reservoir solution contained 100 mM Hepes (pH 7.4) and 20% (vol/vol) PEG 200.

High Performance Liquid Chromatography:

Article Title: ATP-Mediated Activation of RNA Polymerase II Transcription Complexes
Article Snippet: HPLC-purified ribonucleoside 5′-triphosphates were purchased from Sigma. .. Sarkosyl, tRNA, DTT, dATP, and diethylpyrocarbonate (DEPC) were also from Sigma.

Ligation:

Article Title: Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing
Article Snippet: The A-addition master mix consisted of 5 μl 10× NEBuffer 2 (New England Biolabs, Ipswich, MA, USA), 10 μl dATP (1 mM; Sigma Aldrich, MO, USA), and 3 μl Klenow [3′- > 5′ exo-] (Thermo Scientific). .. The A-addition master mix consisted of 5 μl 10× NEBuffer 2 (New England Biolabs, Ipswich, MA, USA), 10 μl dATP (1 mM; Sigma Aldrich, MO, USA), and 3 μl Klenow [3′- > 5′ exo-] (Thermo Scientific).

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: A LIC-modified pMAD vector was constructed in order to enable efficient directional cloning without restriction enzyme digestion or ligation reactions ( ). .. The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C.

other:

Article Title: The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities
Article Snippet: Bovine Cc (catalogue# C3131) and dATP (catalogue# D4788) were purchased from Sigma.

Sequencing:

Article Title: Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing
Article Snippet: Paragraph title: Library preparation and sequencing of DTCs and primary tumors with matched blood ... The A-addition master mix consisted of 5 μl 10× NEBuffer 2 (New England Biolabs, Ipswich, MA, USA), 10 μl dATP (1 mM; Sigma Aldrich, MO, USA), and 3 μl Klenow [3′- > 5′ exo-] (Thermo Scientific).

Labeling:

Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences
Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma. .. IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma.

Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
Article Snippet: Paragraph title: Measuring the Labeling Efficiency of 5-hmC ... Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL.

Purification:

Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences
Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma. .. [γ32 P]-ATP was used to labeled the ends of the Xba I-linearized 3,276-bp pGEM3hom (ldshom ), pGEM3het (ldshet ) or 3,353-bp pGEM3het–ins (ldshet–ins ).

Article Title: Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair
Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes, DNA ligase, etc. were supplied by Roche; and polyethyleneimine, DTT, ATP, dATP, ATPγS and AMP–PNP were from Sigma. .. IPTG was from Calbiochem; DNA restriction enzymes, DNA ligase, etc. were supplied by Roche; and polyethyleneimine, DTT, ATP, dATP, ATPγS and AMP–PNP were from Sigma.

Article Title: Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction
Article Snippet: IPTG was from Calbiochem (Darmstadt, Germany), and polyethyleneimine, DTT, ATP, dATP and ATPγS were from Sigma (Seelze, Germany). .. IPTG was from Calbiochem (Darmstadt, Germany), and polyethyleneimine, DTT, ATP, dATP and ATPγS were from Sigma (Seelze, Germany).

Article Title: Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination
Article Snippet: Isopropyl β- d -thiogalactopyranoside was from Calbiochem, and polyethyleneimine, DTT, ATP, and dATP were from Sigma. .. Isopropyl β- d -thiogalactopyranoside was from Calbiochem, and polyethyleneimine, DTT, ATP, and dATP were from Sigma.

Article Title: Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing
Article Snippet: The sample was column cleaned using a Qiaquick PCR purification Kit (Qiagen, Valencia, CA, USA) and eluted in 36 μl elution buffer (EB), followed by fragment end repair using the End-It™ DNA End-Repair Kit (Epicenter, Madison, WI, USA). .. The A-addition master mix consisted of 5 μl 10× NEBuffer 2 (New England Biolabs, Ipswich, MA, USA), 10 μl dATP (1 mM; Sigma Aldrich, MO, USA), and 3 μl Klenow [3′- > 5′ exo-] (Thermo Scientific).

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: PCR products with complementary overhangs were created using Phusion enzyme (Thermo Scientific) by building appropriate 5′ extensions into the primers. .. The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C. .. To anneal the insert into the pMAD_lic vector, the mix of vector and insert was incubated for 5 min at 22°C and then, EDTA (6.25 mM) was added and a further incubation of 5 min at 22°C was applied.

Protein Purification:

Article Title: Chromosomal transformation in Bacillus subtilis is a non-polar recombination reaction
Article Snippet: Paragraph title: Enzymes, reagents, DNA and protein purification ... IPTG was from Calbiochem (Darmstadt, Germany), and polyethyleneimine, DTT, ATP, dATP and ATPγS were from Sigma (Seelze, Germany).

Article Title: Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination
Article Snippet: Paragraph title: Enzymes, Reagents, DNA, and Protein Purification ... Isopropyl β- d -thiogalactopyranoside was from Calbiochem, and polyethyleneimine, DTT, ATP, and dATP were from Sigma.

Polymerase Chain Reaction:

Article Title: Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing
Article Snippet: The sample was column cleaned using a Qiaquick PCR purification Kit (Qiagen, Valencia, CA, USA) and eluted in 36 μl elution buffer (EB), followed by fragment end repair using the End-It™ DNA End-Repair Kit (Epicenter, Madison, WI, USA). .. The A-addition master mix consisted of 5 μl 10× NEBuffer 2 (New England Biolabs, Ipswich, MA, USA), 10 μl dATP (1 mM; Sigma Aldrich, MO, USA), and 3 μl Klenow [3′- > 5′ exo-] (Thermo Scientific).

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: PCR products with complementary overhangs were created using Phusion enzyme (Thermo Scientific) by building appropriate 5′ extensions into the primers. .. The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C. .. To anneal the insert into the pMAD_lic vector, the mix of vector and insert was incubated for 5 min at 22°C and then, EDTA (6.25 mM) was added and a further incubation of 5 min at 22°C was applied.

Polyacrylamide Gel Electrophoresis:

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β. .. Reactions were carried out at 37°C for 30 min. A portion of reaction product was taken out and incubated with avidin–Sepharose 4B beads, washed and quantified on liquid scintillation analyzer.

SDS Page:

Article Title: An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis
Article Snippet: Standard compounds, dATP (≥97% purity), dADP (≥95% purity), and dAMP (98–100% purity) were purchased from Sigma Aldrich. .. Standard compounds, dATP (≥97% purity), dADP (≥95% purity), and dAMP (98–100% purity) were purchased from Sigma Aldrich.

Plasmid Preparation:

Article Title: Roles of Bacillus subtilis DprA and SsbA in RecA-mediated Genetic Recombination
Article Snippet: Isopropyl β- d -thiogalactopyranoside was from Calbiochem, and polyethyleneimine, DTT, ATP, and dATP were from Sigma. .. Isopropyl β- d -thiogalactopyranoside was from Calbiochem, and polyethyleneimine, DTT, ATP, and dATP were from Sigma.

Article Title: A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus
Article Snippet: To produce specific non-complementary overhangs in the pMAD_lic vector, the ApaI linearized plasmid was treated with T4 DNA Polymerase (Novagen) in the presence of dTTP (Novagen) for 30 min at 22°C. .. The PCR products were purified and then treated with T4 DNA Polymerase in the presence of dATP (Novagen) at 22°C.

Enzyme-linked Immunosorbent Assay:

Article Title: Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells
Article Snippet: The amount of IL-10 in the supernatant was measured by ELISA. .. To assess which purinergic receptor was involved in the microglial IL-10 production, microglia cells were treated with P2 receptor agonists: ATP, ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP-γ-S), adenosine 5'-O-(2-thiodiphosphate) (ADP-β-S), 2-methylthio-ATP (2-meSATP), 2-methylthio-ADP (2-meSADP), α,β-methylene ATP (α,β-meATP), 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), UTP, UDP, or dATP (Sigma, St. Louis, MO); P2 receptor antagonists: trinitrophenyl-substituted ATP (TNP-ATP), adenosine 5'-triphosphate 2',3'-acylic dialcohol (oxidized ATP; oATP), 2'-deoxy-N6 -methyladenosine-3',5'-bisphosphate (MRS2179), 2-methylthioadenisine 5'-monophosphate (2-meSAMP), 5'-O-thiomnophosphate (5'-AMPS) (Sigma, St. Louis, MO); Ca2+ chelators: EGTA (Sigma, St. Louis, MO), bisaminophenoxyethane tetraacetic acid-acetoxymethyl ester (BAPTA-AM; Calbiochem, San Diego, CA); IP3 inhibitor, Xes-C (Sigma, St. Louis, MO); adenylate cyclase inhibitor, SQ22536 (Sigma, St. Louis, MO); PKA inhibitor, H-89 (Sigma, St. Louis, MO); Gi protein inhibitor, pertussis toxin (PTX; Sigma, St. Louis, MO).

Positron Emission Tomography:

Article Title: Bacillus subtilis RarA modulates replication restart
Article Snippet: Disuccinimidyl suberate (DSS), polyethyleneimine, DTT, ATP, dATP and ATPγS were from Sigma. .. Disuccinimidyl suberate (DSS), polyethyleneimine, DTT, ATP, dATP and ATPγS were from Sigma.

In Vitro:

Article Title: Human DNA polymerase ? polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity
Article Snippet: Paragraph title: In vitro polymerase activity assay ... The polymerase activity assay utilized 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 2 mM DTT, 20 mM NaCl, 10% glycerol, 0.1 µM biotin-labeled DNA substrate Pol-GAP (see and A for detail), 50 µM each dATP, dGTP and dTTP (Sigma), 8 µM 2 µCi [α-32 P]-dCTP and 0–10 ng WT or R137Q Pol β.

Article Title: The E295K DNA Polymerase Beta Gastric Cancer-Associated Variant Interferes with Base Excision Repair and Induces Cellular Transformation
Article Snippet: Paragraph title: In vitro primer extension assay. ... Primer extension reactions were conducted in a solution containing 50 mM Tris-Cl buffer (pH 8.0), 10 mM MgCl2 , 2 mM dithiothreitol (DTT), 20 mM NaCl, 10% glycerol, and 50 μM each of dATP, dCTP, dGTP, and dTTP (Sigma).

Activation Assay:

Article Title: Aberrant localization of apoptosis protease activating factor-1 in lipid raft sub-domains of diffuse large B cell lymphomas
Article Snippet: The S-100 pellet was dissolved in 1X RIPA lysis buffer with protease and phosphatase inhibitors and stored at -800 C or used immediately. .. For cell free caspase-3 activation, S-100 cytosol (120μg) from cells treated with MPO (5μM), MPO-Zn (200μM), LY30 (25μM), NaN3 (5μg/ml), etoposide (5μΜ) or daundorubicin (0.5μl/μl) were incubated in the presence or absence of 1mM dATP and 4μM cytochrome C (Sigma, St Louis, MO) for 30min at 370 C. Caspase-3 activity was then measured by using fluorogenic substrate DEVD-AFC as described previously [ ]. .. S-100 cytosol and pellet fractions or lipid raft fractions were denatured in loading buffer and resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membranes.

Concentration Assay:

Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays
Article Snippet: Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL. .. Next, nicked DNA was incubated for 1 h at 72 °C with 15 units of Taq DNA polymerase (New England Biolabs), supplemented with 600 nM of dATP, dGTP, dCTP (Sigma) and atto-532-dUTP (Jena Bioscience), or dATP, dGTP, dTTP (Sigma) and 5hmdCTP (Zymo Research), in the presence of 4.5 μL of 10× thermopol buffer (New England Biolabs) and ultrapure water to a total reaction volume of 45 μL.

DNA Purification:

Article Title: Bacillus subtilis MutS Modulates RecA-Mediated DNA Strand Exchange Between Divergent DNA Sequences
Article Snippet: Paragraph title: Enzymes, Reagents, Protein, and DNA Purification ... IPTG was from Calbiochem; DNA restriction enzymes and DNA ligase were from Roche, and polyethyleneimine, DTT, ATP, dATP were from Sigma.

Article Title: Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair
Article Snippet: Paragraph title: Enzymes, reagents, protein and DNA purification ... IPTG was from Calbiochem; DNA restriction enzymes, DNA ligase, etc. were supplied by Roche; and polyethyleneimine, DTT, ATP, dATP, ATPγS and AMP–PNP were from Sigma.

Standard Deviation:

Article Title: Contractility and kinetics of human fetal and human adult skeletal muscle
Article Snippet: Powdered ATP, dATP, and ADP (Sigma-Aldrich) were dissolved to 100 m m concentrations in H2 O (Barnstead Nanopure; Thermo Scientific) and the pH was adjusted prior to use such that pH components were accounted for in ensuring the ionic strength of the final solutions was maintained at 0.17. .. Powdered ATP, dATP, and ADP (Sigma-Aldrich) were dissolved to 100 m m concentrations in H2 O (Barnstead Nanopure; Thermo Scientific) and the pH was adjusted prior to use such that pH components were accounted for in ensuring the ionic strength of the final solutions was maintained at 0.17.

Lysis:

Article Title: Aberrant localization of apoptosis protease activating factor-1 in lipid raft sub-domains of diffuse large B cell lymphomas
Article Snippet: The S-100 pellet was dissolved in 1X RIPA lysis buffer with protease and phosphatase inhibitors and stored at -800 C or used immediately. .. For cell free caspase-3 activation, S-100 cytosol (120μg) from cells treated with MPO (5μM), MPO-Zn (200μM), LY30 (25μM), NaN3 (5μg/ml), etoposide (5μΜ) or daundorubicin (0.5μl/μl) were incubated in the presence or absence of 1mM dATP and 4μM cytochrome C (Sigma, St Louis, MO) for 30min at 370 C. Caspase-3 activity was then measured by using fluorogenic substrate DEVD-AFC as described previously [ ].

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