t4 pdg  (New England Biolabs)


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    Name:
    T4 PDG T4 Endonuclease V
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    T4 PDG T4 Endonuclease V 2 000 units
    Catalog Number:
    m0308s
    Price:
    76
    Size:
    2 000 units
    Category:
    DNA Glycosylases
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    New England Biolabs t4 pdg
    T4 PDG T4 Endonuclease V
    T4 PDG T4 Endonuclease V 2 000 units
    https://www.bioz.com/result/t4 pdg/product/New England Biolabs
    Average 95 stars, based on 4516 article reviews
    Price from $9.99 to $1999.99
    t4 pdg - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions"

    Article Title: Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09146-5

    In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively
    Figure Legend Snippet: In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

    Techniques Used: In Vitro, Generated, Nucleic Acid Electrophoresis, Irradiation, Labeling

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    Polymerase Chain Reaction:

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
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KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-yeast Rad53 Susan Gasser; Hauer et al., 2017 N/A Rat monoclonal anti-tubulin, clone YL1/2 Sigma-Aldrich Cat# 92092402-1VL, RRID: CVCL_J781 Rabbit polyclonal anti-DNA Ligase 1 Elabscience Cat# E-AB-31210 Mouse monoclonal anti-Chk1, clone 2G1D5 Cell Signaling Technology Cat# 2360, RRID: AB_2080320 Rabbit polyclonal anti-phospho-Chk1 (Ser345) Cell Signaling Technology Cat# 2341, RRID: AB_330023 Goat anti-mouse immunoglobulins, HRP Dako Cat# P0447, RRID: AB_2617137 Goat anti-rabbit immunoglobulins, HRP Dako Cat# P0448, RRID: AB_2617138 Goat anti-rat immunoglobulins, HRP Dako Cat# P0450, RRID: AB_2630354 Goat anti-rat IgG Secondary Antibody, IRDye 800CW LI-COR Cat# 926-32219, RRID: AB_1850025 Chemicals, Peptides, and Recombinant Proteins Zymolyase 20T AMS Biotechnology Cat# 120491-1 T4 DNA ligase, 20,000,000 U/mL New England Biolabs Cat# B0202 Q5 High-Fidelity DNA polymerase New England Biolabs Cat# M0491 RNase A from bovine pancreas Sigma-Aldrich Cat# 10109169001 Proteinase K Roche Cat# 3115801001 Phenylmethanesulfonyl fluoride Sigma-Aldrich Cat# P7626 b-Agarase I, 1000 U/mL New England Biolabs Cat# M0392 Alpha-Factor peptide (WHWLQLKPGQPMY), > 95% ProteoGenix SAS Cat# GM-PT001 BsrDI New England Biolabs Cat# R0574 Nb.BsrDI New England Biolabs Cat# R0648 NotI New England Biolabs Cat# R0189 Nb.BtsI New England Biolabs Cat# R0707 Antarctic Phosphatase New England Biolabs Cat# M0289 T4 Endonuclease V (T4 PDG) New England Biolabs Cat# M0308 APE1 New England Biolabs Cat# M0282 hAAG New England Biolabs Cat# M0313 Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific Cat# 13778150 Benzonase Sigma-Aldrich Cat# E1014-25KU PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat# 04906837001 Opti-MEM Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 Critical Commercial Assays AMPure XP beads Beckman Coulter Cat# A63881 NEBNext Ultra II DNA Library Prep Kit for Illumina New England Biolabs Cat# E7645 Phusion Flash high-fidelity PCR master mix Thermo Fisher Scientific Cat# F-548 Dynabeads MyOne Streptavidin C1 Life Technologies Cat# 65001 High Sensitivity D1000 ScreenTape Agilent Technologies Cat# 5067-5584 High Sensitivity D1000 ScreenTape reagents Agilent Technologies Cat# 5067-5585 RNA ScreenTape Agilent Technologies Cat# 5067-5576 RNA ScreenTape sample buffer Agilent Technologies Cat# 5067-5577 RNA ScreenTape ladder Agilent Technologies Cat# 5067-5578 Qubit dsDNA HS Assay Kit Invitrogen Cat# Q32854 Agilent Bioanalyzer High Sensitivity DNA Kit Agilent Technologies Cat# 5067-4646 (Continued on next page) e1 Molecular Cell 78, 1–11.e1–e7, June 4, 2020 .. 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    Transfection:

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
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    Recombinant:

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-yeast Rad53 Susan Gasser; Hauer et al., 2017 N/A Rat monoclonal anti-tubulin, clone YL1/2 Sigma-Aldrich Cat# 92092402-1VL, RRID: CVCL_J781 Rabbit polyclonal anti-DNA Ligase 1 Elabscience Cat# E-AB-31210 Mouse monoclonal anti-Chk1, clone 2G1D5 Cell Signaling Technology Cat# 2360, RRID: AB_2080320 Rabbit polyclonal anti-phospho-Chk1 (Ser345) Cell Signaling Technology Cat# 2341, RRID: AB_330023 Goat anti-mouse immunoglobulins, HRP Dako Cat# P0447, RRID: AB_2617137 Goat anti-rabbit immunoglobulins, HRP Dako Cat# P0448, RRID: AB_2617138 Goat anti-rat immunoglobulins, HRP Dako Cat# P0450, RRID: AB_2630354 Goat anti-rat IgG Secondary Antibody, IRDye 800CW LI-COR Cat# 926-32219, RRID: AB_1850025 Chemicals, Peptides, and Recombinant Proteins Zymolyase 20T AMS Biotechnology Cat# 120491-1 T4 DNA ligase, 20,000,000 U/mL New England Biolabs Cat# B0202 Q5 High-Fidelity DNA polymerase New England Biolabs Cat# M0491 RNase A from bovine pancreas Sigma-Aldrich Cat# 10109169001 Proteinase K Roche Cat# 3115801001 Phenylmethanesulfonyl fluoride Sigma-Aldrich Cat# P7626 b-Agarase I, 1000 U/mL New England Biolabs Cat# M0392 Alpha-Factor peptide (WHWLQLKPGQPMY), > 95% ProteoGenix SAS Cat# GM-PT001 BsrDI New England Biolabs Cat# R0574 Nb.BsrDI New England Biolabs Cat# R0648 NotI New England Biolabs Cat# R0189 Nb.BtsI New England Biolabs Cat# R0707 Antarctic Phosphatase New England Biolabs Cat# M0289 T4 Endonuclease V (T4 PDG) New England Biolabs Cat# M0308 APE1 New England Biolabs Cat# M0282 hAAG New England Biolabs Cat# M0313 Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific Cat# 13778150 Benzonase Sigma-Aldrich Cat# E1014-25KU PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat# 04906837001 Opti-MEM Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 Critical Commercial Assays AMPure XP beads Beckman Coulter Cat# A63881 NEBNext Ultra II DNA Library Prep Kit for Illumina New England Biolabs Cat# E7645 Phusion Flash high-fidelity PCR master mix Thermo Fisher Scientific Cat# F-548 Dynabeads MyOne Streptavidin C1 Life Technologies Cat# 65001 High Sensitivity D1000 ScreenTape Agilent Technologies Cat# 5067-5584 High Sensitivity D1000 ScreenTape reagents Agilent Technologies Cat# 5067-5585 RNA ScreenTape Agilent Technologies Cat# 5067-5576 RNA ScreenTape sample buffer Agilent Technologies Cat# 5067-5577 RNA ScreenTape ladder Agilent Technologies Cat# 5067-5578 Qubit dsDNA HS Assay Kit Invitrogen Cat# Q32854 Agilent Bioanalyzer High Sensitivity DNA Kit Agilent Technologies Cat# 5067-4646 (Continued on next page) e1 Molecular Cell 78, 1–11.e1–e7, June 4, 2020 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER NextSeq 550 System High-Output Kit Illumina Cat# 20024906 NextSeq 550 System Mid-Output Kit Illumina Cat# 20024904 NuSieve GTG Agarose Lonza Cat# 859081 Yeast Genomic DNA Extraction Kit QIAGEN Cat# 10243 Bio-Rad Protein Assay Dye Reagent Bio-Rad Cat# 500-0006 NuPAGE LDS sample buffer Thermo Fisher Scientific Cat# NP0007 NuPAGE 4-12% Bis-Tris Protein Gels Thermo Fisher Scientific Cat# NP0322 Deposited Data Raw and analyzed data This paper GEO: GSE134225 Human reference genome UCSC GRCh37/hg19 UCSC Genome Browser ftp://hgdownload.soe.ucsc.edu/ goldenPath/hg19/chromosomes/ Yeast reference genome UCSC sacCer3 UCSC Genome Browser ftp://hgdownload.soe.ucsc.edu/ goldenPath/sacCer3/chromosomes/ 30 end maps of Nb.BtsI-digested DNA, WT and pol1-L868M, Fastq (Figure 2A, reanalyzed with GLOE-Pipe) Reijns et al., 2015 GEO: GSM1573437, GSM1573438 Motifs for Abf1, Reb1, Rap1 in yeast, GFF (Figure 4B) MacIsaac et al., 2006 https://downloads.yeastgenome.org/ published_datasets/MacIsaac_2006_ PMID_16522208/MacIsaac_high_ confidence_with_sequence.gff Nucleosome positions in yeast, BED (Figure 4B) Whitehouse et al., 2007 https://static-content.springer.com/ esm/art%3A10.1038%2Fnature06391/ MediaObjects/41586_2007_ BFnature06391_MOESM319_ESM.xls Okazaki fragment maps in Cdc9-depleted yeast, Fastq, rep 1 rep2 (Figure 4C, reanalyzed with GLOE-Pipe) Smith and Whitehouse, 2012 GEO: GSM835650, GSM835651 END-seq data in HCT116, Fastq (Figure 5, reanalyzed with GLOE-Pipe) Tubbs et al., 2018 GEO: GSM3227952 DNase I hypersensitivity data in HCT116, BigWig, rep1 rep2 (Figure 6B) ENCODE Project Consortium, 2012 GEO: GSM736493, GSM736600 OK-Seq and S50 data in HeLa and GM06990, rep1 rep2 (BedGraph, received from Chunlong Chen, chunlong.chen@curie.fr) (Figure 6B) Petryk et al., 2016 GEO SRA: SRP065949 (SRX1427549, SRX1427548, SRX1424659, SRX1424656) Experimental Models: Cell Lines HCT116 Cancer Research UK London Research Institute Cell Services N/A HCT116 LIG3 / :mL3 Oh et al., 2014 N/A Experimental Models: Organisms/Strains S. cerevisiae: strain W303 ATCC ATCC: 201238 S. cerevisiae: strain JKM179 Lee et al., 1998 N/A S. cerevisiae: strain DF5 ATCC ATCC: 200912 S. cerevisiae: strain DF5 apn1::KanMX apn2::KanMX This paper N/A S. cerevisiae: strain cdc9AID Kubota et al., 2013 N/A Oligonucleotides Primer #3898: CTACACGACGCTCTTCCGATCTNNN NN*N-NH2 (*phosphorothioate bond, IDT code *; NH2: 3 0-amino modification, IDT code /3AmMO/) Integrated DNA Technologies N/A Primer #3899: PO4-AGATCGGAAGAGCGTCGTGTAG GGAAAGAGTG TAGATCTCGTTTT-Bio (PO4: 50-phosphorylation, IDT code /5Phos/; T-Bio: 30-biotindT, IDT code /3BiodT/) Integrated DNA Technologies N/A (Continued on next page) Molecular Cell 78, 1–11.e1–e7, June 4, 2020 e2

    Polyacrylamide Gel Electrophoresis:

    Article Title: Genome-wide Nucleotide-Resolution Mapping of DNA Replication Patterns, Single-Strand Breaks, and Lesions by GLOE-Seq.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-yeast Rad53 Susan Gasser; Hauer et al., 2017 N/A Rat monoclonal anti-tubulin, clone YL1/2 Sigma-Aldrich Cat# 92092402-1VL, RRID: CVCL_J781 Rabbit polyclonal anti-DNA Ligase 1 Elabscience Cat# E-AB-31210 Mouse monoclonal anti-Chk1, clone 2G1D5 Cell Signaling Technology Cat# 2360, RRID: AB_2080320 Rabbit polyclonal anti-phospho-Chk1 (Ser345) Cell Signaling Technology Cat# 2341, RRID: AB_330023 Goat anti-mouse immunoglobulins, HRP Dako Cat# P0447, RRID: AB_2617137 Goat anti-rabbit immunoglobulins, HRP Dako Cat# P0448, RRID: AB_2617138 Goat anti-rat immunoglobulins, HRP Dako Cat# P0450, RRID: AB_2630354 Goat anti-rat IgG Secondary Antibody, IRDye 800CW LI-COR Cat# 926-32219, RRID: AB_1850025 Chemicals, Peptides, and Recombinant Proteins Zymolyase 20T AMS Biotechnology Cat# 120491-1 T4 DNA ligase, 20,000,000 U/mL New England Biolabs Cat# B0202 Q5 High-Fidelity DNA polymerase New England Biolabs Cat# M0491 RNase A from bovine pancreas Sigma-Aldrich Cat# 10109169001 Proteinase K Roche Cat# 3115801001 Phenylmethanesulfonyl fluoride Sigma-Aldrich Cat# P7626 b-Agarase I, 1000 U/mL New England Biolabs Cat# M0392 Alpha-Factor peptide (WHWLQLKPGQPMY), > 95% ProteoGenix SAS Cat# GM-PT001 BsrDI New England Biolabs Cat# R0574 Nb.BsrDI New England Biolabs Cat# R0648 NotI New England Biolabs Cat# R0189 Nb.BtsI New England Biolabs Cat# R0707 Antarctic Phosphatase New England Biolabs Cat# M0289 T4 Endonuclease V (T4 PDG) New England Biolabs Cat# M0308 APE1 New England Biolabs Cat# M0282 hAAG New England Biolabs Cat# M0313 Lipofectamine RNAiMAX Transfection Reagent Thermo Fisher Scientific Cat# 13778150 Benzonase Sigma-Aldrich Cat# E1014-25KU PhosSTOP Phosphatase Inhibitor Sigma-Aldrich Cat# 04906837001 Opti-MEM Reduced Serum Medium Thermo Fisher Scientific Cat# 11058021 Critical Commercial Assays AMPure XP beads Beckman Coulter Cat# A63881 NEBNext Ultra II DNA Library Prep Kit for Illumina New England Biolabs Cat# E7645 Phusion Flash high-fidelity PCR master mix Thermo Fisher Scientific Cat# F-548 Dynabeads MyOne Streptavidin C1 Life Technologies Cat# 65001 High Sensitivity D1000 ScreenTape Agilent Technologies Cat# 5067-5584 High Sensitivity D1000 ScreenTape reagents Agilent Technologies Cat# 5067-5585 RNA ScreenTape Agilent Technologies Cat# 5067-5576 RNA ScreenTape sample buffer Agilent Technologies Cat# 5067-5577 RNA ScreenTape ladder Agilent Technologies Cat# 5067-5578 Qubit dsDNA HS Assay Kit Invitrogen Cat# Q32854 Agilent Bioanalyzer High Sensitivity DNA Kit Agilent Technologies Cat# 5067-4646 (Continued on next page) e1 Molecular Cell 78, 1–11.e1–e7, June 4, 2020 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER NextSeq 550 System High-Output Kit Illumina Cat# 20024906 NextSeq 550 System Mid-Output Kit Illumina Cat# 20024904 NuSieve GTG Agarose Lonza Cat# 859081 Yeast Genomic DNA Extraction Kit QIAGEN Cat# 10243 Bio-Rad Protein Assay Dye Reagent Bio-Rad Cat# 500-0006 NuPAGE LDS sample buffer Thermo Fisher Scientific Cat# NP0007 NuPAGE 4-12% Bis-Tris Protein Gels Thermo Fisher Scientific Cat# NP0322 Deposited Data Raw and analyzed data This paper GEO: GSE134225 Human reference genome UCSC GRCh37/hg19 UCSC Genome Browser ftp://hgdownload.soe.ucsc.edu/ goldenPath/hg19/chromosomes/ Yeast reference genome UCSC sacCer3 UCSC Genome Browser ftp://hgdownload.soe.ucsc.edu/ goldenPath/sacCer3/chromosomes/ 30 end maps of Nb.BtsI-digested DNA, WT and pol1-L868M, Fastq (Figure 2A, reanalyzed with GLOE-Pipe) Reijns et al., 2015 GEO: GSM1573437, GSM1573438 Motifs for Abf1, Reb1, Rap1 in yeast, GFF (Figure 4B) MacIsaac et al., 2006 https://downloads.yeastgenome.org/ published_datasets/MacIsaac_2006_ PMID_16522208/MacIsaac_high_ confidence_with_sequence.gff Nucleosome positions in yeast, BED (Figure 4B) Whitehouse et al., 2007 https://static-content.springer.com/ esm/art%3A10.1038%2Fnature06391/ MediaObjects/41586_2007_ BFnature06391_MOESM319_ESM.xls Okazaki fragment maps in Cdc9-depleted yeast, Fastq, rep 1 rep2 (Figure 4C, reanalyzed with GLOE-Pipe) Smith and Whitehouse, 2012 GEO: GSM835650, GSM835651 END-seq data in HCT116, Fastq (Figure 5, reanalyzed with GLOE-Pipe) Tubbs et al., 2018 GEO: GSM3227952 DNase I hypersensitivity data in HCT116, BigWig, rep1 rep2 (Figure 6B) ENCODE Project Consortium, 2012 GEO: GSM736493, GSM736600 OK-Seq and S50 data in HeLa and GM06990, rep1 rep2 (BedGraph, received from Chunlong Chen, chunlong.chen@curie.fr) (Figure 6B) Petryk et al., 2016 GEO SRA: SRP065949 (SRX1427549, SRX1427548, SRX1424659, SRX1424656) Experimental Models: Cell Lines HCT116 Cancer Research UK London Research Institute Cell Services N/A HCT116 LIG3 / :mL3 Oh et al., 2014 N/A Experimental Models: Organisms/Strains S. cerevisiae: strain W303 ATCC ATCC: 201238 S. cerevisiae: strain JKM179 Lee et al., 1998 N/A S. cerevisiae: strain DF5 ATCC ATCC: 200912 S. cerevisiae: strain DF5 apn1::KanMX apn2::KanMX This paper N/A S. cerevisiae: strain cdc9AID Kubota et al., 2013 N/A Oligonucleotides Primer #3898: CTACACGACGCTCTTCCGATCTNNN NN*N-NH2 (*phosphorothioate bond, IDT code *; NH2: 3 0-amino modification, IDT code /3AmMO/) Integrated DNA Technologies N/A Primer #3899: PO4-AGATCGGAAGAGCGTCGTGTAG GGAAAGAGTG TAGATCTCGTTTT-Bio (PO4: 50-phosphorylation, IDT code /5Phos/; T-Bio: 30-biotindT, IDT code /3BiodT/) Integrated DNA Technologies N/A (Continued on next page) Molecular Cell 78, 1–11.e1–e7, June 4, 2020 e2

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    New England Biolabs t4 pdg
    In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by <t>T4-PDG.</t> Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively
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    In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

    Journal: Nature Communications

    Article Title: Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions

    doi: 10.1038/s41467-019-09146-5

    Figure Lengend Snippet: In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively

    Article Snippet: We confirmed the existence of these adducts by the sensitivity of this damaged template to an enzyme called, T4-PDG (from bacteriophage T4, pyrimidine dimer glycosylase; NEB) (Fig. , third lane).

    Techniques: In Vitro, Generated, Nucleic Acid Electrophoresis, Irradiation, Labeling