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  • 85
    Jena Bioscience datp
    Synthesis of <t>7-deaza-7-modified</t> <t>dATP.</t>
    Datp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 85/100, based on 232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs datp
    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with <t>dATP</t> and fluorescence-labeled dUTP (such as <t>TMR-dUTP;</t> marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).
    Datp, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2795 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher datp
    Model of the intrinsic pathway of apoptosis during brain maturation. In the intrinsic pathway of apoptosis, <t>cyt</t> c is released to the cytosol, where in the presence of <t>dATP</t> it assembles with Apaf-1 and procaspase-9 (pC9). Caspase 9 (C9) activates procaspase-3
    Datp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 7636 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer datp
    Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c <t>(hcytc-</t> His6 ), DC3 His6 , and DC4 His6 . Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3 His6 , DC4 His6 , and hcytc- His6 . White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3 His6 , DC4 His6 , or hcytc His6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM <t>dATP</t> and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.
    Datp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare α 32 p datp
    RTA binds to ISRE. A. The purified RTA protein binds to K14 ISRE. The probe was labeled with [α- 32 <t>P]dATP.</t> K14 ISRE, mK14 ISRE, ISG15 ISRE, Tap-2 ISRE, and mTap2-ISRE were used as cold competitors. Cold competitors were added at a 50-fold molar excess over hot probe. Various amounts of Ni 2+ -NTA agarose beads (1.5, 5, and 15 μl) were used to remove the histidine-tagged RTA proteins in an EMSA. Fifteen microliters of GST beads was used as a control. Rabbit polyclonal anti-RTA or K15 serum was also used. ns, nonspecific. B. RTA binds to known ISREs. The probe was ISRE-1 from the vIL-6 promoter. The cold competitors, Ni 2+ -NTA agarose beads, GST beads, and antisera used are shown. Specific protein-DNA complexes are shown.
    α 32 P Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher biotin 14 datp
    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, <t>biotin-14-dCTP,</t> and <t>biotin-14-dATP,</t> are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.
    Biotin 14 Datp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore datp
    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 <t>ATP</t> or <t>dATP.</t> 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.
    Datp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1397 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    datp  (TaKaRa)
    98
    TaKaRa datp
    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 <t>ATP</t> or <t>dATP.</t> 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.
    Datp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 98/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare α 35 s datp
    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 <t>ATP</t> or <t>dATP.</t> 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.
    α 35 S Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare p datp
    Summary of the genetic organization, expression and assembly of the fragmented class Ia RNR from phage Aeh1. The map of the Aeh1 RNR operon (gDNA) shows an early phage promoter (right facing arrow) that drives expression of the operon ( 32 ). The <t>NrdA-a,</t> NrdA-b and NrdB polypeptides are independently translated from this message (mRNA) ( 8 ), while expression of MobE is inhibited by an RNA secondary structure ( 33 ). Our current data indicate that the NrdA-a and NrdA-b polypeptides self assemble to form the α a /α b heterodimer (active site residues depicted as spheres), while dimerization to form the (α a /α b ) 2 large subunit is stimulated by <t>dATP.</t> The ΔT mutants are defective in dimerization of the α a /α b heterodimer. Holoenzyme formation is stimulated by dATP and includes the small subunit dimer, β 2 . The structures of the individual polypeptides were modified from the E. coli NrdA (PBD file 4R1R) and NrdB (PDB file 1AV8) structures. In the holoenzyme model [based on Ref. ( 34 )], the (α a /α b ) 2 large subunit is rotated 90° vertically relative to the free large subunit and the β 2 subunit is rotated 90° horizontally relative to the free dimer.
    P Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 506 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare s datp
    Reproducibility of the method of double radioactive labelling of probes on a microarray. ( A ) The results of two independent hybridisation experiments, each using 100 ng [ 35 <t>S]dATP-labelled</t> probe and 100 ng [ 3 <t>H]dCTP-labelled</t> probe from two different tissue samples (total brain of adult rat versus cortex of 12-day-old rat) were compared. For each of the experiments, ratios of the 3 H signal intensity to the 35 S signal intensity were calculated for each element of the microarray and normalised with respect to an external standard (luciferase cDNA sequence). The values from one experiment were plotted against those of the other. ( B ) Comparison of two independent hybridisations, each using 100 ng [ 35 S]dATP-labelled probe and 100 ng [ 3 H]dCTP-labelled probe from two different tissue samples. For one experiment, 10 7 molecules of transcribed luciferase RNA were added to 100 ng mRNA used for probe synthesis (experiment 1) and 10 8 molecules of luciferase RNA were added for the other experiment (experiment 2). The 3 H signal intensities (number of counts in 24 h) for each element of the microarray of one experiment were plotted against those of the other. The values of signal intensity are statistically reliable from 100 counts for 24 h. The clone shown by an arrow corresponds to the luciferase control. The ratio of the 3 H signal intensity of the luciferase clone in experiment 2 to that in experiment 1 is ∼10, which is as expected.
    S Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours α 32 p datp
    Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 <t>P]dATP.</t> The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.
    α 32 P Datp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega datp
    Potential routes for cellular production and metabolism of <t>N6-methyl-dATP</t> and N6-methyl-ATP. N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by <t>MTH1</t> to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.
    Datp, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 2266 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours s datp
    Potential routes for cellular production and metabolism of <t>N6-methyl-dATP</t> and N6-methyl-ATP. N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by <t>MTH1</t> to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.
    S Datp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Valiant α 32 p datp
    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 <t>P]dATP.</t> DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .
    α 32 P Datp, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega set of datp dctp dgtp dttp
    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 <t>P]dATP.</t> DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .
    Set Of Datp Dctp Dgtp Dttp, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    NEN Life Science α 32 p datp
    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 <t>P]dATP-labeled</t> DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.
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    Image Search Results


    Synthesis of 7-deaza-7-modified dATP.

    Journal: Nucleic Acids Research

    Article Title: Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

    doi: 10.1093/nar/gkp845

    Figure Lengend Snippet: Synthesis of 7-deaza-7-modified dATP.

    Article Snippet: Kinetics of cleavage of unmodified and modifided DNA by KpnI and SacI The reaction mixture (70 μl) contained Vent(exo-) DNA polymerase (New England Biolabs, 0.1 U), natural dNTPs (dGTP, dCTP and TTP, Fermentas, 0.2 mM), modified surrogates of dATP (derivatives of 7-deaza-dATP (7-deaza dATP, Jena Bioscience), 0.2 mM, 8-modified dATP 1 mM), primer (Sigma-Aldrich oligoes, sequence see and , 0.15 μM), template (Sigma-Aldrich oligoes, sequence see and , 0.225 μM) in 1 × ThermoPol reaction buffer.

    Techniques: Modification

    tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).

    Journal: bioRxiv

    Article Title: OTTER, a new method quantifying absolute amounts of tRNAs

    doi: 10.1101/2020.05.18.101501

    Figure Lengend Snippet: tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).

    Article Snippet: After adding final concentration of 10 μM TMR-dUTP (Roche Diagnostics, Basel, Switzerland), 250 μM dATP, 10 mM MgCl2 , and 2.0 units of Klenow Fragment (3’-5’ exo− )(New England Biolabs, Ipswich, Massachusetts, USA), total 10 μl of the mixture was incubate at 37°C for 90 min.

    Techniques: Fluorescence, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay, Northern Blot

    Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c /ATP. ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by dATP or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited

    Journal: Cell Death & Disease

    Article Title: Cell death regulation during influenza A virus infection by matrix (M1) protein: a model of viral control over the cellular survival pathway

    doi: 10.1038/cddis.2011.75

    Figure Lengend Snippet: Amino acid 128–165 is responsible for direct binding with Hsp70 to prevent Apaf-1 binding and to aid in caspase-9 processing induced by cytochrome c /ATP. ( a ) His-tagged M1or M1Δ128–165 (10 μ g) was immobilized on Ni +2 overnight in HBS at 4 °C, and then washed and incubated with recombinant Hsp70 (10 μ g) for 4 h at 4 °C. Associated Hsp70 was detected by immunoblotting. Wild-type Hsp70 associated with native M1 but not with M1Δ128–165. ( b ) Jurkat cell extracts were Hsp70 immunodepleted, before recombinant Hsp70 and M1 or M1Δ128–165 proteins were added to it incubated and Apaf-1 was immunoprecipitated. Hsp70 did not associate with Apaf-1 when native M1 was present, but did associate in the absence of M1 or in the presence of M1Δ128–165 protein. ( c ) Co-addition of recombinant M1 enhances processing of procaspase-9 induced by dATP or ATP and cytochrome c in Jurkat cell extracts in the presence of wild-type Hsp70, whereas in the presence of M1Δ128–165 protein, caspase-9 activation is inhibited

    Article Snippet: ATP (NEB, P0756S) or dATP (NEB, N0440S) was used at 1 mM/ml.

    Techniques: Binding Assay, Incubation, Recombinant, Immunoprecipitation, Activation Assay

    Model of the intrinsic pathway of apoptosis during brain maturation. In the intrinsic pathway of apoptosis, cyt c is released to the cytosol, where in the presence of dATP it assembles with Apaf-1 and procaspase-9 (pC9). Caspase 9 (C9) activates procaspase-3

    Journal:

    Article Title: Differential Regulation of Smac/DIABLO and Hsp-70 during Brain Maturation

    doi: 10.1007/s12017-007-8007-9

    Figure Lengend Snippet: Model of the intrinsic pathway of apoptosis during brain maturation. In the intrinsic pathway of apoptosis, cyt c is released to the cytosol, where in the presence of dATP it assembles with Apaf-1 and procaspase-9 (pC9). Caspase 9 (C9) activates procaspase-3

    Article Snippet: Briefly, 50 µg of cell-free extracts of mouse brain at different stages of maturation were activated with 10 µM horse heart cyt c (Sigma, #C7752) and 1 mM dATP (Invitrogen, #10216-018) at 37°C for 60 min. Control samples in the absence of cyt c and dATP were run under the same conditions.

    Techniques:

    Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc- His6 ), DC3 His6 , and DC4 His6 . Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3 His6 , DC4 His6 , and hcytc- His6 . White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3 His6 , DC4 His6 , or hcytc His6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells

    doi: 10.1083/jcb.200408054

    Figure Lengend Snippet: Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc- His6 ), DC3 His6 , and DC4 His6 . Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3 His6 , DC4 His6 , and hcytc- His6 . White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3 His6 , DC4 His6 , or hcytc His6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.

    Article Snippet: Where indicated, S100 lysates were incubated with 10 μM of purified DC3His6 , DC4His6 , or human cytochrome c (hcytc ), and 1 mM dATP, in caspase assay buffer ( ) at RT and activity was measured over time using a fluorometric plate reader (PerkinElmer) (excitation 385 nm, emission 460 nm).

    Techniques: Recombinant, Activity Assay, Expressing, Purification, Incubation

    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

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

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

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

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

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

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

    Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

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

    Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

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

    A) Sequencing electropherogram of m.1555A > G mutation. B) Heteroplasmy analysis of m.1555A > G patient and mother was conducted by 35 S-dATP labeled RFLP using Alw26I . The fragment of m.1555A > G mtDNA remains uncut by Alw26I and produces a 319 bp band. The smaller 189 bp digested band is the wild type mtDNA part. 1. Patient 2. Mother.

    Journal: Audiology Research

    Article Title: Mitochondrial hearing loss mutations among Finnish preterm and term-born infants

    doi: 10.4081/audiores.2017.189

    Figure Lengend Snippet: A) Sequencing electropherogram of m.1555A > G mutation. B) Heteroplasmy analysis of m.1555A > G patient and mother was conducted by 35 S-dATP labeled RFLP using Alw26I . The fragment of m.1555A > G mtDNA remains uncut by Alw26I and produces a 319 bp band. The smaller 189 bp digested band is the wild type mtDNA part. 1. Patient 2. Mother.

    Article Snippet: Mutation heteroplasmy quantification was determined using 35 S-dATP (Perkin-Elmer, Wellesley, MA, USA) labeled RFLP protocol.

    Techniques: Sequencing, Mutagenesis, Labeling

    Electrophoretic mobility shift assays using DNA probes with the -309 SNP in PTPRCAP . A nuclear extract (8 µ g) from MKN28 cells was incubated with a 29-bp [γ- 32 P]dATP-labeled DNA probe with either major (G) or minor (T) allele at the SNP-corresponding

    Journal:

    Article Title: A Regulatory Polymorphism at Position -309 in PTPRCAP Is Associated with Susceptibility to Diffuse-type Gastric Cancer and Gene Expression 1

    doi:

    Figure Lengend Snippet: Electrophoretic mobility shift assays using DNA probes with the -309 SNP in PTPRCAP . A nuclear extract (8 µ g) from MKN28 cells was incubated with a 29-bp [γ- 32 P]dATP-labeled DNA probe with either major (G) or minor (T) allele at the SNP-corresponding

    Article Snippet: Double-stranded probes were radioactively labeled with [γ-32 P]dATP (Perkin Elmer, Wellesley, MA) at the 5′ ends using the T4 polynucleotide kinase (Takara Bio, Shiga, Japan), and unincorporated [γ-32 P]dATP was removed using the PROBER probe DNA purifying system (iNtRON Biotechnology, Seongnam, Korea).

    Techniques: Electrophoretic Mobility Shift Assay, Incubation, Labeling

    Biophysical measurement of interaction between PAR-binding protein domain WWE and PAR of defined chain length. . (b) Filter binding assay of RNF146 WWE domain binding to 10- and 20-mer PAR, which were radiolabeled using ELTA and 32 P-dATP. (c) MST analysis of RNF146 WWE domain binding to 20-mer PAR, which was labeled using ELTA and Cy5-dATP.

    Journal: Molecular cell

    Article Title: ELTA: Enzymatic Labeling of Terminal ADP-ribose

    doi: 10.1016/j.molcel.2018.12.022

    Figure Lengend Snippet: Biophysical measurement of interaction between PAR-binding protein domain WWE and PAR of defined chain length. . (b) Filter binding assay of RNF146 WWE domain binding to 10- and 20-mer PAR, which were radiolabeled using ELTA and 32 P-dATP. (c) MST analysis of RNF146 WWE domain binding to 20-mer PAR, which was labeled using ELTA and Cy5-dATP.

    Article Snippet: For radiolabeling, ADP-ribose, NAD+ , iso -ADP-ribose (10 μM each), or PAR (25 μM each) were reacted with 5 μCi α−32 P-dATP (Perkin-Elmer), 50 μg/mL low molecular weight (LMW) poly(I:C) (Invivogen).

    Techniques: Binding Assay, Filter-binding Assay, Labeling

    Detection of ADP-ribose length from individual proteins and cells using ELTA. (a) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose monomer (lane 1) and PAR of mixed length (lane 2), as well as ADP-ribose monomers and polymers isolated from PARP1 automodification reactions with 1 mM NAD + for 0 (lane 3), 10 (lane 4), or 30 min (lane 5) that were labeled by OAS1 and 32 P-dATP. As a comparison, the ADP-ribose isolated from PARP1 automodification reaction in the same time frame with 1 mM NAD + with a trace of 32 P-NAD + were loaded in lanes 6–8. We note that 50-fold less of the reaction were loaded in lanes 3–5 compared with lanes 6–8. (b) 15% urea-PAGE analyses of ELTA labeling reaction of ADP-ribose monomers and polymers isolated from automodification of PARP1 along with either BSA and HPF1. The first lane contained ELTA-labeling of an equal mole of 5-, 10- and 20-mer PAR. The reaction in the PARP1+BSA lane was diluted 15 times in water prior to ELTA labeling. (c) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose isolated from in vitro modified ha PARP (lane 1), from untreated HaCaT cells (lane 2), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min (lane 3), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min, but pre-treated the cells with 20 μM PARP inhibitor Olaparib for 2 h (lane 4), or pre-treated with 1 μM PARG inhibitor PDD00017273 for 2 h (lane 5). Corresponding lysates of cells from lanes 2–5 were probed with β-actin.

    Journal: Molecular cell

    Article Title: ELTA: Enzymatic Labeling of Terminal ADP-ribose

    doi: 10.1016/j.molcel.2018.12.022

    Figure Lengend Snippet: Detection of ADP-ribose length from individual proteins and cells using ELTA. (a) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose monomer (lane 1) and PAR of mixed length (lane 2), as well as ADP-ribose monomers and polymers isolated from PARP1 automodification reactions with 1 mM NAD + for 0 (lane 3), 10 (lane 4), or 30 min (lane 5) that were labeled by OAS1 and 32 P-dATP. As a comparison, the ADP-ribose isolated from PARP1 automodification reaction in the same time frame with 1 mM NAD + with a trace of 32 P-NAD + were loaded in lanes 6–8. We note that 50-fold less of the reaction were loaded in lanes 3–5 compared with lanes 6–8. (b) 15% urea-PAGE analyses of ELTA labeling reaction of ADP-ribose monomers and polymers isolated from automodification of PARP1 along with either BSA and HPF1. The first lane contained ELTA-labeling of an equal mole of 5-, 10- and 20-mer PAR. The reaction in the PARP1+BSA lane was diluted 15 times in water prior to ELTA labeling. (c) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose isolated from in vitro modified ha PARP (lane 1), from untreated HaCaT cells (lane 2), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min (lane 3), from HaCaT cells treated with 1 mM H 2 O 2 for 10 min, but pre-treated the cells with 20 μM PARP inhibitor Olaparib for 2 h (lane 4), or pre-treated with 1 μM PARG inhibitor PDD00017273 for 2 h (lane 5). Corresponding lysates of cells from lanes 2–5 were probed with β-actin.

    Article Snippet: For radiolabeling, ADP-ribose, NAD+ , iso -ADP-ribose (10 μM each), or PAR (25 μM each) were reacted with 5 μCi α−32 P-dATP (Perkin-Elmer), 50 μg/mL low molecular weight (LMW) poly(I:C) (Invivogen).

    Techniques: Polyacrylamide Gel Electrophoresis, Labeling, Isolation, In Vitro, Modification

    RTA binds to ISRE. A. The purified RTA protein binds to K14 ISRE. The probe was labeled with [α- 32 P]dATP. K14 ISRE, mK14 ISRE, ISG15 ISRE, Tap-2 ISRE, and mTap2-ISRE were used as cold competitors. Cold competitors were added at a 50-fold molar excess over hot probe. Various amounts of Ni 2+ -NTA agarose beads (1.5, 5, and 15 μl) were used to remove the histidine-tagged RTA proteins in an EMSA. Fifteen microliters of GST beads was used as a control. Rabbit polyclonal anti-RTA or K15 serum was also used. ns, nonspecific. B. RTA binds to known ISREs. The probe was ISRE-1 from the vIL-6 promoter. The cold competitors, Ni 2+ -NTA agarose beads, GST beads, and antisera used are shown. Specific protein-DNA complexes are shown.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Replication and Transcription Activator Regulates Viral and Cellular Genes via Interferon-Stimulated Response Elements

    doi: 10.1128/JVI.79.9.5640-5652.2005

    Figure Lengend Snippet: RTA binds to ISRE. A. The purified RTA protein binds to K14 ISRE. The probe was labeled with [α- 32 P]dATP. K14 ISRE, mK14 ISRE, ISG15 ISRE, Tap-2 ISRE, and mTap2-ISRE were used as cold competitors. Cold competitors were added at a 50-fold molar excess over hot probe. Various amounts of Ni 2+ -NTA agarose beads (1.5, 5, and 15 μl) were used to remove the histidine-tagged RTA proteins in an EMSA. Fifteen microliters of GST beads was used as a control. Rabbit polyclonal anti-RTA or K15 serum was also used. ns, nonspecific. B. RTA binds to known ISREs. The probe was ISRE-1 from the vIL-6 promoter. The cold competitors, Ni 2+ -NTA agarose beads, GST beads, and antisera used are shown. Specific protein-DNA complexes are shown.

    Article Snippet: The probes were obtained by first annealing complementary oligonucleotides and then labeling them with [α-32 P]dATP (Amersham) using DNA polymerase Klenow fragment (Fermentas).

    Techniques: Purification, Labeling

    Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    doi: 10.3389/fbioe.2019.00401

    Figure Lengend Snippet: Schematic illustration of the principle of antarctic thermal-sensitive uracil-DNA-glycosylase-supplemented polymerase spiral reaction (ATSU-PSR) technique for eliminating carryover contamination. Two steps are needed for ATSU-PSR technique for removing carryover contamination. During the first stage, all PSR complicons labeled with dUTP in the presence of Bst 2.0 enzyme and dUTP. During the second stage, all subsequent ATSU-PSR amplifications are digested using ATSU, which specifically cleave carryover contaminants by removing uracil in amplicons from previous reactions. Importantly, ATSU is heat inactivated during the PSR amplification stage (63°C), and the digested contaminants are degraded, ensuring that only the target templates are amplified. In addition, three components, including fluorescein isothiocyanate (FITC)-labeled primer, biotin-14-dCTP, and biotin-14-dATP, are added into ATSU-PSR mixtures for forming the biotin- and FITC-attached duplex products.

    Article Snippet: Biotin-14-dCTP and biotin-14-dATP were obtained from Thermo Scientific Co., Ltd. (Shanghai, China).

    Techniques: Labeling, Amplification

    Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

    doi: 10.3389/fbioe.2019.00401

    Figure Lengend Snippet: Outline of nanoparticle-based biosensor-supplemented polymerase spiral reaction assay (NB-PSR). (A) Outline of PSR with Ft* primer, biotin-14-dCTP, and biotin-14-dATP. (B) The detailed structure of NB. (C) The schematic illustration of the principle of NB for visualization of PSR products. (D) Interpretation of the results: (I) positive (two red bands, including test line and control line, appeared on the visual region of NB); (II) negative (only the control line region showed a red band).

    Article Snippet: Biotin-14-dCTP and biotin-14-dATP were obtained from Thermo Scientific Co., Ltd. (Shanghai, China).

    Techniques:

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkv545

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkv545

    Figure Lengend 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.

    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.

    Techniques: 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).

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkv545

    Figure Lengend 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).

    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.

    Techniques: Incubation, Generated, Plasmid Preparation

    Summary of the genetic organization, expression and assembly of the fragmented class Ia RNR from phage Aeh1. The map of the Aeh1 RNR operon (gDNA) shows an early phage promoter (right facing arrow) that drives expression of the operon ( 32 ). The NrdA-a, NrdA-b and NrdB polypeptides are independently translated from this message (mRNA) ( 8 ), while expression of MobE is inhibited by an RNA secondary structure ( 33 ). Our current data indicate that the NrdA-a and NrdA-b polypeptides self assemble to form the α a /α b heterodimer (active site residues depicted as spheres), while dimerization to form the (α a /α b ) 2 large subunit is stimulated by dATP. The ΔT mutants are defective in dimerization of the α a /α b heterodimer. Holoenzyme formation is stimulated by dATP and includes the small subunit dimer, β 2 . The structures of the individual polypeptides were modified from the E. coli NrdA (PBD file 4R1R) and NrdB (PDB file 1AV8) structures. In the holoenzyme model [based on Ref. ( 34 )], the (α a /α b ) 2 large subunit is rotated 90° vertically relative to the free large subunit and the β 2 subunit is rotated 90° horizontally relative to the free dimer.

    Journal: Nucleic Acids Research

    Article Title: Assembly of a fragmented ribonucleotide reductase by protein interaction domains derived from a mobile genetic element

    doi: 10.1093/nar/gkq924

    Figure Lengend Snippet: Summary of the genetic organization, expression and assembly of the fragmented class Ia RNR from phage Aeh1. The map of the Aeh1 RNR operon (gDNA) shows an early phage promoter (right facing arrow) that drives expression of the operon ( 32 ). The NrdA-a, NrdA-b and NrdB polypeptides are independently translated from this message (mRNA) ( 8 ), while expression of MobE is inhibited by an RNA secondary structure ( 33 ). Our current data indicate that the NrdA-a and NrdA-b polypeptides self assemble to form the α a /α b heterodimer (active site residues depicted as spheres), while dimerization to form the (α a /α b ) 2 large subunit is stimulated by dATP. The ΔT mutants are defective in dimerization of the α a /α b heterodimer. Holoenzyme formation is stimulated by dATP and includes the small subunit dimer, β 2 . The structures of the individual polypeptides were modified from the E. coli NrdA (PBD file 4R1R) and NrdB (PDB file 1AV8) structures. In the holoenzyme model [based on Ref. ( 34 )], the (α a /α b ) 2 large subunit is rotated 90° vertically relative to the free large subunit and the β 2 subunit is rotated 90° horizontally relative to the free dimer.

    Article Snippet: The interactions between the mutant NrdA and the NrdB protein were analyzed using the same protocol but with 0.1–3.5 µM NrdA in the absence of dATP and 0.25–6 µM NrdA in the presence of 1 mM dATP (GE Healthcare).

    Techniques: Expressing, IA, Modification

    Reproducibility of the method of double radioactive labelling of probes on a microarray. ( A ) The results of two independent hybridisation experiments, each using 100 ng [ 35 S]dATP-labelled probe and 100 ng [ 3 H]dCTP-labelled probe from two different tissue samples (total brain of adult rat versus cortex of 12-day-old rat) were compared. For each of the experiments, ratios of the 3 H signal intensity to the 35 S signal intensity were calculated for each element of the microarray and normalised with respect to an external standard (luciferase cDNA sequence). The values from one experiment were plotted against those of the other. ( B ) Comparison of two independent hybridisations, each using 100 ng [ 35 S]dATP-labelled probe and 100 ng [ 3 H]dCTP-labelled probe from two different tissue samples. For one experiment, 10 7 molecules of transcribed luciferase RNA were added to 100 ng mRNA used for probe synthesis (experiment 1) and 10 8 molecules of luciferase RNA were added for the other experiment (experiment 2). The 3 H signal intensities (number of counts in 24 h) for each element of the microarray of one experiment were plotted against those of the other. The values of signal intensity are statistically reliable from 100 counts for 24 h. The clone shown by an arrow corresponds to the luciferase control. The ratio of the 3 H signal intensity of the luciferase clone in experiment 2 to that in experiment 1 is ∼10, which is as expected.

    Journal: Nucleic Acids Research

    Article Title: A novel sensitive microarray approach for differential screening using probes labelled with two different radioelements

    doi:

    Figure Lengend Snippet: Reproducibility of the method of double radioactive labelling of probes on a microarray. ( A ) The results of two independent hybridisation experiments, each using 100 ng [ 35 S]dATP-labelled probe and 100 ng [ 3 H]dCTP-labelled probe from two different tissue samples (total brain of adult rat versus cortex of 12-day-old rat) were compared. For each of the experiments, ratios of the 3 H signal intensity to the 35 S signal intensity were calculated for each element of the microarray and normalised with respect to an external standard (luciferase cDNA sequence). The values from one experiment were plotted against those of the other. ( B ) Comparison of two independent hybridisations, each using 100 ng [ 35 S]dATP-labelled probe and 100 ng [ 3 H]dCTP-labelled probe from two different tissue samples. For one experiment, 10 7 molecules of transcribed luciferase RNA were added to 100 ng mRNA used for probe synthesis (experiment 1) and 10 8 molecules of luciferase RNA were added for the other experiment (experiment 2). The 3 H signal intensities (number of counts in 24 h) for each element of the microarray of one experiment were plotted against those of the other. The values of signal intensity are statistically reliable from 100 counts for 24 h. The clone shown by an arrow corresponds to the luciferase control. The ratio of the 3 H signal intensity of the luciferase clone in experiment 2 to that in experiment 1 is ∼10, which is as expected.

    Article Snippet: Probe synthesis and labelling were then performed in the presence of 5 mM MgCl2 , 1× reverse transcription buffer (Life Technologies), 10 mM dithiothreitol, 100 U RNaseOUT RNase inhibitor (Life Technologies), 0.05 mM ddTTP, 0.5 mM dGTP and dTTP, 100 U Superscript II reverse transcriptase (Life Technologies) and 10 µCi [35 S]dATP (Amersham) and 0.5 mM dCTP or 20 µCi [3 H]dCTP (Amersham) and 0.5 mM dATP for the phosphorylated and tritiated probes, respectively, by incubation of the mixtures at 42°C for 50 min. RNA was eliminated by heating at 70°C for 15 min and treatment with 2 U RNase H (Life Technologies) at 37°C for 20 min. Unincorporated nucleotides were removed by passage through a P10 column (Bio-Rad).

    Techniques: Microarray, Hybridization, Luciferase, Sequencing

    Visualisation (arbitrary colours) of the results of double radioactive labelling of probes on a microarray. Hybridisation images obtained with 100 ng [ 35 S]dATP-labelled probe and 100 ng [ 3 H]dCTP-labelled probe from two different tissue samples. Targets were PCR products of 300–1500 bp spotted onto polylysine-coated slides. ( A ) Simultaneous visualisation of both 3 H and 35 S labelling. The 3 H labelling is represented in green, the 35 S labelling in red and overlapping of the two in shades of yellow. ( B ) Visualisation of only 3 H labelling. ( C ) Visualisation of only 35 S labelling. Above the three microarray images, a spot of 3 H, one of a mix of 3 H and 35 S and another of 35 S were set down on the microarray as controls for filtering, allowing segregation of 35 S β from 3 H β disintegrations.

    Journal: Nucleic Acids Research

    Article Title: A novel sensitive microarray approach for differential screening using probes labelled with two different radioelements

    doi:

    Figure Lengend Snippet: Visualisation (arbitrary colours) of the results of double radioactive labelling of probes on a microarray. Hybridisation images obtained with 100 ng [ 35 S]dATP-labelled probe and 100 ng [ 3 H]dCTP-labelled probe from two different tissue samples. Targets were PCR products of 300–1500 bp spotted onto polylysine-coated slides. ( A ) Simultaneous visualisation of both 3 H and 35 S labelling. The 3 H labelling is represented in green, the 35 S labelling in red and overlapping of the two in shades of yellow. ( B ) Visualisation of only 3 H labelling. ( C ) Visualisation of only 35 S labelling. Above the three microarray images, a spot of 3 H, one of a mix of 3 H and 35 S and another of 35 S were set down on the microarray as controls for filtering, allowing segregation of 35 S β from 3 H β disintegrations.

    Article Snippet: Probe synthesis and labelling were then performed in the presence of 5 mM MgCl2 , 1× reverse transcription buffer (Life Technologies), 10 mM dithiothreitol, 100 U RNaseOUT RNase inhibitor (Life Technologies), 0.05 mM ddTTP, 0.5 mM dGTP and dTTP, 100 U Superscript II reverse transcriptase (Life Technologies) and 10 µCi [35 S]dATP (Amersham) and 0.5 mM dCTP or 20 µCi [3 H]dCTP (Amersham) and 0.5 mM dATP for the phosphorylated and tritiated probes, respectively, by incubation of the mixtures at 42°C for 50 min. RNA was eliminated by heating at 70°C for 15 min and treatment with 2 U RNase H (Life Technologies) at 37°C for 20 min. Unincorporated nucleotides were removed by passage through a P10 column (Bio-Rad).

    Techniques: Microarray, Hybridization, Polymerase Chain Reaction

    Apoptosome contains exogenously added dATP. ( A ) Indicated samples were subjected to glycerol gradient separation after incubation as described in Materials and Methods . Fractions from the glycerol gradient were subjected to SDS/PAGE followed by Western blotting using anti-Apaf-1 antibody. ( B ) The same glycerol gradient fractions as in A were subjected to caspase-3 activity assay. Aliquots (14 μl) of glycerol gradient fractions were incubated with 50 nM procaspase-9, 50 nM procaspase-3, 10 μM dATP, 100 nM cytochrome c , and 10 μM fluorogenic DEVD caspase substrate in a final volume of 20 μl. Samples were mixed in the test tubes and transferred to 384-well microplates, and caspase-3 activity was measured by using the XFluor4 spectrometry reader (Tecan). ( C ) dATP incorporation was measured by using [α- 33 P]dATP. A total of 10 μCi of [α- 33 P]dATP plus 10 μM of dATP were incubated with Apaf-1 and cytochrome c at 30°C for 3 h (blue diamond). For another sample, 10 μCi of [α- 33 P] dATP plus 10 μM of dATP was added to Apaf-1 and cytochrome c mixture after they were preincubated for 1.5 h. The sample was incubated for additional 1.5 h before being subjected to glycerol gradient (pink square). The fractions were then collected as in A , and a 4-μl aliquot of each fraction was counted for radioactivity by a liquid scintillation counter. ( D ) For the active apoptosome, 10 ng of Apaf-1, 5 ng of cytochrome c , and 10 μCi of [α- 33 P]dATP plus 10 μM of dATP were incubated at 30°C for 3 h before being subjected to a Superpose 6 chromatography column as described in Materials and Methods . For the inactive apoptosome sample, 10 ng of Apaf-1 and 5 ng of cytochrome c were preincubated at 30°C for 1.5 h. Then, 10 μCi of [α- 33 P]dATP plus 10 μM of dATP were added to the sample, and incubation was continued for 1.5 h before the sample was subjected to the Superpose 6 gel-filtration column. An aliquot of 3 μl of peak Apaf-1 fraction was spotted on a TLC plate and analyzed as described in Materials and Methods .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1

    doi: 10.1073/pnas.0507900102

    Figure Lengend Snippet: Apoptosome contains exogenously added dATP. ( A ) Indicated samples were subjected to glycerol gradient separation after incubation as described in Materials and Methods . Fractions from the glycerol gradient were subjected to SDS/PAGE followed by Western blotting using anti-Apaf-1 antibody. ( B ) The same glycerol gradient fractions as in A were subjected to caspase-3 activity assay. Aliquots (14 μl) of glycerol gradient fractions were incubated with 50 nM procaspase-9, 50 nM procaspase-3, 10 μM dATP, 100 nM cytochrome c , and 10 μM fluorogenic DEVD caspase substrate in a final volume of 20 μl. Samples were mixed in the test tubes and transferred to 384-well microplates, and caspase-3 activity was measured by using the XFluor4 spectrometry reader (Tecan). ( C ) dATP incorporation was measured by using [α- 33 P]dATP. A total of 10 μCi of [α- 33 P]dATP plus 10 μM of dATP were incubated with Apaf-1 and cytochrome c at 30°C for 3 h (blue diamond). For another sample, 10 μCi of [α- 33 P] dATP plus 10 μM of dATP was added to Apaf-1 and cytochrome c mixture after they were preincubated for 1.5 h. The sample was incubated for additional 1.5 h before being subjected to glycerol gradient (pink square). The fractions were then collected as in A , and a 4-μl aliquot of each fraction was counted for radioactivity by a liquid scintillation counter. ( D ) For the active apoptosome, 10 ng of Apaf-1, 5 ng of cytochrome c , and 10 μCi of [α- 33 P]dATP plus 10 μM of dATP were incubated at 30°C for 3 h before being subjected to a Superpose 6 chromatography column as described in Materials and Methods . For the inactive apoptosome sample, 10 ng of Apaf-1 and 5 ng of cytochrome c were preincubated at 30°C for 1.5 h. Then, 10 μCi of [α- 33 P]dATP plus 10 μM of dATP were added to the sample, and incubation was continued for 1.5 h before the sample was subjected to the Superpose 6 gel-filtration column. An aliquot of 3 μl of peak Apaf-1 fraction was spotted on a TLC plate and analyzed as described in Materials and Methods .

    Article Snippet: Nucleotides dATP and dADP were purchased from Amersham Pharmacia; [α-33 P]dATP was obtained from MP Bioscience.

    Techniques: Incubation, SDS Page, Western Blot, Caspase-3 Activity Assay, Activity Assay, Radioactivity, Chromatography, Filtration, Thin Layer Chromatography

    Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 P]dATP. The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir

    doi:

    Figure Lengend Snippet: Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 P]dATP. The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.

    Article Snippet: For standard EPAs , WHV virions or immunocomplexed HBV capsids were resuspended in 50 μl of EPA buffer (50 mM Tris hydrochloride [pH 7.4], 75 mM NH4 Cl, 1 mM EDTA, 20 mM MgCl2 , 0.1 mM β-mercaptoethanol, 0.5% Tween 20) supplemented with 50 μM (or in some reactions 12.5 μM) unlabeled dNTPs (dGTP, dCTP, and TTP) and 33 nM [α-32 P]dATP (3,000 Ci/mmol; NEN-Dupont, Boston, Mass.).

    Techniques: DNA Synthesis, Purification, Infection, Isolation, Autoradiography, Inhibition, Generated

    Effect of guanosine versus cytosine analogs on DHBV priming. In vitro-translated DHBV Pol was incubated with 250 nM [α- 32 P]dGTP; 220 nM unlabeled dCTP, dATP, and TTP; and increasing concentrations of the indicated analog-TPs. (A) The radiolabeled Pol-oligonucleotide adducts were analyzed by conventional SDS-PAGE and autoradiography. The migration positions of the 35 S-labeled DHBV Pol (lane 35 S) are indicated at the sides. Substr., substrate. (B) Titration curves were generated by phosphorimaging.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir

    doi:

    Figure Lengend Snippet: Effect of guanosine versus cytosine analogs on DHBV priming. In vitro-translated DHBV Pol was incubated with 250 nM [α- 32 P]dGTP; 220 nM unlabeled dCTP, dATP, and TTP; and increasing concentrations of the indicated analog-TPs. (A) The radiolabeled Pol-oligonucleotide adducts were analyzed by conventional SDS-PAGE and autoradiography. The migration positions of the 35 S-labeled DHBV Pol (lane 35 S) are indicated at the sides. Substr., substrate. (B) Titration curves were generated by phosphorimaging.

    Article Snippet: For standard EPAs , WHV virions or immunocomplexed HBV capsids were resuspended in 50 μl of EPA buffer (50 mM Tris hydrochloride [pH 7.4], 75 mM NH4 Cl, 1 mM EDTA, 20 mM MgCl2 , 0.1 mM β-mercaptoethanol, 0.5% Tween 20) supplemented with 50 μM (or in some reactions 12.5 μM) unlabeled dNTPs (dGTP, dCTP, and TTP) and 33 nM [α-32 P]dATP (3,000 Ci/mmol; NEN-Dupont, Boston, Mass.).

    Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Migration, Labeling, Titration, Generated

    Potential routes for cellular production and metabolism of N6-methyl-dATP and N6-methyl-ATP. N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by MTH1 to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: Potential routes for cellular production and metabolism of N6-methyl-dATP and N6-methyl-ATP. N6-methyl-dATP and N6-methyl-ATP may be produced from N6-methyl-dAMP and N6-methyl-AMP formed upon DNA and RNA degradation, respectively. This may occur through the consecutive actions of adenylate kinase ( AK ) and nucleoside diphosphate kinase or through nonspecific methylation by S -adenosylmethionine ( SAM ), the N6-adenosine-methyltransferase METTL3 or N6-adenine–specific DNA methyltransferase 1 ( N6AMT1 ). N6-methyl-dATP and N6-methyl-ATP are hydrolyzed by MTH1 to their corresponding monophosphates and further metabolized by ADAL1 to dIMP and IMP that can then enter the nucleotide salvage pathway. Abbreviations used in the figure: NDPK , nucleoside diphosphate kinase; RNR, ribonucleotide reductase; METTL3, N6-adenosine methyltransferase; N6AMT1, N6-adenine-specific DNA methyltransferase 1.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Produced, Methylation

    MTH1 catalyzes the hydrolysis of N6-methyl-dATP. A, MTH1 catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and PP i . B, time course hydrolysis of N6-methyl-dATP (1 m m ) catalyzed by MTH1 (20 n m ) was monitored by separation of reaction samples incubated 0–40 min at 22 °C on a Hypercarb column using HPLC coupled to MS. Reaction substrate and product was detected at 254 nm and the mass of the product N6-methyl-dAMP was clearly observed by mass detection. C, graph showing the fraction of N6-methyl-dATP and N6-methyl-dAMP in percent after various times of hydrolysis based on the respective area under the curve of the peaks in the corresponding HPLC chromatogram.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: MTH1 catalyzes the hydrolysis of N6-methyl-dATP. A, MTH1 catalyzes the hydrolysis of N6-methyl-dATP to N6-methyl-dAMP and PP i . B, time course hydrolysis of N6-methyl-dATP (1 m m ) catalyzed by MTH1 (20 n m ) was monitored by separation of reaction samples incubated 0–40 min at 22 °C on a Hypercarb column using HPLC coupled to MS. Reaction substrate and product was detected at 254 nm and the mass of the product N6-methyl-dAMP was clearly observed by mass detection. C, graph showing the fraction of N6-methyl-dATP and N6-methyl-dAMP in percent after various times of hydrolysis based on the respective area under the curve of the peaks in the corresponding HPLC chromatogram.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Incubation, High Performance Liquid Chromatography

    Activity with N6-methyl-dATP is unique to MTH1 within human NUDIX subfamily. Activities of human NUDIX enzyme (MTH1, NUDT15, NUDT17, and NUDT18) were assayed with data points in quadruplicate with 50 μ m dATP or N6-methyl-dATP at 20 and 200 n m enzyme in MTH1 reaction buffer (pH 8.0). 0.2 units/ml of PPase was used to convert formed PP i to P i that was detected using malachite green reagent and measurement of absorbance at 630 nm. Graph shows mean ± S.D. from one experiment performed in triplicate.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: Activity with N6-methyl-dATP is unique to MTH1 within human NUDIX subfamily. Activities of human NUDIX enzyme (MTH1, NUDT15, NUDT17, and NUDT18) were assayed with data points in quadruplicate with 50 μ m dATP or N6-methyl-dATP at 20 and 200 n m enzyme in MTH1 reaction buffer (pH 8.0). 0.2 units/ml of PPase was used to convert formed PP i to P i that was detected using malachite green reagent and measurement of absorbance at 630 nm. Graph shows mean ± S.D. from one experiment performed in triplicate.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Activity Assay

    Activity with N6-methyl-dATP is evolutionary conserved among vertebrates. MutT homologues (MTH1 and NUDT1) from different animal species as well as E. coli MutT and NUDT1 from the plant A. thaliana were screened for hydrolysis activity with N6-methyl-dATP. Enzyme (1.25 n m ) was incubated with 50 μ m N6-methyl-dATP in MTH1 reaction buffer (pH 7.5) with 0.4 units/ml of PPase for 20 min at 22 °C in triplicates. P i was detected using Biomol Green (Enzo Life Sciences). Absorbance at 630 nm was read after 20 min. A P i standard curve was included on the plate and used to convert the assay signal to produced PP i . Data are shown as hydrolyzed N6-methyl-dATP (μ m ) divided by concentration of NUDT1 enzyme (μ m ) per second. The graph shows the mean ± S.D. from an experiment performed in triplicate.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: Activity with N6-methyl-dATP is evolutionary conserved among vertebrates. MutT homologues (MTH1 and NUDT1) from different animal species as well as E. coli MutT and NUDT1 from the plant A. thaliana were screened for hydrolysis activity with N6-methyl-dATP. Enzyme (1.25 n m ) was incubated with 50 μ m N6-methyl-dATP in MTH1 reaction buffer (pH 7.5) with 0.4 units/ml of PPase for 20 min at 22 °C in triplicates. P i was detected using Biomol Green (Enzo Life Sciences). Absorbance at 630 nm was read after 20 min. A P i standard curve was included on the plate and used to convert the assay signal to produced PP i . Data are shown as hydrolyzed N6-methyl-dATP (μ m ) divided by concentration of NUDT1 enzyme (μ m ) per second. The graph shows the mean ± S.D. from an experiment performed in triplicate.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Activity Assay, Incubation, Produced, Concentration Assay

    Kinetic characterization of MTH1-catalyzed hydrolysis of dATP, N6-methyl-dATP, N6-methyl-ATP, dGTP, and O6-methyl-dGTP. A, substrate saturation curves of MTH1 (1 n m ) were produced using MTH1 reaction buffer (pH 7.5). Initial rates were determined at dATP concentrations varied between 0 and 200 μ m and between 0 and 300 μ m for N6-methyl-dATP and N6-methyl-ATP, respectively, and for dGTP and O6-methyl-dGTP ( B ) using 0–300 and 0–200 μ m , respectively. Formed PP i was detected using PPiLight TM Inorganic Pyrophosphate Assay (Lonza) and the assay signal was converted to concentration of PP i by including a PP i standard curve on the assay plate.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: Kinetic characterization of MTH1-catalyzed hydrolysis of dATP, N6-methyl-dATP, N6-methyl-ATP, dGTP, and O6-methyl-dGTP. A, substrate saturation curves of MTH1 (1 n m ) were produced using MTH1 reaction buffer (pH 7.5). Initial rates were determined at dATP concentrations varied between 0 and 200 μ m and between 0 and 300 μ m for N6-methyl-dATP and N6-methyl-ATP, respectively, and for dGTP and O6-methyl-dGTP ( B ) using 0–300 and 0–200 μ m , respectively. Formed PP i was detected using PPiLight TM Inorganic Pyrophosphate Assay (Lonza) and the assay signal was converted to concentration of PP i by including a PP i standard curve on the assay plate.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Produced, Pyrophosphate Assay, Concentration Assay

    MTH1 activity with N6-methyl-dATP and N6-methyl-ATP compared with dATP. Activity of 1 and 5 n m MTH1 was tested with 50 μ m N6-methyl-dATP, dATP, ATP, and N6-methyl-ATP in MTH1 reaction buffer (pH 8.0) at 22 °C. Reaction time was 30 min and 0.2 units/ml of PPase was used to generate P i from produced PP i . P i was detected by addition of malachite green reagent followed by measurement of the absorbance at 630 nm. Controls with PPase only was included and background signal was subtracted from the assay data. A P i standard curve was included on the plate enabling determination of the concentration of formed PP i . Graph shows mean ± S.D. from one experiment performed in quadruplicate.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: MTH1 activity with N6-methyl-dATP and N6-methyl-ATP compared with dATP. Activity of 1 and 5 n m MTH1 was tested with 50 μ m N6-methyl-dATP, dATP, ATP, and N6-methyl-ATP in MTH1 reaction buffer (pH 8.0) at 22 °C. Reaction time was 30 min and 0.2 units/ml of PPase was used to generate P i from produced PP i . P i was detected by addition of malachite green reagent followed by measurement of the absorbance at 630 nm. Controls with PPase only was included and background signal was subtracted from the assay data. A P i standard curve was included on the plate enabling determination of the concentration of formed PP i . Graph shows mean ± S.D. from one experiment performed in quadruplicate.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Activity Assay, Produced, Concentration Assay

    Schematic representation of the recognition of nucleotides by hMTH1. Hydrogen bond interactions of ( A ) N6-methyl-dAMP (N6-metA), ( B ) 2-oxo-dATP (2-oxoA) ( 23 ), ( C ) O6-methyl-dGMP (O6-metG) ( 10 ), and ( D ) 8-oxo-dGMP (8-oxoG) ( 24 ). Hydrogen bonds are shown as dashed lines and bond distances are given in Angstroms (Å). Deprotonated aspartates, which act as hydrogen bond acceptors, are indicated by the minus sign , where this is unambiguous.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: Schematic representation of the recognition of nucleotides by hMTH1. Hydrogen bond interactions of ( A ) N6-methyl-dAMP (N6-metA), ( B ) 2-oxo-dATP (2-oxoA) ( 23 ), ( C ) O6-methyl-dGMP (O6-metG) ( 10 ), and ( D ) 8-oxo-dGMP (8-oxoG) ( 24 ). Hydrogen bonds are shown as dashed lines and bond distances are given in Angstroms (Å). Deprotonated aspartates, which act as hydrogen bond acceptors, are indicated by the minus sign , where this is unambiguous.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques:

    N6-methyl-dATP is incorporated into DNA in an MTH1-dependent manner. DNA was extracted from zebrafish MTH1KO and MTH1WT embryos developed from fertilized zebrafish eggs microinjected with N6-methyl-dATP or left untreated. DNA was analyzed for N6-methyl-dA content using LC-MS/MS. N6-methyl-dA content was normalized to N6-methyldA levels in untreated MTH1KO and MTH1WT zebrafish embryos, respectively. N6-methyl-dATP microinjected MTH1KO zebrafish embryos display a 2-fold higher N6-methyl-dA level compared with untreated embryos, whereas N6-methyl-dA DNA levels in MTH1WT zebrafish did not differ between untreated and N6-methyl-dA–microinjected embryos. This suggests that N6-methyl-dATP is incorporated into DNA and incorporation can be prevented by MTH1. The graph shows mean ± S.D., n = 2.

    Journal: The Journal of Biological Chemistry

    Article Title: MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool

    doi: 10.1074/jbc.RA120.012636

    Figure Lengend Snippet: N6-methyl-dATP is incorporated into DNA in an MTH1-dependent manner. DNA was extracted from zebrafish MTH1KO and MTH1WT embryos developed from fertilized zebrafish eggs microinjected with N6-methyl-dATP or left untreated. DNA was analyzed for N6-methyl-dA content using LC-MS/MS. N6-methyl-dA content was normalized to N6-methyldA levels in untreated MTH1KO and MTH1WT zebrafish embryos, respectively. N6-methyl-dATP microinjected MTH1KO zebrafish embryos display a 2-fold higher N6-methyl-dA level compared with untreated embryos, whereas N6-methyl-dA DNA levels in MTH1WT zebrafish did not differ between untreated and N6-methyl-dA–microinjected embryos. This suggests that N6-methyl-dATP is incorporated into DNA and incorporation can be prevented by MTH1. The graph shows mean ± S.D., n = 2.

    Article Snippet: MTH1 activity assay Activity of MTH1 (5 or 1 nm ) with 50 μm dATP (Promega), N6-methyl-dATP (Jena Bioscience), ATP (Promega), and N6-methyl-ATP (Jena Bioscience) was assayed in MTH1 reaction buffer (Tris acetate, pH 8.0, 40 mm sodium chloride, 10 mm magnesium acetate).

    Techniques: Liquid Chromatography with Mass Spectroscopy

    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 P]dATP. DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .

    Journal: Nucleic Acids Research

    Article Title: Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding

    doi: 10.1093/nar/gkr345

    Figure Lengend Snippet: External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 P]dATP. DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .

    Article Snippet: [γ-32 P]ATP and [α-32 P]dATP were purchased from MP Biomedicals and used with PNK/Optikinase or TdT to 32 P label the 5′- or 3′-ends of DNA, respectively.

    Techniques: Mutagenesis, Labeling

    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 P]dATP-labeled DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: The Candida dubliniensis CdCDR1 Gene Is Not Essential for Fluconazole Resistance

    doi: 10.1128/AAC.46.9.2829-2841.2002

    Figure Lengend Snippet: Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 P]dATP-labeled DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.

    Article Snippet: RNA was hybridized at 42°C with DNA probes homologous to CdCDR1 , CdMDR1 , and CdTEF3 labeled with [α-32 P]dATP (6,000 Ci/mmol, 220 TBq/mmol; NEN Life Sciences, Boston, Mass.) by random primer labeling as described by Moran et al. ( ).

    Techniques: Northern Blot, Expressing, In Vitro, Generated, Hybridization, Labeling