Journal: Nucleic Acids Research

Article Title: Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

doi: 10.1093/nar/gkx633

Figure Lengend Snippet: Fluorometric assay to detect RNA-dependent Hs PrimPol DNA primase/polymerase activity. ( A ) Fluorescence kinetics as a measure of de novo DNA synthesis by purified Hs PrimPol (450 nM), in combination with different components as indicated. An increase in fluorescence as a function of time occurred only when dATP and dGTP (100 μM each), the GTCC template (1 μM; see Supplementary Table S1 ), and MnCl 2 (1 mM) were added. The use of the ATCC template (1 μM; Supplementary Table S1 ), or MgCl 2 as metal donor, did not render a measurable fluorescence signal. ( B ) Fluorescence kinetics using the optimal conditions described in A, but comparing GTCC and GUCC templates (see Supplementary Table S1 ). A negative control assay using GTCC template in the absence of dNTPs is represented as a comparison. Note that the scale of arbitrary units in the y-axis is different with respect to A. ( C ) Representative electropherogram of a priming experiment carried out with purified Hs PrimPol (450 nM) and 16 nM [γ-32P]ATP, 100 μM dGTP and either GTCC or GUCC as template. The position of the labeled ‘5′-AdG-3′ dimer is indicated. Detailed experimental conditions, as well as templates sequences are described in Materials and Methods.

Article Snippet: Dpn I Restriction enzyme, polynucleotide kinase (PNK), bovine serum albumin (BSA) and 100 mM stock solutions of dATP, dTTP, dGTP and dCTP were purchased from New England Biolabs.

Techniques: Activity Assay, Fluorescence, DNA Synthesis, Purification, Negative Control, Labeling

Journal: Nucleic Acids Research

Article Title: Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

doi: 10.1093/nar/gkx633

Figure Lengend Snippet: RNA-dependent DNA polymerase activity shown by WT HsPrimPol and Y89R variant. ( A ) Names and sequences of oligonucleotides used as template/primer hybrids to measure primer-dependent dNTP incorporation by Hs PrimPol; RNA sequences are indicated in italics; ‘*’ indicates the radiolabeled oligonucleotide used as primer. ( B ) Polymerization reactions by WT or Y89R variant Hs PrimPol was evaluated using the indicated combinations of dNTPs (100 μM each) and either DNA (T13T/Spc1) or RNA ( T13U /Spc1) templates. As shown by electrophoretic analysis, primers (15-mer) elongated in 2, 4, 6 or 13 residues represent the expected products in the presence of either dATP, dATP+dGTP, dATP+dGTP+dTTP, or the four dNTPs, respectively.

Article Snippet: Dpn I Restriction enzyme, polynucleotide kinase (PNK), bovine serum albumin (BSA) and 100 mM stock solutions of dATP, dTTP, dGTP and dCTP were purchased from New England Biolabs.

Techniques: Activity Assay, Variant Assay

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

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

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

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

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:

Journal: The Journal of Experimental Medicine

Article Title: 4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses

doi: 10.1084/jem.20171068

Figure Lengend Snippet: 4-1BB costimulation promotes PGC1α-dependent mitochondrial function. (A) MitoTracker Deep Red FM staining of CD8 + T cells activated with immobilized anti-CD3 (3 µg/ml plate-bound) in the presence of anti-CD28 (2 µg/ml soluble), anti–4-1BB (10 µg/ml soluble), or both for 24 h, then expanded with 25 U/ml IL-2 for 7 d. Shaded histogram indicates unstained control. (B) Baseline OCR and respiratory capacity of T cells expanded from CD28 or 4-1BB cultures. (C) Tabulated ATP and ADP concentrations from cells activated as in A but for 24 h. (D) Representative OCR trace of CD8 + T cells sorted from C57/BL6 mice treated with isotype control, anti–PD-1, anti–4-1BB, or both for 3 d. (E) Tabulated OCR (left) and spare respiratory capacity (right) T cells as in D. (F) Tabulated ATP and ADP concentrations from T cells as in D. (G) PGC1α intracellular staining (left) of cells treated as in A. (H) MitoTracker Deep Red FM staining of cells from Ppargc1a f/f or Ppargc1a f/f Cd4 Cre mice treated as in A. (I and J) OCR trace (I) and respiratory capacity (J) of T cells from Ppargc1a f/f or Ppargc1a f/f Cd4 Cre mice treated as in A. Data represent three to five (A, C, and I) or represent the mean (B, D–H, and J) of three to five independent experiments. *, P

Article Snippet: For ATP and ADP determination, CD8+ T cells were analyzed from C57/BL6 mice activated with anti-CD3 (2C11, 3 µg/ml) immobilized on tissue culture plates in the presence of anti-CD28 (37.51, 2 µg/ml), anti–4-1BB (3H3, 10 µg/ml), or both or from cells isolated from C57/BL6 mice treated with 50 µg anti–4-1BB (clone 3H3; Bio X-Cell) or respective rat isotype control, intraperitoneally every other day for 3 d. ATP and ADP concentrations were measured by using the ATP Determination kit (from Life Technologies) and the ADP Assay kit (from Sigma-Aldrich) according to the manufacturer’s instructions.

Techniques: Staining, Mouse Assay

Journal: Genome Biology

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

doi: 10.1186/s13059-018-1407-3

Figure Lengend Snippet: Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

Article Snippet: This RT was stopped at 70 °C for 15 min. We added 5 μL of ExoIB solution (1.6× Exonuclease I buffer, 3.2× PCR buffer, 16 mM DTT) and 20 μL of TdT solution (1× PCR buffer, 3 mM dATP, 0.0384 units/μL RNase H (Invitrogen), 33.6 units/μL terminal transferase (Roche)) into 20 μL of extracted cDNA using a pipette at 0 °C.

Techniques: Lysis, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Sequencing, Binding Assay, Labeling, Centrifugation, Purification, Amplification, Synthesized, Derivative Assay

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

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

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

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

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

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

Journal: Journal of Bacteriology

Article Title: Crystal Structures of Mycobacterium smegmatis RecA and Its Nucleotide Complexes

doi: 10.1128/JB.185.14.4280-4284.2003

Figure Lengend Snippet: (a) Superposition of the nucleotides and their surroundings in the complexes with ADP (red), ATPγS (green), and dATP (blue). The protein atoms that interact with the nucleotide in all or most structures of RecA Ms and RecA Mt are shown. Where this atom belongs to the side chain, the α-carbon position is also indicated. The tyrosyl side chain, which stacks against the base in addition to interacting with the sugar, is shown in full. (b) Comparable views, based on superposition of bases, of nucleotides in RecA Ms (left) and RecA Mt (right). ADP, ATPγS, and dATP are shown in red, green, and blue, respectively.

Article Snippet: ADP, ATPγS, and dATP were obtained from Amersham Biosciences.

Techniques: Mass Spectrometry

Journal: Molecular and Cellular Biology

Article Title: Eukaryotic Translation Initiation Factor 4G Is Targeted for Proteolytic Cleavage by Caspase 3 during Inhibition of Translation in Apoptotic Cells

doi:

Figure Lengend Snippet: Cytochrome c - and dATP-dependent activation of the eIF4G cleavage activity in vitro. (A) Induction of eIF4G cleavage in HeLa S100 extracts. HeLa S100 extract (40 μl) was incubated with 400 nM cytochrome c and 1 mM dATP for the time periods indicated at 37°C. Reactions were stopped by addition of SDS-PAGE sample buffer, subjected to SDS-PAGE, and analyzed by immunoblotting. eIF4G and eIF4GcpN are indicated on the right. The arrow and arrowhead indicate intermediate cleavage products of eIF4G. (B) Activation of caspase 3 in HeLa S100 extracts. Reaction mixtures as described for panel A were loaded on 13% acrylamide gels and immunoblotted for caspase 3. The immunoblots in panels A and B were scanned with an Artec Viewstation A6000C, and the resulting images were labeled with Adobe Photoshop version 3.0.

Article Snippet: HeLa S100 lysates were incubated with 400 nM cytochrome c (Sigma) and 1 mM dATP (Boehringer Mannheim) at 37°C for the time indicated in the figure legends.

Techniques: Activation Assay, Activity Assay, In Vitro, Incubation, SDS Page, Western Blot, Labeling

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

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: