supercoiled dna ladder  (New England Biolabs)


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    Name:
    Supercoiled DNA Ladder
    Description:
    Supercoiled DNA Ladder 100 gel lanes
    Catalog Number:
    n0472s
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    105
    Size:
    100 gel lanes
    Category:
    DNA Ladders
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    New England Biolabs supercoiled dna ladder
    Supercoiled DNA Ladder
    Supercoiled DNA Ladder 100 gel lanes
    https://www.bioz.com/result/supercoiled dna ladder/product/New England Biolabs
    Average 94 stars, based on 117 article reviews
    Price from $9.99 to $1999.99
    supercoiled dna ladder - by Bioz Stars, 2020-10
    94/100 stars

    Images

    1) Product Images from "Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea"

    Article Title: Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea

    Journal: Biomolecules

    doi: 10.3390/biom10071045

    Addition of PCNA stimulates DNA extension by DNA polymerases on recombination intermediates. ( A ) Schematic representation for DNA extension by family-B polymerase (PolB) or family-D polymerase (PolD) following the D-loop formation by RadA described in Figure 1 A. ( B ) Recombination-associated DNA synthesis assay. An amount of 25 nM of labeled ssDNA was first incubated with 1.6 µM RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. D-loop provided by RadA strand exchange activity was extended by 675 nM of PolB or PolD for 1 hr at 65 °C. DNA products were separated on a 1.2% native agarose gel. Same DNA products from ( B ) were separated as well in 5% denaturing acrylamide gel ( C ) or 1% denaturing alkaline agarose gel ( D ). When indicated, 675 nM of PCNA was added together with DNA polymerases. DNA products were revealed by fluorescence for HiLyte TM 647 labeled DNA. The denaturing alkaline agarose gel ( D ) was also stained by SYBR Gold to detect the DNA ladder (lane 1) and pUC19 plasmid (lane 2). For all the experiments, controls were treated as the assays (volume and incubation time), when a protein was absent it was replaced by the corresponding buffer. The two bands at the top of the gel in lanes 3 to 12 are non-specific products corresponding to incomplete denaturation of pUC19 plasmid with residual labelled ssDNA fixed on melted regions.
    Figure Legend Snippet: Addition of PCNA stimulates DNA extension by DNA polymerases on recombination intermediates. ( A ) Schematic representation for DNA extension by family-B polymerase (PolB) or family-D polymerase (PolD) following the D-loop formation by RadA described in Figure 1 A. ( B ) Recombination-associated DNA synthesis assay. An amount of 25 nM of labeled ssDNA was first incubated with 1.6 µM RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. D-loop provided by RadA strand exchange activity was extended by 675 nM of PolB or PolD for 1 hr at 65 °C. DNA products were separated on a 1.2% native agarose gel. Same DNA products from ( B ) were separated as well in 5% denaturing acrylamide gel ( C ) or 1% denaturing alkaline agarose gel ( D ). When indicated, 675 nM of PCNA was added together with DNA polymerases. DNA products were revealed by fluorescence for HiLyte TM 647 labeled DNA. The denaturing alkaline agarose gel ( D ) was also stained by SYBR Gold to detect the DNA ladder (lane 1) and pUC19 plasmid (lane 2). For all the experiments, controls were treated as the assays (volume and incubation time), when a protein was absent it was replaced by the corresponding buffer. The two bands at the top of the gel in lanes 3 to 12 are non-specific products corresponding to incomplete denaturation of pUC19 plasmid with residual labelled ssDNA fixed on melted regions.

    Techniques Used: DNA Synthesis, Labeling, Incubation, Purification, Activity Assay, Agarose Gel Electrophoresis, Acrylamide Gel Assay, Fluorescence, Staining, Plasmid Preparation

    P. abyssi RadA recombinase activity catalyzed displacement-loop (D-loop) formation. ( A ) Schematic representation for the D-loop formation assay. Labeled linear ssDNA (93 nt) was incubated first with RadA to form nucleoprotein filaments before adding the purified supercoiled plasmid pUC19 for further homology search. ( B ) D-loop formation assay with increased quantity of RadA. An amount of 25 nM of labeled ssDNA (93 nt) was incubated with RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. DNA products were separated on a 1.2% native agarose gel and visualized by fluorescence. ( C ) Histogram representation of the D-loop formation assays for a range of RadA as observed in ( B ). RadA-dependent D-loop (%), densitometry measurement of formed D-loop as a percentage of total lane densitometry after data normalization and the D-loop background from lane 3 was subtracted. Experiments were performed in triplicate.
    Figure Legend Snippet: P. abyssi RadA recombinase activity catalyzed displacement-loop (D-loop) formation. ( A ) Schematic representation for the D-loop formation assay. Labeled linear ssDNA (93 nt) was incubated first with RadA to form nucleoprotein filaments before adding the purified supercoiled plasmid pUC19 for further homology search. ( B ) D-loop formation assay with increased quantity of RadA. An amount of 25 nM of labeled ssDNA (93 nt) was incubated with RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. DNA products were separated on a 1.2% native agarose gel and visualized by fluorescence. ( C ) Histogram representation of the D-loop formation assays for a range of RadA as observed in ( B ). RadA-dependent D-loop (%), densitometry measurement of formed D-loop as a percentage of total lane densitometry after data normalization and the D-loop background from lane 3 was subtracted. Experiments were performed in triplicate.

    Techniques Used: Activity Assay, Tube Formation Assay, Labeling, Incubation, Purification, Plasmid Preparation, Agarose Gel Electrophoresis, Fluorescence

    2) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    3) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.
    Figure Legend Snippet: Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Hybridization, Staining, Generated, Irradiation

    4) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    5) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    6) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    7) Product Images from "Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2"

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt315

    Analytical gel filtration analysis of Csn2 and Csn2-DNA complexes performed with a Superdex 200 PC 3.2/30 column is shown. Elution profiles of 20 µM Csn2 ( A ), 30 nM linearized pCR001 plasmid ( B ) and Csn2-DNA complexes ( C ) are shown. In ( D ) the elution profiles of 30 nM supercoiled pCR001 alone (upper part) or in the presence of 20 µM Csn2 (lower part) are shown. 100 µl fractions were collected starting at an elution volume of 0.8 ml. Aliquots of the fractions 1 to 8, indicated by the green lines below the elution profiles, were analyzed on 10% SDS gels (A, C) and on 1% agarose gels (B, C).
    Figure Legend Snippet: Analytical gel filtration analysis of Csn2 and Csn2-DNA complexes performed with a Superdex 200 PC 3.2/30 column is shown. Elution profiles of 20 µM Csn2 ( A ), 30 nM linearized pCR001 plasmid ( B ) and Csn2-DNA complexes ( C ) are shown. In ( D ) the elution profiles of 30 nM supercoiled pCR001 alone (upper part) or in the presence of 20 µM Csn2 (lower part) are shown. 100 µl fractions were collected starting at an elution volume of 0.8 ml. Aliquots of the fractions 1 to 8, indicated by the green lines below the elution profiles, were analyzed on 10% SDS gels (A, C) and on 1% agarose gels (B, C).

    Techniques Used: Filtration, Plasmid Preparation

    Electrophoretic mobility shift assays of a radiolabeled 155 bp DNA fragment with Csn2 either in the absence ( A ) or in the presence of competitor DNA ( B and C ) are presented. In each reaction 2 nM 32 P-labeled DNA, 20 ng/µl heparin, and 10 mM CaCl 2 were employed. (A) Titration of Csn2 in the range of 0 to 1 µM is shown. (B) Csn2 binding to the radiolabeled DNA fragment was competed with indicated amounts of 2915 bp unlabeled plasmid DNA either in ScaI -linearized (lanes 3–6), supercoiled (lanes 7–10) or in relaxed (lanes 11–14) form. The concentration of Csn2 was constant at 60 nM in lanes 2–14. Lanes 1 and 2 show the control reactions, performed either in the absence of Csn2 (lane 1) or in the absence of competitor DNA (lane 2). (C) The same competition experiment as in (B) but with PvuII -, AvaII -, or HaeIII -cleaved competitor plasmid. The numbers of cleavage sites of the different endonucleases are given in the brackets. The black arrowheads indicate intermediate Csn2-DNA complexes, resulting from decomposition of the fully occupied complexes.
    Figure Legend Snippet: Electrophoretic mobility shift assays of a radiolabeled 155 bp DNA fragment with Csn2 either in the absence ( A ) or in the presence of competitor DNA ( B and C ) are presented. In each reaction 2 nM 32 P-labeled DNA, 20 ng/µl heparin, and 10 mM CaCl 2 were employed. (A) Titration of Csn2 in the range of 0 to 1 µM is shown. (B) Csn2 binding to the radiolabeled DNA fragment was competed with indicated amounts of 2915 bp unlabeled plasmid DNA either in ScaI -linearized (lanes 3–6), supercoiled (lanes 7–10) or in relaxed (lanes 11–14) form. The concentration of Csn2 was constant at 60 nM in lanes 2–14. Lanes 1 and 2 show the control reactions, performed either in the absence of Csn2 (lane 1) or in the absence of competitor DNA (lane 2). (C) The same competition experiment as in (B) but with PvuII -, AvaII -, or HaeIII -cleaved competitor plasmid. The numbers of cleavage sites of the different endonucleases are given in the brackets. The black arrowheads indicate intermediate Csn2-DNA complexes, resulting from decomposition of the fully occupied complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Labeling, Titration, Binding Assay, Plasmid Preparation, Concentration Assay

    8) Product Images from "Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR"

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2016.19

    Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.
    Figure Legend Snippet: Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Techniques Used: Plasmid Preparation, CTG Assay, Agarose Gel Electrophoresis, Electrophoresis, Concentration Assay, Spectrophotometry

    9) Product Images from "The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9"

    Article Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9

    Journal: ACS synthetic biology

    doi: 10.1021/acssynbio.7b00050

    Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.
    Figure Legend Snippet: Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    10) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.
    Figure Legend Snippet: Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Hybridization, Staining, Generated, Irradiation

    11) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    12) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    13) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    14) Product Images from "Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed"

    Article Title: Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh124

    Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).
    Figure Legend Snippet: Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).

    Techniques Used: Purification, Plasmid Preparation, Migration, Concentration Assay, Activity Assay, Incubation, Produced

    15) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    16) Product Images from "Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed"

    Article Title: Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh124

    Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).
    Figure Legend Snippet: Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).

    Techniques Used: Purification, Plasmid Preparation, Migration, Concentration Assay, Activity Assay, Incubation, Produced

    17) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    18) Product Images from "Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2"

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt315

    Analytical gel filtration analysis of Csn2 and Csn2-DNA complexes performed with a Superdex 200 PC 3.2/30 column is shown. Elution profiles of 20 µM Csn2 ( A ), 30 nM linearized pCR001 plasmid ( B ) and Csn2-DNA complexes ( C ) are shown. In ( D ) the elution profiles of 30 nM supercoiled pCR001 alone (upper part) or in the presence of 20 µM Csn2 (lower part) are shown. 100 µl fractions were collected starting at an elution volume of 0.8 ml. Aliquots of the fractions 1 to 8, indicated by the green lines below the elution profiles, were analyzed on 10% SDS gels (A, C) and on 1% agarose gels (B, C).
    Figure Legend Snippet: Analytical gel filtration analysis of Csn2 and Csn2-DNA complexes performed with a Superdex 200 PC 3.2/30 column is shown. Elution profiles of 20 µM Csn2 ( A ), 30 nM linearized pCR001 plasmid ( B ) and Csn2-DNA complexes ( C ) are shown. In ( D ) the elution profiles of 30 nM supercoiled pCR001 alone (upper part) or in the presence of 20 µM Csn2 (lower part) are shown. 100 µl fractions were collected starting at an elution volume of 0.8 ml. Aliquots of the fractions 1 to 8, indicated by the green lines below the elution profiles, were analyzed on 10% SDS gels (A, C) and on 1% agarose gels (B, C).

    Techniques Used: Filtration, Plasmid Preparation

    Electrophoretic mobility shift assays of a radiolabeled 155 bp DNA fragment with Csn2 either in the absence ( A ) or in the presence of competitor DNA ( B and C ) are presented. In each reaction 2 nM 32 P-labeled DNA, 20 ng/µl heparin, and 10 mM CaCl 2 were employed. (A) Titration of Csn2 in the range of 0 to 1 µM is shown. (B) Csn2 binding to the radiolabeled DNA fragment was competed with indicated amounts of 2915 bp unlabeled plasmid DNA either in ScaI -linearized (lanes 3–6), supercoiled (lanes 7–10) or in relaxed (lanes 11–14) form. The concentration of Csn2 was constant at 60 nM in lanes 2–14. Lanes 1 and 2 show the control reactions, performed either in the absence of Csn2 (lane 1) or in the absence of competitor DNA (lane 2). (C) The same competition experiment as in (B) but with PvuII -, AvaII -, or HaeIII -cleaved competitor plasmid. The numbers of cleavage sites of the different endonucleases are given in the brackets. The black arrowheads indicate intermediate Csn2-DNA complexes, resulting from decomposition of the fully occupied complexes.
    Figure Legend Snippet: Electrophoretic mobility shift assays of a radiolabeled 155 bp DNA fragment with Csn2 either in the absence ( A ) or in the presence of competitor DNA ( B and C ) are presented. In each reaction 2 nM 32 P-labeled DNA, 20 ng/µl heparin, and 10 mM CaCl 2 were employed. (A) Titration of Csn2 in the range of 0 to 1 µM is shown. (B) Csn2 binding to the radiolabeled DNA fragment was competed with indicated amounts of 2915 bp unlabeled plasmid DNA either in ScaI -linearized (lanes 3–6), supercoiled (lanes 7–10) or in relaxed (lanes 11–14) form. The concentration of Csn2 was constant at 60 nM in lanes 2–14. Lanes 1 and 2 show the control reactions, performed either in the absence of Csn2 (lane 1) or in the absence of competitor DNA (lane 2). (C) The same competition experiment as in (B) but with PvuII -, AvaII -, or HaeIII -cleaved competitor plasmid. The numbers of cleavage sites of the different endonucleases are given in the brackets. The black arrowheads indicate intermediate Csn2-DNA complexes, resulting from decomposition of the fully occupied complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Labeling, Titration, Binding Assay, Plasmid Preparation, Concentration Assay

    19) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    20) Product Images from "The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9"

    Article Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9

    Journal: ACS synthetic biology

    doi: 10.1021/acssynbio.7b00050

    Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.
    Figure Legend Snippet: Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    21) Product Images from "Single-Strand DNA Breaks Cause Replisome Disassembly"

    Article Title: Single-Strand DNA Breaks Cause Replisome Disassembly

    Journal: bioRxiv

    doi: 10.1101/2020.08.17.254235

    Lagging strand fork collapse in Xenopus extracts using the Tet-nick strategy. (A) Depiction of structures generated during replication of a top strand (lag collapse) plasmid in the presence of TetR. Inset, detailed representation of the three nicks and flanking tetO sites. (B) Plasmid with a bottom strand (lead) nick or top strand (lag) nick was replicated in the presence of LacR +/−TetR using egg extracts. In the absence of TetR and LacR (lanes 1-3), nicks were ligated, and replication went to completion, generating the expected open circular and supercoiled products. In the presence of LacR, but no TetR (lanes 4-6), the unprotected nicks were ligated, and a prominent theta band was generated from forks converging on the LacR array (as in (A), bottom route). In the presence of both LacR and TetR (lanes 7-9), the nicks were protected, and lag collapse occurred (as in (A), top route), generating the same collapsed product detected from lead collapse (lanes 10-12 and Figure S1D ). (C) Same as Figure 3B , except that lead products for all three nicks are shown for model iv. (D) Same as Figure 3D , except that DNA was digested with AflII (35 nt away from first nick) and run on a 10% polyacrylamide gel to improve the resolution. A significant fraction of the leading strands were extended approximately 3 nt beyond the nick site, consistent with limited strand displacement synthesis.
    Figure Legend Snippet: Lagging strand fork collapse in Xenopus extracts using the Tet-nick strategy. (A) Depiction of structures generated during replication of a top strand (lag collapse) plasmid in the presence of TetR. Inset, detailed representation of the three nicks and flanking tetO sites. (B) Plasmid with a bottom strand (lead) nick or top strand (lag) nick was replicated in the presence of LacR +/−TetR using egg extracts. In the absence of TetR and LacR (lanes 1-3), nicks were ligated, and replication went to completion, generating the expected open circular and supercoiled products. In the presence of LacR, but no TetR (lanes 4-6), the unprotected nicks were ligated, and a prominent theta band was generated from forks converging on the LacR array (as in (A), bottom route). In the presence of both LacR and TetR (lanes 7-9), the nicks were protected, and lag collapse occurred (as in (A), top route), generating the same collapsed product detected from lead collapse (lanes 10-12 and Figure S1D ). (C) Same as Figure 3B , except that lead products for all three nicks are shown for model iv. (D) Same as Figure 3D , except that DNA was digested with AflII (35 nt away from first nick) and run on a 10% polyacrylamide gel to improve the resolution. A significant fraction of the leading strands were extended approximately 3 nt beyond the nick site, consistent with limited strand displacement synthesis.

    Techniques Used: Generated, Plasmid Preparation

    22) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    23) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    24) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

    25) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Related Articles

    Concentration Assay:

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: .. CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA. .. CcdB stabilized the A592 B2 cleavage complex and inhibited the relaxation of negatively supercoiled DNA by the enzyme ( ).

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR
    Article Snippet: .. One hundred ng of each supercoiled and linearized plasmids were analyzed on a 1% native agarose gel alongside a 2-log DNA ladder and supercoiled ladder (New England BioLabs Cat N3200S and N0472S, Ipswich, MA) to confirm complete digestion and purity, and to confirm concentration. .. The concentration determined for each of these templates using the nanophotometer was used to calculate the number of copies of DNA present in each series of dilutions.

    Incubation:

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: .. Ten micrograms of supercoiled plasmid DNA were incubated in 1 × NEBuffer 4 (50 mM potassium acetate, 20 mM Tris acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT) and 1 µg ml−1 bovine serum albumin in a total volume of 30 µl. .. Five units E. coli Topoismerase I were added and the reaction mixture was incubated for 1 h at 37°C to allow complete relaxation.

    other:

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: With linear DNA as substrate, CcdB stabilization of cleavage complexes is ATP-dependent; with negatively supercoiled DNA, the presence of at least the non-hydrolysable analogue of ATP, ADPNP (5′-adenylyl β,γ-imidodiphosphate), is required ( ).

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: Gyrase with a truncated GyrA (A592 B2 ) can still perform relaxation of negatively supercoiled DNA in both an ATP-independent ( ) and ATP-dependent manner ( ) manner.

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: We suggest that the earlier observations are a consequence of the ‘bottom-up’ strand passage that occurs during relaxation of negatively supercoiled DNA by gyrase, generating intermediate 5 from 7 in .

    Agarose Gel Electrophoresis:

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR
    Article Snippet: .. One hundred ng of each supercoiled and linearized plasmids were analyzed on a 1% native agarose gel alongside a 2-log DNA ladder and supercoiled ladder (New England BioLabs Cat N3200S and N0472S, Ipswich, MA) to confirm complete digestion and purity, and to confirm concentration. .. The concentration determined for each of these templates using the nanophotometer was used to calculate the number of copies of DNA present in each series of dilutions.

    Plasmid Preparation:

    Article Title: Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2
    Article Snippet: .. Ten micrograms of supercoiled plasmid DNA were incubated in 1 × NEBuffer 4 (50 mM potassium acetate, 20 mM Tris acetate, pH 7.9, 10 mM magnesium acetate, 1 mM DTT) and 1 µg ml−1 bovine serum albumin in a total volume of 30 µl. .. Five units E. coli Topoismerase I were added and the reaction mixture was incubated for 1 h at 37°C to allow complete relaxation.

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    New England Biolabs supercoiled dna ladder
    Addition of PCNA stimulates <t>DNA</t> extension by DNA polymerases on recombination intermediates. ( A ) Schematic representation for DNA extension by family-B polymerase (PolB) or family-D polymerase (PolD) following the D-loop formation by RadA described in Figure 1 A. ( B ) Recombination-associated DNA synthesis assay. An amount of 25 nM of labeled ssDNA was first incubated with 1.6 µM RadA for 10 min at 65 °C. Then, 25 nM of purified <t>supercoiled</t> pUC19 was added and incubated for another 10 min. D-loop provided by RadA strand exchange activity was extended by 675 nM of PolB or PolD for 1 hr at 65 °C. DNA products were separated on a 1.2% native agarose gel. Same DNA products from ( B ) were separated as well in 5% denaturing acrylamide gel ( C ) or 1% denaturing alkaline agarose gel ( D ). When indicated, 675 nM of PCNA was added together with DNA polymerases. DNA products were revealed by fluorescence for HiLyte TM 647 labeled DNA. The denaturing alkaline agarose gel ( D ) was also stained by SYBR Gold to detect the DNA ladder (lane 1) and pUC19 plasmid (lane 2). For all the experiments, controls were treated as the assays (volume and incubation time), when a protein was absent it was replaced by the corresponding buffer. The two bands at the top of the gel in lanes 3 to 12 are non-specific products corresponding to incomplete denaturation of pUC19 plasmid with residual labelled ssDNA fixed on melted regions.
    Supercoiled Dna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addition of PCNA stimulates DNA extension by DNA polymerases on recombination intermediates. ( A ) Schematic representation for DNA extension by family-B polymerase (PolB) or family-D polymerase (PolD) following the D-loop formation by RadA described in Figure 1 A. ( B ) Recombination-associated DNA synthesis assay. An amount of 25 nM of labeled ssDNA was first incubated with 1.6 µM RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. D-loop provided by RadA strand exchange activity was extended by 675 nM of PolB or PolD for 1 hr at 65 °C. DNA products were separated on a 1.2% native agarose gel. Same DNA products from ( B ) were separated as well in 5% denaturing acrylamide gel ( C ) or 1% denaturing alkaline agarose gel ( D ). When indicated, 675 nM of PCNA was added together with DNA polymerases. DNA products were revealed by fluorescence for HiLyte TM 647 labeled DNA. The denaturing alkaline agarose gel ( D ) was also stained by SYBR Gold to detect the DNA ladder (lane 1) and pUC19 plasmid (lane 2). For all the experiments, controls were treated as the assays (volume and incubation time), when a protein was absent it was replaced by the corresponding buffer. The two bands at the top of the gel in lanes 3 to 12 are non-specific products corresponding to incomplete denaturation of pUC19 plasmid with residual labelled ssDNA fixed on melted regions.

    Journal: Biomolecules

    Article Title: Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea

    doi: 10.3390/biom10071045

    Figure Lengend Snippet: Addition of PCNA stimulates DNA extension by DNA polymerases on recombination intermediates. ( A ) Schematic representation for DNA extension by family-B polymerase (PolB) or family-D polymerase (PolD) following the D-loop formation by RadA described in Figure 1 A. ( B ) Recombination-associated DNA synthesis assay. An amount of 25 nM of labeled ssDNA was first incubated with 1.6 µM RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. D-loop provided by RadA strand exchange activity was extended by 675 nM of PolB or PolD for 1 hr at 65 °C. DNA products were separated on a 1.2% native agarose gel. Same DNA products from ( B ) were separated as well in 5% denaturing acrylamide gel ( C ) or 1% denaturing alkaline agarose gel ( D ). When indicated, 675 nM of PCNA was added together with DNA polymerases. DNA products were revealed by fluorescence for HiLyte TM 647 labeled DNA. The denaturing alkaline agarose gel ( D ) was also stained by SYBR Gold to detect the DNA ladder (lane 1) and pUC19 plasmid (lane 2). For all the experiments, controls were treated as the assays (volume and incubation time), when a protein was absent it was replaced by the corresponding buffer. The two bands at the top of the gel in lanes 3 to 12 are non-specific products corresponding to incomplete denaturation of pUC19 plasmid with residual labelled ssDNA fixed on melted regions.

    Article Snippet: The supercoiled DNA ladder (N0472S, NEB, Ipswich, MA, USA) was used to identify the supercoiled form of pUC19 (2686 bp) and extracted the corresponding band.

    Techniques: DNA Synthesis, Labeling, Incubation, Purification, Activity Assay, Agarose Gel Electrophoresis, Acrylamide Gel Assay, Fluorescence, Staining, Plasmid Preparation

    P. abyssi RadA recombinase activity catalyzed displacement-loop (D-loop) formation. ( A ) Schematic representation for the D-loop formation assay. Labeled linear ssDNA (93 nt) was incubated first with RadA to form nucleoprotein filaments before adding the purified supercoiled plasmid pUC19 for further homology search. ( B ) D-loop formation assay with increased quantity of RadA. An amount of 25 nM of labeled ssDNA (93 nt) was incubated with RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. DNA products were separated on a 1.2% native agarose gel and visualized by fluorescence. ( C ) Histogram representation of the D-loop formation assays for a range of RadA as observed in ( B ). RadA-dependent D-loop (%), densitometry measurement of formed D-loop as a percentage of total lane densitometry after data normalization and the D-loop background from lane 3 was subtracted. Experiments were performed in triplicate.

    Journal: Biomolecules

    Article Title: Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea

    doi: 10.3390/biom10071045

    Figure Lengend Snippet: P. abyssi RadA recombinase activity catalyzed displacement-loop (D-loop) formation. ( A ) Schematic representation for the D-loop formation assay. Labeled linear ssDNA (93 nt) was incubated first with RadA to form nucleoprotein filaments before adding the purified supercoiled plasmid pUC19 for further homology search. ( B ) D-loop formation assay with increased quantity of RadA. An amount of 25 nM of labeled ssDNA (93 nt) was incubated with RadA for 10 min at 65 °C. Then, 25 nM of purified supercoiled pUC19 was added and incubated for another 10 min. DNA products were separated on a 1.2% native agarose gel and visualized by fluorescence. ( C ) Histogram representation of the D-loop formation assays for a range of RadA as observed in ( B ). RadA-dependent D-loop (%), densitometry measurement of formed D-loop as a percentage of total lane densitometry after data normalization and the D-loop background from lane 3 was subtracted. Experiments were performed in triplicate.

    Article Snippet: The supercoiled DNA ladder (N0472S, NEB, Ipswich, MA, USA) was used to identify the supercoiled form of pUC19 (2686 bp) and extracted the corresponding band.

    Techniques: Activity Assay, Tube Formation Assay, Labeling, Incubation, Purification, Plasmid Preparation, Agarose Gel Electrophoresis, Fluorescence

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation