exoiii  (New England Biolabs)


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    Name:
    Exonuclease III E coli
    Description:
    Exonuclease III E coli 25 000 units
    Catalog Number:
    M0206L
    Price:
    248
    Category:
    Exonucleases
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    25 000 units
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    Structured Review

    New England Biolabs exoiii
    Exonuclease III E coli
    Exonuclease III E coli 25 000 units
    https://www.bioz.com/result/exoiii/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exoiii - by Bioz Stars, 2021-07
    99/100 stars

    Images

    1) Product Images from "Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure"

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku150

    Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.
    Figure Legend Snippet: Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

    Techniques Used: Purification, Standard Deviation

    2) Product Images from "Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase"

    Article Title: Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv586

    p7 destabilizes ECs and favours forward translocation of RNAP. ( A ) Stability of elongation complex with 11 nt-long transcript (EC11 A1long ) on A1long (see also Supplementary Figure S2 for stability of EC20 A1long ) After incubation in TB with 1M KCl in the absence (blue plot) or the presence of p7 (red plot) for the indicated periods of time, fractions of the ECs that remained on the beads after washing were measured. Data are mean and error bars are standard deviation from two independent experiments. ( B ) ExoIII footprinting of the front edge of RNAP in the EC11 A1long formed on A1long template. Superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown below the gels. Here and after, profiles are normalized to total radioactivity in each lane. ( C ) ExoIII footprinting of the front edge of RNAP and kinetics of the intrinsic transcript hydrolysis in the absence or the presence of p7, in EC14 ScTerm and EC22 ScTerm formed on scaffold ScTerm . Superimposed densitometry profiles of the ExoIII footprinting without p7 (blue) and with p7 (red) are shown below the gels. Lesser signal in 15 min lane with p7 is due to loading defect. ( D ) ExoIII footprinting of the front edge of RNAP, intrinsic hydrolysis (30 min) and pyrophosphorolysis (10 min) in the absence or presence of p7, in EC13 Sc1 formed on scaffold Sc1 . Superimposed densitometry profiles of the Exo III footprinting without p7 (blue) and with p7 (red) are shown below the gels.
    Figure Legend Snippet: p7 destabilizes ECs and favours forward translocation of RNAP. ( A ) Stability of elongation complex with 11 nt-long transcript (EC11 A1long ) on A1long (see also Supplementary Figure S2 for stability of EC20 A1long ) After incubation in TB with 1M KCl in the absence (blue plot) or the presence of p7 (red plot) for the indicated periods of time, fractions of the ECs that remained on the beads after washing were measured. Data are mean and error bars are standard deviation from two independent experiments. ( B ) ExoIII footprinting of the front edge of RNAP in the EC11 A1long formed on A1long template. Superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown below the gels. Here and after, profiles are normalized to total radioactivity in each lane. ( C ) ExoIII footprinting of the front edge of RNAP and kinetics of the intrinsic transcript hydrolysis in the absence or the presence of p7, in EC14 ScTerm and EC22 ScTerm formed on scaffold ScTerm . Superimposed densitometry profiles of the ExoIII footprinting without p7 (blue) and with p7 (red) are shown below the gels. Lesser signal in 15 min lane with p7 is due to loading defect. ( D ) ExoIII footprinting of the front edge of RNAP, intrinsic hydrolysis (30 min) and pyrophosphorolysis (10 min) in the absence or presence of p7, in EC13 Sc1 formed on scaffold Sc1 . Superimposed densitometry profiles of the Exo III footprinting without p7 (blue) and with p7 (red) are shown below the gels.

    Techniques Used: Translocation Assay, Incubation, Standard Deviation, Footprinting, Radioactivity

    p7 induces more efficient rewinding of the upstream DNA duplex that results in bias towards more forward translocated complexes and destabilization of EC. ( A ) ExoIII footprinting of the front edge of RNAP in the EC16 Sc1 in the absence or presence of p7. Superimposed densitometry profiles of the lanes (normalized as above) without p7 (blue) and with p7 (red) are shown below the gel. ( B ) KMnO 4 probing of elongation complex EC16 Sc1 in the absence or presence of p7 (probing of non-template strand in EC13 Sc1 is shown as a control for comparison of opening of T22 and T33; dashed line in densitometry profile). Non-template (left panel) or template (right panel) strand of the DNA was 5′ radiolabelled. Schemes of the complexes and superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown to the side of the gels. Profiles were normalized to total radioactivity in each lane in the gel. ( C ) EMSA of different DNA substrates in the absence or presence of increasing concentration of p7 ( Escherichia coli SSB used as a positive control). ( D ) Stability of EC13 formed on scaffolds with the different state of the upstream DNA duplex; fully complementary template and non-template ( Sc1 ), carrying mismatches of different lengths just upstream of the RNA–DNA hybrid, or lacking upstream portion of the non-template strand. The schemes of the assembled complexes are shown below the graph and sequences in Supplementary Figure S1. Measured is the fraction of ECs that remained bound to beads in the absence or the presence of p7 after 30 min incubation in TB with 1 M KCl and washing. Data are mean and error bars are standard deviation from three independent experiments. ( E ) Effect of mismatch in the upstream DNA duplex on termination pattern in the absence or the presence of p7. Transcription from EC14 ScTerm with fully complementary template and non-template DNA strands and EC14 ScTerm/mm4 carrying 4 nt long mismatch just upstream of the RNA–DNA hybrid of the termination complex. The scheme of scaffold with mismatch is shown next to the gel, asterisks indicate termination positions.
    Figure Legend Snippet: p7 induces more efficient rewinding of the upstream DNA duplex that results in bias towards more forward translocated complexes and destabilization of EC. ( A ) ExoIII footprinting of the front edge of RNAP in the EC16 Sc1 in the absence or presence of p7. Superimposed densitometry profiles of the lanes (normalized as above) without p7 (blue) and with p7 (red) are shown below the gel. ( B ) KMnO 4 probing of elongation complex EC16 Sc1 in the absence or presence of p7 (probing of non-template strand in EC13 Sc1 is shown as a control for comparison of opening of T22 and T33; dashed line in densitometry profile). Non-template (left panel) or template (right panel) strand of the DNA was 5′ radiolabelled. Schemes of the complexes and superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown to the side of the gels. Profiles were normalized to total radioactivity in each lane in the gel. ( C ) EMSA of different DNA substrates in the absence or presence of increasing concentration of p7 ( Escherichia coli SSB used as a positive control). ( D ) Stability of EC13 formed on scaffolds with the different state of the upstream DNA duplex; fully complementary template and non-template ( Sc1 ), carrying mismatches of different lengths just upstream of the RNA–DNA hybrid, or lacking upstream portion of the non-template strand. The schemes of the assembled complexes are shown below the graph and sequences in Supplementary Figure S1. Measured is the fraction of ECs that remained bound to beads in the absence or the presence of p7 after 30 min incubation in TB with 1 M KCl and washing. Data are mean and error bars are standard deviation from three independent experiments. ( E ) Effect of mismatch in the upstream DNA duplex on termination pattern in the absence or the presence of p7. Transcription from EC14 ScTerm with fully complementary template and non-template DNA strands and EC14 ScTerm/mm4 carrying 4 nt long mismatch just upstream of the RNA–DNA hybrid of the termination complex. The scheme of scaffold with mismatch is shown next to the gel, asterisks indicate termination positions.

    Techniques Used: Footprinting, Radioactivity, Concentration Assay, Positive Control, Incubation, Standard Deviation

    3) Product Images from "DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞"

    Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞

    Journal:

    doi: 10.1074/jbc.M806809200

    Verification of the ExoIII digestion method with sequence-selective G oxidation by and riboflavin. 32 P-Labeled double-stranded oligodeoxynucleotides containing four representative G sequence contexts () were treated with ( A ) or photo-activated
    Figure Legend Snippet: Verification of the ExoIII digestion method with sequence-selective G oxidation by and riboflavin. 32 P-Labeled double-stranded oligodeoxynucleotides containing four representative G sequence contexts () were treated with ( A ) or photo-activated

    Techniques Used: Sequencing, Labeling

    4) Product Images from "Activity of FEN1 Endonuclease on Nucleosome Substrates Is Dependent upon DNA Sequence but Not Flap Orientation *"

    Article Title: Activity of FEN1 Endonuclease on Nucleosome Substrates Is Dependent upon DNA Sequence but Not Flap Orientation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.229658

    ExoIII analysis of flap-out and flap-in 154-bp 5 S nucleosome substrates. Nucleosomes were purified and digested with ExoIII as described in the text, and cleavage products were analyzed by sequencing gels and phosphorimagery. Shown are lanes corresponding
    Figure Legend Snippet: ExoIII analysis of flap-out and flap-in 154-bp 5 S nucleosome substrates. Nucleosomes were purified and digested with ExoIII as described in the text, and cleavage products were analyzed by sequencing gels and phosphorimagery. Shown are lanes corresponding

    Techniques Used: Purification, Sequencing

    Related Articles

    Incubation:

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. The circularized DNA was concentrated to 1 μl, then mixed with 9 μl Sample buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30).

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION
    Article Snippet: To measure the amount of replicated pFLORI31 or pFLORI40, 25 μl of total genomic DNA was digested with 10 units of DpnI (New England Biolabs) for 16 hrs in a final volume of 30 μl. .. The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes. .. The primers used to amplify an 80 bp-portion of the firefly luciferase gene present on the pFLORI40 (SV40 ori), pFLORI31 (HPV31 ori) or pCI-Fluc (No ori) plasmids as well as the probe used to detect the amplicon were synthesized according to unpublished sequences validated by Dr Iain Morgan (personal communication).

    Hybridization:

    Article Title: The replication of plastid minicircles involves rolling circle intermediates
    Article Snippet: Therefore, treated DNA was then tested on the sensitivity to Exo III. .. Total H. triquetra DNA (which contained minicircle DNA as detected in the Southern hybridization) were sequentially treated with Klenow, T4 DNA ligase and Exo III. .. Presence of minicircle DNA was revealed by Southern blotting by the psbA NCR probe.

    Transfection:

    Article Title: The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA
    Article Snippet: The insoluble materials containing viral replicative intermediates and most of the cellular DNA were removed by centrifugation, and the resultant supernatant containing viral cccDNA was extracted with phenol. .. To remove the transfected linear viral DNA, the nucleic acid obtained was treated with Exonuclease III (New England Biolabs) at 37°C for 1 h, followed by treatment with mung bean nuclease (New England Biolabs) at 37°C for 30 min. ..

    other:

    Article Title: The replication of plastid minicircles involves rolling circle intermediates
    Article Snippet: Exo III catalyzes a stepwise removal of mononucleotides from the 3′-hydroxyl termini of linear duplex DNA.

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  • 86
    New England Biolabs dna preparationwt exoiii
    Coordinated gap processing and gap filling by <t>ExoIII</t> and pol I. ( A ) Representative fluorescence intensity (top, green for donor and red for acceptor) and FRET efficiency time trajectories (blue) with the characteristic features of degradation (orange region), polymerase binding (violet region), and polymerization (green region). The experiments were performed by adding ExoIII and pol I to <t>AP-DNA</t> in a solution containing Mg 2+ and dNTPs. ( B ) Representative FRET time trajectory (top two panels) and histograms (bottom) before (black) and after (gray) degradation. The FRET efficiency shift caused by degradation in the presence of pol I indicates that the gap size is approximately 5 to 6 nt long. ( C ) Representative smFRET time trajectories taken from polymerization reactions with various dNTP concentrations after the degradation reaction performed in (B). ( D ) Degradation rate and polymerization rate as a function of dNTP concentration. The degradation rate does not change, whereas the polymerization rate is strongly dependent on the dNTP concentration. The polymerization rate follows Michaelis-Menten kinetics with a maximum velocity of ~12.5 nt/s and a K m value of ~513 nM dNTP. How reaction rates are calculated is described in the Supplementary Materials.
    Dna Preparationwt Exoiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs exoiii
    Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and <t>ExoIII</t> nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.
    Exoiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exoiii/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exoiii - by Bioz Stars, 2021-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Coordinated gap processing and gap filling by ExoIII and pol I. ( A ) Representative fluorescence intensity (top, green for donor and red for acceptor) and FRET efficiency time trajectories (blue) with the characteristic features of degradation (orange region), polymerase binding (violet region), and polymerization (green region). The experiments were performed by adding ExoIII and pol I to AP-DNA in a solution containing Mg 2+ and dNTPs. ( B ) Representative FRET time trajectory (top two panels) and histograms (bottom) before (black) and after (gray) degradation. The FRET efficiency shift caused by degradation in the presence of pol I indicates that the gap size is approximately 5 to 6 nt long. ( C ) Representative smFRET time trajectories taken from polymerization reactions with various dNTP concentrations after the degradation reaction performed in (B). ( D ) Degradation rate and polymerization rate as a function of dNTP concentration. The degradation rate does not change, whereas the polymerization rate is strongly dependent on the dNTP concentration. The polymerization rate follows Michaelis-Menten kinetics with a maximum velocity of ~12.5 nt/s and a K m value of ~513 nM dNTP. How reaction rates are calculated is described in the Supplementary Materials.

    Journal: Science Advances

    Article Title: The mechanism of gap creation by a multifunctional nuclease during base excision repair

    doi: 10.1126/sciadv.abg0076

    Figure Lengend Snippet: Coordinated gap processing and gap filling by ExoIII and pol I. ( A ) Representative fluorescence intensity (top, green for donor and red for acceptor) and FRET efficiency time trajectories (blue) with the characteristic features of degradation (orange region), polymerase binding (violet region), and polymerization (green region). The experiments were performed by adding ExoIII and pol I to AP-DNA in a solution containing Mg 2+ and dNTPs. ( B ) Representative FRET time trajectory (top two panels) and histograms (bottom) before (black) and after (gray) degradation. The FRET efficiency shift caused by degradation in the presence of pol I indicates that the gap size is approximately 5 to 6 nt long. ( C ) Representative smFRET time trajectories taken from polymerization reactions with various dNTP concentrations after the degradation reaction performed in (B). ( D ) Degradation rate and polymerization rate as a function of dNTP concentration. The degradation rate does not change, whereas the polymerization rate is strongly dependent on the dNTP concentration. The polymerization rate follows Michaelis-Menten kinetics with a maximum velocity of ~12.5 nt/s and a K m value of ~513 nM dNTP. How reaction rates are calculated is described in the Supplementary Materials.

    Article Snippet: Protein and DNA preparationWT ExoIII was purchased from New England Biolabs (NEB) (catalog #M0206S), and all mutants and fluorescently labeled WT ExoIII were purified (Supplementary Materials).

    Techniques: Fluorescence, Binding Assay, Concentration Assay

    Structure of ExoIII and smFRET assay. ( A ) Twofold symmetric topology of ExoIII (PDB entry: 1AKO). ( B ) Modeled DNA-enzyme complex reconstituted in silico from ExoIII (PDB entry: 1AKO) and DNA (PDB entry: 1DE8), showing the active site (green), the binding of the protruding α helix into the DNA major groove (blue), and AP site–specific binding (red). ( C ) Experimental schematics before (left) and after (middle) degradation by ExoIII and an ssDNA mimicking the degradation product (right). ExoIII converts the dsDNA between the donor (green) and the acceptor (red) to ssDNA, resulting in an increase in FRET efficiency. ( D ) A representative FRET time trajectory under 80 nM ExoIII and 10 mM Mg 2+ , showing how the degradation time is measured. The total intensity is the sum of the donor and acceptor intensities (black, top). Green and red curves represent the donor and acceptor intensities, respectively (middle), and the blue curve represents the calculated FRET efficiency (bottom). a.u., arbitrary units. ( E ) smFRET histograms before (black) and after a 2-min reaction (red). ( F ) smFRET histograms of dsDNA (black) and ssDNA (blue) as degradation products in the absence of ExoIII.

    Journal: Science Advances

    Article Title: The mechanism of gap creation by a multifunctional nuclease during base excision repair

    doi: 10.1126/sciadv.abg0076

    Figure Lengend Snippet: Structure of ExoIII and smFRET assay. ( A ) Twofold symmetric topology of ExoIII (PDB entry: 1AKO). ( B ) Modeled DNA-enzyme complex reconstituted in silico from ExoIII (PDB entry: 1AKO) and DNA (PDB entry: 1DE8), showing the active site (green), the binding of the protruding α helix into the DNA major groove (blue), and AP site–specific binding (red). ( C ) Experimental schematics before (left) and after (middle) degradation by ExoIII and an ssDNA mimicking the degradation product (right). ExoIII converts the dsDNA between the donor (green) and the acceptor (red) to ssDNA, resulting in an increase in FRET efficiency. ( D ) A representative FRET time trajectory under 80 nM ExoIII and 10 mM Mg 2+ , showing how the degradation time is measured. The total intensity is the sum of the donor and acceptor intensities (black, top). Green and red curves represent the donor and acceptor intensities, respectively (middle), and the blue curve represents the calculated FRET efficiency (bottom). a.u., arbitrary units. ( E ) smFRET histograms before (black) and after a 2-min reaction (red). ( F ) smFRET histograms of dsDNA (black) and ssDNA (blue) as degradation products in the absence of ExoIII.

    Article Snippet: Protein and DNA preparationWT ExoIII was purchased from New England Biolabs (NEB) (catalog #M0206S), and all mutants and fluorescently labeled WT ExoIII were purified (Supplementary Materials).

    Techniques: Smfret Assay, In Silico, Binding Assay

    In the presence of Mg 2+ , ExoIII processively degrades DNA substrates containing an AP site, whereas it performs distributive degradation on dsDNA substrates lacking an AP site (fig. S2). ( A to D ) Schematics of different substrates (top), smFRET histograms [middle, before (black) and after (red) reaction], and a representative FRET time trajectory (bottom) for different substrates. ExoIII (5 nM) and Mg 2+ (10 mM) were added for the degradation reaction. ( E ) Average degradation time per nucleotide for various DNA substrates with SEM. Degradation times were determined during which FRET increases from the minimum to the maximum values. ( F ) Fraction degraded versus ExoIII concentration and single-exponential growth fits (fig. S5) for the three different substrates (AP-DNA, nicked DNA, and blunt-ended DNA), demonstrating ExoIII’s strong affinity for the AP site. The tau (τ 1 ) is a characteristic time (fig. S5). ( G ) Fraction degraded versus NaCl concentration for the three different substrates (AP-DNA, nicked AP-DNA, and nicked DNA), showing a strong ExoIII affinity for the AP site even at high NaCl concentrations. ( H ) A gel-based degradation assay for blunt-ended DNA used for the single-molecule experiments in Fig. 1 and this figure. ( I ) In the presence of Mg 2+ , the enzyme nicks by its endonuclease activity and subsequently degrades downstream of the DNA substrate by its exonuclease activity (lanes 2 to 5). Product 1 is a fragment produced by AP endonucleolytic cleavage at the AP site, whereas product 2 is the final product by exonucleolytic degradation after cleavage at the AP site. In the presence of Ca 2+ (lane 6), the enzyme cleaves at the AP site without significant further degradation.

    Journal: Science Advances

    Article Title: The mechanism of gap creation by a multifunctional nuclease during base excision repair

    doi: 10.1126/sciadv.abg0076

    Figure Lengend Snippet: In the presence of Mg 2+ , ExoIII processively degrades DNA substrates containing an AP site, whereas it performs distributive degradation on dsDNA substrates lacking an AP site (fig. S2). ( A to D ) Schematics of different substrates (top), smFRET histograms [middle, before (black) and after (red) reaction], and a representative FRET time trajectory (bottom) for different substrates. ExoIII (5 nM) and Mg 2+ (10 mM) were added for the degradation reaction. ( E ) Average degradation time per nucleotide for various DNA substrates with SEM. Degradation times were determined during which FRET increases from the minimum to the maximum values. ( F ) Fraction degraded versus ExoIII concentration and single-exponential growth fits (fig. S5) for the three different substrates (AP-DNA, nicked DNA, and blunt-ended DNA), demonstrating ExoIII’s strong affinity for the AP site. The tau (τ 1 ) is a characteristic time (fig. S5). ( G ) Fraction degraded versus NaCl concentration for the three different substrates (AP-DNA, nicked AP-DNA, and nicked DNA), showing a strong ExoIII affinity for the AP site even at high NaCl concentrations. ( H ) A gel-based degradation assay for blunt-ended DNA used for the single-molecule experiments in Fig. 1 and this figure. ( I ) In the presence of Mg 2+ , the enzyme nicks by its endonuclease activity and subsequently degrades downstream of the DNA substrate by its exonuclease activity (lanes 2 to 5). Product 1 is a fragment produced by AP endonucleolytic cleavage at the AP site, whereas product 2 is the final product by exonucleolytic degradation after cleavage at the AP site. In the presence of Ca 2+ (lane 6), the enzyme cleaves at the AP site without significant further degradation.

    Article Snippet: Protein and DNA preparationWT ExoIII was purchased from New England Biolabs (NEB) (catalog #M0206S), and all mutants and fluorescently labeled WT ExoIII were purified (Supplementary Materials).

    Techniques: Concentration Assay, Degradation Assay, Activity Assay, Produced

    ExoIII processively degrades the downstream DNA from the AP site without dissociating, and its degree of degradation strongly depends on salt concentration. ( A ) Single-turnover degradation reactions, in which no degradation is caused by free proteins rebinding from the solution, using various substrates. During the reaction, free proteins were not present, only bound proteins contributed to the reaction, and the reaction stopped if the bound proteins dissociated from the DNA. This reaction allows us to measure how many nucleotides the enzyme degrades per binding (fig. S6). ( B ) Single-turnover degradation reactions at various NaCl concentrations. Experiments were performed in the presence of 20 nM ExoIII unless otherwise mentioned. ( C ) Calibration curve of FRET efficiency and gap size measured at various salt concentrations with 10 mM Mg 2+ . ( D ) FRET values were determined by fitting degradation FRET peaks at various NaCl concentrations, as presented in (B), and the gap size was interpolated from the FRET versus gap size curve of (C).

    Journal: Science Advances

    Article Title: The mechanism of gap creation by a multifunctional nuclease during base excision repair

    doi: 10.1126/sciadv.abg0076

    Figure Lengend Snippet: ExoIII processively degrades the downstream DNA from the AP site without dissociating, and its degree of degradation strongly depends on salt concentration. ( A ) Single-turnover degradation reactions, in which no degradation is caused by free proteins rebinding from the solution, using various substrates. During the reaction, free proteins were not present, only bound proteins contributed to the reaction, and the reaction stopped if the bound proteins dissociated from the DNA. This reaction allows us to measure how many nucleotides the enzyme degrades per binding (fig. S6). ( B ) Single-turnover degradation reactions at various NaCl concentrations. Experiments were performed in the presence of 20 nM ExoIII unless otherwise mentioned. ( C ) Calibration curve of FRET efficiency and gap size measured at various salt concentrations with 10 mM Mg 2+ . ( D ) FRET values were determined by fitting degradation FRET peaks at various NaCl concentrations, as presented in (B), and the gap size was interpolated from the FRET versus gap size curve of (C).

    Article Snippet: Protein and DNA preparationWT ExoIII was purchased from New England Biolabs (NEB) (catalog #M0206S), and all mutants and fluorescently labeled WT ExoIII were purified (Supplementary Materials).

    Techniques: Concentration Assay, Binding Assay

    Single-molecule protein imaging shows that the AP site provides a strong affinity to the DNA substrate and that there is no significant bending of DNA after AP-endonucleolytic cleavage. ( A ) A catalytic mutant of ExoIII (D151N) is labeled with a Cy3 fluorophore (green star) at the N terminus via site-specific labeling by sortase. ( B ) Experimental setup for monitoring direct protein binding, where Cy3-labeled ExoIII was added to a Cy5-labeled AP-DNA substrate immobilized on a fluorescence imaging surface. ( C ) A representative trace exhibiting distinct recognition of the AP site. ( D ) FRET histogram obtained from binding events to the AP site of DNA. ( E ) FRET evolutionary tendency calibrated as a function of gap size generated during gap processing ( x stands for the gap size in number of nucleotides). ( F ) Progress in binding affinity (dissociation constant, K d ) as a function of gap size during gap creation. K d is calculated as K d = k off / k on , where k off and k on are obtained from single-exponential decay fitting on the distributions of binding and dissociation times.

    Journal: Science Advances

    Article Title: The mechanism of gap creation by a multifunctional nuclease during base excision repair

    doi: 10.1126/sciadv.abg0076

    Figure Lengend Snippet: Single-molecule protein imaging shows that the AP site provides a strong affinity to the DNA substrate and that there is no significant bending of DNA after AP-endonucleolytic cleavage. ( A ) A catalytic mutant of ExoIII (D151N) is labeled with a Cy3 fluorophore (green star) at the N terminus via site-specific labeling by sortase. ( B ) Experimental setup for monitoring direct protein binding, where Cy3-labeled ExoIII was added to a Cy5-labeled AP-DNA substrate immobilized on a fluorescence imaging surface. ( C ) A representative trace exhibiting distinct recognition of the AP site. ( D ) FRET histogram obtained from binding events to the AP site of DNA. ( E ) FRET evolutionary tendency calibrated as a function of gap size generated during gap processing ( x stands for the gap size in number of nucleotides). ( F ) Progress in binding affinity (dissociation constant, K d ) as a function of gap size during gap creation. K d is calculated as K d = k off / k on , where k off and k on are obtained from single-exponential decay fitting on the distributions of binding and dissociation times.

    Article Snippet: Protein and DNA preparationWT ExoIII was purchased from New England Biolabs (NEB) (catalog #M0206S), and all mutants and fluorescently labeled WT ExoIII were purified (Supplementary Materials).

    Techniques: Imaging, Mutagenesis, Labeling, Protein Binding, Fluorescence, Binding Assay, Generated

    Model of AP site–specific anchoring mechanism. ( A ) In silico model of ExoIII and a DNA complex showing AP site–specific binding, in which the tryptophan (Trp 212 ) indole ring stacks on the nucleotide sugar ring at the AP site (red) (fig. S14C). ( B ) The catalytic residues (green) and the coordination of the phosphate group by a metal ion induce a kink at the P─O3′ bond (red circle), inducing a nucleophilic attack on the P─O3′ bond during the enzymatic cleavage reaction. ( C ) Molecular basis of exonuclease activity. Two charged residues (Lys 121 and Asn 153 ) around the active site provide an attraction force to pull in the negatively charged phosphates along the hydrolyzable strand for a successive cleavage reaction. ( D ) ExoIII tightly anchors itself to the AP site, cleaves 5′ to the AP site, and processively digests DNA in the 3′-to-5′ direction without dissociating. The physical constraint of being at the AP site limits the gap size. ( E ) Model of BER. (a) ExoIII binds to the AP site via the tryptophan (Trp 212 ) indole ring. (b) The exonuclease anchors itself at the AP site, cleaves at the AP site, and processively digests the downstream DNA, yielding an intermediate ssDNA loop during degradation. The physical constraint of the enzyme to bind at the AP site limits the size of the gap created, and the ssDNA loop may confer resistance against enzyme activity. (c and d) Upon the first release of the 3′ end from ExoIII, pol I and ExoIII compete to occupy the 3′ end, but pol I eventually wins because of the nature of ExoIII’s distributive exonuclease activity. Thus, ExoIII achieves processive degradation from the AP site, whereas it performs distributive degradation at the 3′ side of the gap. (e) Pol I fills the gap upon loading the 3′ end and removes the AP site through its 5′ dRP lyase activity. (f) DNA ligase seals the nick.

    Journal: Science Advances

    Article Title: The mechanism of gap creation by a multifunctional nuclease during base excision repair

    doi: 10.1126/sciadv.abg0076

    Figure Lengend Snippet: Model of AP site–specific anchoring mechanism. ( A ) In silico model of ExoIII and a DNA complex showing AP site–specific binding, in which the tryptophan (Trp 212 ) indole ring stacks on the nucleotide sugar ring at the AP site (red) (fig. S14C). ( B ) The catalytic residues (green) and the coordination of the phosphate group by a metal ion induce a kink at the P─O3′ bond (red circle), inducing a nucleophilic attack on the P─O3′ bond during the enzymatic cleavage reaction. ( C ) Molecular basis of exonuclease activity. Two charged residues (Lys 121 and Asn 153 ) around the active site provide an attraction force to pull in the negatively charged phosphates along the hydrolyzable strand for a successive cleavage reaction. ( D ) ExoIII tightly anchors itself to the AP site, cleaves 5′ to the AP site, and processively digests DNA in the 3′-to-5′ direction without dissociating. The physical constraint of being at the AP site limits the gap size. ( E ) Model of BER. (a) ExoIII binds to the AP site via the tryptophan (Trp 212 ) indole ring. (b) The exonuclease anchors itself at the AP site, cleaves at the AP site, and processively digests the downstream DNA, yielding an intermediate ssDNA loop during degradation. The physical constraint of the enzyme to bind at the AP site limits the size of the gap created, and the ssDNA loop may confer resistance against enzyme activity. (c and d) Upon the first release of the 3′ end from ExoIII, pol I and ExoIII compete to occupy the 3′ end, but pol I eventually wins because of the nature of ExoIII’s distributive exonuclease activity. Thus, ExoIII achieves processive degradation from the AP site, whereas it performs distributive degradation at the 3′ side of the gap. (e) Pol I fills the gap upon loading the 3′ end and removes the AP site through its 5′ dRP lyase activity. (f) DNA ligase seals the nick.

    Article Snippet: Protein and DNA preparationWT ExoIII was purchased from New England Biolabs (NEB) (catalog #M0206S), and all mutants and fluorescently labeled WT ExoIII were purified (Supplementary Materials).

    Techniques: In Silico, Binding Assay, Activity Assay

    Processive degradation in the presence of Mg 2+ , directly monitored by Cy3-labeled WT ExoIII and Cy5-labeled AP-DNA. ( A ) Representative fluorescence intensity (top, green for donor and red for acceptor) and FRET efficiency (second and below) time trajectories show a gradual FRET increase during gap creation. ( B ) Histogram obtained from the final FRET values. ( C ) Histogram of total degradation times. ( D ) Histogram of binding times.

    Journal: Science Advances

    Article Title: The mechanism of gap creation by a multifunctional nuclease during base excision repair

    doi: 10.1126/sciadv.abg0076

    Figure Lengend Snippet: Processive degradation in the presence of Mg 2+ , directly monitored by Cy3-labeled WT ExoIII and Cy5-labeled AP-DNA. ( A ) Representative fluorescence intensity (top, green for donor and red for acceptor) and FRET efficiency (second and below) time trajectories show a gradual FRET increase during gap creation. ( B ) Histogram obtained from the final FRET values. ( C ) Histogram of total degradation times. ( D ) Histogram of binding times.

    Article Snippet: Protein and DNA preparationWT ExoIII was purchased from New England Biolabs (NEB) (catalog #M0206S), and all mutants and fluorescently labeled WT ExoIII were purified (Supplementary Materials).

    Techniques: Labeling, Fluorescence, Binding Assay

    Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    doi: 10.1093/nar/gku150

    Figure Lengend Snippet: Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

    Article Snippet: Reactions were carried out in an initial volume of 50 μl with 10 nM nucleosomes and 50 U/ml of ExoIII (New England Biolabs) in 20 mM Tris, 0.5 mM MgCl2 , pH 8.0, at 16°C.

    Techniques: Purification, Standard Deviation

    p7 destabilizes ECs and favours forward translocation of RNAP. ( A ) Stability of elongation complex with 11 nt-long transcript (EC11 A1long ) on A1long (see also Supplementary Figure S2 for stability of EC20 A1long ) After incubation in TB with 1M KCl in the absence (blue plot) or the presence of p7 (red plot) for the indicated periods of time, fractions of the ECs that remained on the beads after washing were measured. Data are mean and error bars are standard deviation from two independent experiments. ( B ) ExoIII footprinting of the front edge of RNAP in the EC11 A1long formed on A1long template. Superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown below the gels. Here and after, profiles are normalized to total radioactivity in each lane. ( C ) ExoIII footprinting of the front edge of RNAP and kinetics of the intrinsic transcript hydrolysis in the absence or the presence of p7, in EC14 ScTerm and EC22 ScTerm formed on scaffold ScTerm . Superimposed densitometry profiles of the ExoIII footprinting without p7 (blue) and with p7 (red) are shown below the gels. Lesser signal in 15 min lane with p7 is due to loading defect. ( D ) ExoIII footprinting of the front edge of RNAP, intrinsic hydrolysis (30 min) and pyrophosphorolysis (10 min) in the absence or presence of p7, in EC13 Sc1 formed on scaffold Sc1 . Superimposed densitometry profiles of the Exo III footprinting without p7 (blue) and with p7 (red) are shown below the gels.

    Journal: Nucleic Acids Research

    Article Title: Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase

    doi: 10.1093/nar/gkv586

    Figure Lengend Snippet: p7 destabilizes ECs and favours forward translocation of RNAP. ( A ) Stability of elongation complex with 11 nt-long transcript (EC11 A1long ) on A1long (see also Supplementary Figure S2 for stability of EC20 A1long ) After incubation in TB with 1M KCl in the absence (blue plot) or the presence of p7 (red plot) for the indicated periods of time, fractions of the ECs that remained on the beads after washing were measured. Data are mean and error bars are standard deviation from two independent experiments. ( B ) ExoIII footprinting of the front edge of RNAP in the EC11 A1long formed on A1long template. Superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown below the gels. Here and after, profiles are normalized to total radioactivity in each lane. ( C ) ExoIII footprinting of the front edge of RNAP and kinetics of the intrinsic transcript hydrolysis in the absence or the presence of p7, in EC14 ScTerm and EC22 ScTerm formed on scaffold ScTerm . Superimposed densitometry profiles of the ExoIII footprinting without p7 (blue) and with p7 (red) are shown below the gels. Lesser signal in 15 min lane with p7 is due to loading defect. ( D ) ExoIII footprinting of the front edge of RNAP, intrinsic hydrolysis (30 min) and pyrophosphorolysis (10 min) in the absence or presence of p7, in EC13 Sc1 formed on scaffold Sc1 . Superimposed densitometry profiles of the Exo III footprinting without p7 (blue) and with p7 (red) are shown below the gels.

    Article Snippet: ECs obtained as above were treated with 100 units of ExoIII (New England Biolabs) 15 μl of TB for 10 min (A1long ) or 1 (scaffolds) min.

    Techniques: Translocation Assay, Incubation, Standard Deviation, Footprinting, Radioactivity

    p7 induces more efficient rewinding of the upstream DNA duplex that results in bias towards more forward translocated complexes and destabilization of EC. ( A ) ExoIII footprinting of the front edge of RNAP in the EC16 Sc1 in the absence or presence of p7. Superimposed densitometry profiles of the lanes (normalized as above) without p7 (blue) and with p7 (red) are shown below the gel. ( B ) KMnO 4 probing of elongation complex EC16 Sc1 in the absence or presence of p7 (probing of non-template strand in EC13 Sc1 is shown as a control for comparison of opening of T22 and T33; dashed line in densitometry profile). Non-template (left panel) or template (right panel) strand of the DNA was 5′ radiolabelled. Schemes of the complexes and superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown to the side of the gels. Profiles were normalized to total radioactivity in each lane in the gel. ( C ) EMSA of different DNA substrates in the absence or presence of increasing concentration of p7 ( Escherichia coli SSB used as a positive control). ( D ) Stability of EC13 formed on scaffolds with the different state of the upstream DNA duplex; fully complementary template and non-template ( Sc1 ), carrying mismatches of different lengths just upstream of the RNA–DNA hybrid, or lacking upstream portion of the non-template strand. The schemes of the assembled complexes are shown below the graph and sequences in Supplementary Figure S1. Measured is the fraction of ECs that remained bound to beads in the absence or the presence of p7 after 30 min incubation in TB with 1 M KCl and washing. Data are mean and error bars are standard deviation from three independent experiments. ( E ) Effect of mismatch in the upstream DNA duplex on termination pattern in the absence or the presence of p7. Transcription from EC14 ScTerm with fully complementary template and non-template DNA strands and EC14 ScTerm/mm4 carrying 4 nt long mismatch just upstream of the RNA–DNA hybrid of the termination complex. The scheme of scaffold with mismatch is shown next to the gel, asterisks indicate termination positions.

    Journal: Nucleic Acids Research

    Article Title: Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase

    doi: 10.1093/nar/gkv586

    Figure Lengend Snippet: p7 induces more efficient rewinding of the upstream DNA duplex that results in bias towards more forward translocated complexes and destabilization of EC. ( A ) ExoIII footprinting of the front edge of RNAP in the EC16 Sc1 in the absence or presence of p7. Superimposed densitometry profiles of the lanes (normalized as above) without p7 (blue) and with p7 (red) are shown below the gel. ( B ) KMnO 4 probing of elongation complex EC16 Sc1 in the absence or presence of p7 (probing of non-template strand in EC13 Sc1 is shown as a control for comparison of opening of T22 and T33; dashed line in densitometry profile). Non-template (left panel) or template (right panel) strand of the DNA was 5′ radiolabelled. Schemes of the complexes and superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown to the side of the gels. Profiles were normalized to total radioactivity in each lane in the gel. ( C ) EMSA of different DNA substrates in the absence or presence of increasing concentration of p7 ( Escherichia coli SSB used as a positive control). ( D ) Stability of EC13 formed on scaffolds with the different state of the upstream DNA duplex; fully complementary template and non-template ( Sc1 ), carrying mismatches of different lengths just upstream of the RNA–DNA hybrid, or lacking upstream portion of the non-template strand. The schemes of the assembled complexes are shown below the graph and sequences in Supplementary Figure S1. Measured is the fraction of ECs that remained bound to beads in the absence or the presence of p7 after 30 min incubation in TB with 1 M KCl and washing. Data are mean and error bars are standard deviation from three independent experiments. ( E ) Effect of mismatch in the upstream DNA duplex on termination pattern in the absence or the presence of p7. Transcription from EC14 ScTerm with fully complementary template and non-template DNA strands and EC14 ScTerm/mm4 carrying 4 nt long mismatch just upstream of the RNA–DNA hybrid of the termination complex. The scheme of scaffold with mismatch is shown next to the gel, asterisks indicate termination positions.

    Article Snippet: ECs obtained as above were treated with 100 units of ExoIII (New England Biolabs) 15 μl of TB for 10 min (A1long ) or 1 (scaffolds) min.

    Techniques: Footprinting, Radioactivity, Concentration Assay, Positive Control, Incubation, Standard Deviation

    RSC1 and RSC2 slide Widom 601 H3 R40A mononucleosomes similarly. (A) Comparative sliding of RSC1 and RSC2 complexes (10 nM) on H3 R40A 205 bp Widom 601 yeast mononucleosomes (20 nM). The nucleosomal Start (green), Slid (blue), and free DNA (grey) bands were quantified and reported as a percent of the total signal. (B) Mapping the positions of the wild-type and H3 R40A 205 bp Widom 601 yeast mononucleosomes by ExoIII and S1 treatment as in Figure 3 -figure supplement 3. Partially unwrapped nucleosome fragments (≤120 bp) are depicted in blue. More fully wrapped nucleosome fragments (≥128 bp) are in black. Averages from two technical replicates. Figure 4 - figure supplement 5B - Source Data 1. 205 bp Widom 601 nucleosome mapping.

    Journal: bioRxiv

    Article Title: Specialization of the Chromatin Remodeler RSC to Mobilize Partially-Unwrapped Nucleosomes

    doi: 10.1101/799361

    Figure Lengend Snippet: RSC1 and RSC2 slide Widom 601 H3 R40A mononucleosomes similarly. (A) Comparative sliding of RSC1 and RSC2 complexes (10 nM) on H3 R40A 205 bp Widom 601 yeast mononucleosomes (20 nM). The nucleosomal Start (green), Slid (blue), and free DNA (grey) bands were quantified and reported as a percent of the total signal. (B) Mapping the positions of the wild-type and H3 R40A 205 bp Widom 601 yeast mononucleosomes by ExoIII and S1 treatment as in Figure 3 -figure supplement 3. Partially unwrapped nucleosome fragments (≤120 bp) are depicted in blue. More fully wrapped nucleosome fragments (≥128 bp) are in black. Averages from two technical replicates. Figure 4 - figure supplement 5B - Source Data 1. 205 bp Widom 601 nucleosome mapping.

    Article Snippet: Approximately 400-800 fmol of nucleosomes purified from sucrose gradients was digested with a 5-25U titration of ExoIII enzyme (New England Biolabs) for 1, 2, or 3 min at 37°C in ExoIII Buffer (10 mM Tris [pH 8], 50 mM NaCl, 3 mM MgCl2).

    Techniques:

    Mapping the positions of the wt and H3 R40A 174 bp sea urchin 5S yeast mononucleosomes. Nucleo- some mapping by ExoIII and S1 treatment followed by paired-end next-generation sequencing. Experiment conducted once. Partially wrapped nucleosome fragments (≤120 bp) are depicted in blue. More fully wrapped nucleosome fragments (≥128 bp) are in black. Figure 3 - figure supplement 5 - Source Data 1. 174 bp 5S nucleosome mapping.

    Journal: bioRxiv

    Article Title: Specialization of the Chromatin Remodeler RSC to Mobilize Partially-Unwrapped Nucleosomes

    doi: 10.1101/799361

    Figure Lengend Snippet: Mapping the positions of the wt and H3 R40A 174 bp sea urchin 5S yeast mononucleosomes. Nucleo- some mapping by ExoIII and S1 treatment followed by paired-end next-generation sequencing. Experiment conducted once. Partially wrapped nucleosome fragments (≤120 bp) are depicted in blue. More fully wrapped nucleosome fragments (≥128 bp) are in black. Figure 3 - figure supplement 5 - Source Data 1. 174 bp 5S nucleosome mapping.

    Article Snippet: Approximately 400-800 fmol of nucleosomes purified from sucrose gradients was digested with a 5-25U titration of ExoIII enzyme (New England Biolabs) for 1, 2, or 3 min at 37°C in ExoIII Buffer (10 mM Tris [pH 8], 50 mM NaCl, 3 mM MgCl2).

    Techniques: Next-Generation Sequencing