supercoiled dna  (New England Biolabs)


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    Structured Review

    New England Biolabs supercoiled dna
    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 <t>supercoiled</t> 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 <t>DNA</t> 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.
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    Images

    1) 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

    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 "Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines"

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006445

    Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P
    Figure Legend Snippet: Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P

    Techniques Used: BAC Assay, Sequencing, Activity Assay, Luciferase, Marker, Cell Culture, Transfection, Standard Deviation

    The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.
    Figure Legend Snippet: The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Techniques Used: Selection, Marker, FACS, Transfection, BAC Assay, Electroporation, Incubation, Fluorescence, Flow Cytometry, Cytometry

    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 domain insertion in Escherichia coli GyrB adopts a novel fold that plays a critical role in gyrase function"

    Article Title: A domain insertion in Escherichia coli GyrB adopts a novel fold that plays a critical role in gyrase function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq665

    Comparison of wild-type and GyrB Δinsert gyrase activities. Negative supercoiling assays ( A ), positive supercoil relaxation assays ( B ), negative supercoil relaxation assays ( C ) and ciprofloxacin-dependent DNA cleavage assays ( D ) were carried out as described (‘Materials and Methods’ section). The enzyme (wild-type or GyrB Δinsert gyrase) used in each titration is shown at left. Positions of relaxed and supercoiled species are indicated at right. Lanes with negatively supercoiled (–SC) or BamHI-linearized (L) plasmid standards are indicated, as are the concentrations of gyrase tetramer (in nM). Asterisks indicate lanes in each pair of gels with comparable activities. In the cleavage assay ( D ), the rightmost lane contains a DNA ladder (O’GeneRuler, Fermentas) with the standard sizes (in kb) indicated.
    Figure Legend Snippet: Comparison of wild-type and GyrB Δinsert gyrase activities. Negative supercoiling assays ( A ), positive supercoil relaxation assays ( B ), negative supercoil relaxation assays ( C ) and ciprofloxacin-dependent DNA cleavage assays ( D ) were carried out as described (‘Materials and Methods’ section). The enzyme (wild-type or GyrB Δinsert gyrase) used in each titration is shown at left. Positions of relaxed and supercoiled species are indicated at right. Lanes with negatively supercoiled (–SC) or BamHI-linearized (L) plasmid standards are indicated, as are the concentrations of gyrase tetramer (in nM). Asterisks indicate lanes in each pair of gels with comparable activities. In the cleavage assay ( D ), the rightmost lane contains a DNA ladder (O’GeneRuler, Fermentas) with the standard sizes (in kb) indicated.

    Techniques Used: Titration, Plasmid Preparation, Cleavage Assay

    GyrB H669E activities. Negative supercoiling assays ( A ) and ciprofloxacin-dependent DNA cleavage assays ( B ) are shown and labeled with the enzyme (wild-type or GyrB-H669E gyrase) used in each titration. Positions of relaxed and supercoiled species are indicated, as are lanes with negatively supercoiled (–SC) or BamHI-linearized (L) plasmid standards. The concentrations of gyrase tetramer (in nM) are also shown. Asterisks indicate lanes with comparable activity for each pair of gels. In the cleavage assay (B), the rightmost lane contains a DNA ladder (O’GeneRuler, Fermentas) with the standard sizes (in kb) indicated. ( C ) DNA binding. Arrows indicate the positions of free DNA (F) and protein-DNA complex (C) in the EMSA. Values indicate the amount of tetramer in nM. ( D ) Basal ATPase activity. The ATPase activities of wild-type (circles) and GyrB–H669E (triangles) gyrase are plotted as a function of ATP concentration and fit to a standard Michaelis–Menten kinetic model. ( E ) The ATPase activities of wild-type (circles) and GyrB–H669E (triangles) gyrase at a constant ATP concentration are plotted as a function of increasing amounts of sheared salmon sperm DNA. Data are fit to a single-site binding equation.
    Figure Legend Snippet: GyrB H669E activities. Negative supercoiling assays ( A ) and ciprofloxacin-dependent DNA cleavage assays ( B ) are shown and labeled with the enzyme (wild-type or GyrB-H669E gyrase) used in each titration. Positions of relaxed and supercoiled species are indicated, as are lanes with negatively supercoiled (–SC) or BamHI-linearized (L) plasmid standards. The concentrations of gyrase tetramer (in nM) are also shown. Asterisks indicate lanes with comparable activity for each pair of gels. In the cleavage assay (B), the rightmost lane contains a DNA ladder (O’GeneRuler, Fermentas) with the standard sizes (in kb) indicated. ( C ) DNA binding. Arrows indicate the positions of free DNA (F) and protein-DNA complex (C) in the EMSA. Values indicate the amount of tetramer in nM. ( D ) Basal ATPase activity. The ATPase activities of wild-type (circles) and GyrB–H669E (triangles) gyrase are plotted as a function of ATP concentration and fit to a standard Michaelis–Menten kinetic model. ( E ) The ATPase activities of wild-type (circles) and GyrB–H669E (triangles) gyrase at a constant ATP concentration are plotted as a function of increasing amounts of sheared salmon sperm DNA. Data are fit to a single-site binding equation.

    Techniques Used: Labeling, Titration, Plasmid Preparation, Activity Assay, Cleavage Assay, Binding Assay, Concentration Assay

    6) 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

    7) 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

    8) 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

    9) Product Images from "Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues"

    Article Title: Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2022.e08879

    Electrophoretic evaluation of plasmids. (a) Electrophoresis of 10 μL of minipep eluates from first to fifth passage linearized with Not I restriction enzyme. Plasmid lengths: pORF-mIL-12 (p40p35) - 4833 bp, pORF-mIL-12-ORT - 4380 bp, pORF-caIL-12-ORT - 4293 bp, pORF-hIL-12-ORT - 4515 bp. 1% agarose pre-stained with 1× SYBR tm Safe, 100 V, 1 h, LL, linear ladder: GeneRuler DNA ladder mix. Lanes 1–5, passages. (b) Electrophoresis of 200 ng of uncut plasmids isolated from first to fifth passage. 1% agarose, 100 V, 1 h, stained in 1× SYBR tm Gold. SCL, supercoiled ladder: Supercoiled DNA Ladder. SCm, supercoiled monomer. SCd, supercoiled dimer, SCt, supercoiled topoisomers. Lanes 1–5, passages (c) pORF-hIL-12-ORT plasmid incubated with increasing concentrations of SYBR tm Safe (0, 0.5×, 1× and 2×). SCL, supercoiled ladder: Supercoiled DNA Ladder.
    Figure Legend Snippet: Electrophoretic evaluation of plasmids. (a) Electrophoresis of 10 μL of minipep eluates from first to fifth passage linearized with Not I restriction enzyme. Plasmid lengths: pORF-mIL-12 (p40p35) - 4833 bp, pORF-mIL-12-ORT - 4380 bp, pORF-caIL-12-ORT - 4293 bp, pORF-hIL-12-ORT - 4515 bp. 1% agarose pre-stained with 1× SYBR tm Safe, 100 V, 1 h, LL, linear ladder: GeneRuler DNA ladder mix. Lanes 1–5, passages. (b) Electrophoresis of 200 ng of uncut plasmids isolated from first to fifth passage. 1% agarose, 100 V, 1 h, stained in 1× SYBR tm Gold. SCL, supercoiled ladder: Supercoiled DNA Ladder. SCm, supercoiled monomer. SCd, supercoiled dimer, SCt, supercoiled topoisomers. Lanes 1–5, passages (c) pORF-hIL-12-ORT plasmid incubated with increasing concentrations of SYBR tm Safe (0, 0.5×, 1× and 2×). SCL, supercoiled ladder: Supercoiled DNA Ladder.

    Techniques Used: Electrophoresis, Plasmid Preparation, Staining, Isolation, Incubation

    10) 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

    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 "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

    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 "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

    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 "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

    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 "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

    21) 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

    22) Product Images from "Non-Clinical In Vitro Evaluation of Antibiotic Resistance Gene-Free Plasmids Encoding Human or Murine IL-12 Intended for First-in-Human Clinical Study"

    Article Title: Non-Clinical In Vitro Evaluation of Antibiotic Resistance Gene-Free Plasmids Encoding Human or Murine IL-12 Intended for First-in-Human Clinical Study

    Journal: Pharmaceutics

    doi: 10.3390/pharmaceutics13101739

    Construction and confirmation of the p21-mIL-12-ORT plasmid. ( a ) Cloning plan created using the SnapGene software: the expression cassette carrying the murine IL-12 sequence under the EF1/HTLV promoter was cut out of the pORF-mIL-12 (p40:p35) plasmid with the Not I and Swa I (blunt end) restriction enzymes and ligated to the pCR-blunt psiCAT plasmid cut with Not I and Pml I (blunt end). In the resulting plasmid, the promoter region was replaced with the p21 promoter from the p21-hIL-12-ORT plasmid using the Not I and Sal I restriction enzymes. The chloramphenicol antibiotic resistance gene (CmR) was then removed from the p21-mIL-12-Xmark plasmid using the ORT technology, resulting in the p21-mIL-12-ORT plasmid. ( b ) Annotated plasmid map: p21 promoter–promoter region from the native human p21 (CDKN1A) gene, mIL-12 (p40:p35) murine IL-12 intronless open reading frame consisting of the IL-12b (p40, beta subunit) and IL-12a (p35, alpha subunit) genes, SV40 polyA-simian virus 40 late polyadenylation signal, ORI- E. coli origin of replication, LacP lactose operon promoter with the lacO operator. ( c , d ) Restriction analysis: ( c ) the plasmid was cut with different combinations of restriction enzymes and its identity was confirmed by positive matching of the pattern of bands on the electrophoresis gel to the expected ( d ) pattern obtained by means of a simulation experiment using the SnapGene software. For the uncut plasmid, the simulated band pattern differs from the actual pattern because simulation can only be done for a supercoiled monomer, while other forms (supercoiled dimer, open circular, linear, nicked) can also be seen on the electrophoretic gel. Electrophoresis details: 1% agarose (Sigma-Aldrich), run for 45 min at 100 V/cm, stained in 1× Sybr Gold (Thermo Fisher Scientific). LL (linear DNA ladder): GeneRuler™ 1 kb Plus DNA Ladder (Thermo Fisher Scientific), lane 1: HindIII + MunI (2338 bp, 1792 bp, 1338 bp, 409 bp, 79 bp, 19 bp), lane 2: HindIII (3130 bp, 2338 bp, 409 bp, 79 bp, 19 bp), lane 3: KpnI (3936 bp, 2039 bp), lane 4: NcoI + Alw44I (3633 bp, 2342 bp), lane 5: BamHI + XbaI (3150 bp, 1652 bp, 1173 bp), lane 6: KpnI + Alw44I (3936 bp, 1314 bp, 725 bp), lane 7: NotI + Alw44I (4787 bp, 1188 bp), lane 8: uncut (supercoiled 5975 bp), SC (supercoiled DNA ladder): Supercoiled DNA Ladder (New England BioLabs, Ipswich, MA, USA).
    Figure Legend Snippet: Construction and confirmation of the p21-mIL-12-ORT plasmid. ( a ) Cloning plan created using the SnapGene software: the expression cassette carrying the murine IL-12 sequence under the EF1/HTLV promoter was cut out of the pORF-mIL-12 (p40:p35) plasmid with the Not I and Swa I (blunt end) restriction enzymes and ligated to the pCR-blunt psiCAT plasmid cut with Not I and Pml I (blunt end). In the resulting plasmid, the promoter region was replaced with the p21 promoter from the p21-hIL-12-ORT plasmid using the Not I and Sal I restriction enzymes. The chloramphenicol antibiotic resistance gene (CmR) was then removed from the p21-mIL-12-Xmark plasmid using the ORT technology, resulting in the p21-mIL-12-ORT plasmid. ( b ) Annotated plasmid map: p21 promoter–promoter region from the native human p21 (CDKN1A) gene, mIL-12 (p40:p35) murine IL-12 intronless open reading frame consisting of the IL-12b (p40, beta subunit) and IL-12a (p35, alpha subunit) genes, SV40 polyA-simian virus 40 late polyadenylation signal, ORI- E. coli origin of replication, LacP lactose operon promoter with the lacO operator. ( c , d ) Restriction analysis: ( c ) the plasmid was cut with different combinations of restriction enzymes and its identity was confirmed by positive matching of the pattern of bands on the electrophoresis gel to the expected ( d ) pattern obtained by means of a simulation experiment using the SnapGene software. For the uncut plasmid, the simulated band pattern differs from the actual pattern because simulation can only be done for a supercoiled monomer, while other forms (supercoiled dimer, open circular, linear, nicked) can also be seen on the electrophoretic gel. Electrophoresis details: 1% agarose (Sigma-Aldrich), run for 45 min at 100 V/cm, stained in 1× Sybr Gold (Thermo Fisher Scientific). LL (linear DNA ladder): GeneRuler™ 1 kb Plus DNA Ladder (Thermo Fisher Scientific), lane 1: HindIII + MunI (2338 bp, 1792 bp, 1338 bp, 409 bp, 79 bp, 19 bp), lane 2: HindIII (3130 bp, 2338 bp, 409 bp, 79 bp, 19 bp), lane 3: KpnI (3936 bp, 2039 bp), lane 4: NcoI + Alw44I (3633 bp, 2342 bp), lane 5: BamHI + XbaI (3150 bp, 1652 bp, 1173 bp), lane 6: KpnI + Alw44I (3936 bp, 1314 bp, 725 bp), lane 7: NotI + Alw44I (4787 bp, 1188 bp), lane 8: uncut (supercoiled 5975 bp), SC (supercoiled DNA ladder): Supercoiled DNA Ladder (New England BioLabs, Ipswich, MA, USA).

    Techniques Used: Plasmid Preparation, Clone Assay, Software, Expressing, Sequencing, Polymerase Chain Reaction, Electrophoresis, Nucleic Acid Electrophoresis, Staining

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    New England Biolabs supercoiled dna
    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 <t>supercoiled</t> 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 <t>DNA</t> 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.
    Supercoiled Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: bioRxiv

    Article Title: Single-Strand DNA Breaks Cause Replisome Disassembly

    doi: 10.1101/2020.08.17.254235

    Figure Lengend 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.

    Article Snippet: Plasmids were then run on 0.8% agarose gels and the supercoiled DNA was extracted via electroelution.

    Techniques: Generated, Plasmid Preparation

    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: Gyrase with the GyrB ATPase domain truncated (A2 B472 ) can still relax negatively supercoiled DNA in an ATP-independent reaction ( , ).

    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: Gyrase with the GyrB ATPase domain truncated (A2 B472 ) can still relax negatively supercoiled DNA in an ATP-independent reaction ( , ).

    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: Gyrase with the GyrB ATPase domain truncated (A2 B472 ) can still relax negatively supercoiled DNA in an ATP-independent reaction ( , ).

    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: Gyrase with the GyrB ATPase domain truncated (A2 B472 ) can still relax negatively supercoiled DNA in an ATP-independent reaction ( , ).

    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: Gyrase with the GyrB ATPase domain truncated (A2 B472 ) can still relax negatively supercoiled DNA in an ATP-independent reaction ( , ).

    Techniques: Incubation

    Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P

    Journal: PLoS ONE

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    doi: 10.1371/journal.pone.0006445

    Figure Lengend Snippet: Tetracycline inducibility of BAC E11-IGR-β-catenin-ERα. As a gene of interest, the coding sequence of β-catenin-ERα fusion protein is engineered into BAC. (A) The activity of firefly luciferase, a surrogate marker is assayed to reflect the activity of P tet bi. HTB56 cells cultured in 6-well plates (300,000 per well) were cotransfected with 1 µg supercoiled BAC E11-IGR-β-catenin-ERα and 0.1 µg of renilla luciferase-encoding pRLSV40 DNA by lipofectamine reagent. Cells were left untreated or exposed to indicated concentrations of Tet or Dox 24 hours after transfection and luciferase activity was measured in cell extracts 24 hours later. Data from 3 independent experiments are represented as means plus standard deviation (SD). The SD values are too small to be visible in the first five bars. The relative luciferase activity of Tet and Dox untreated cells were set as 1 arbitrarily (* P

    Article Snippet: Before transfection into 32D cells, the supercoiled BAC DNA was linearized by digestion with I-Sce I restriction enzyme (New England Biolabs, Ipswich, MA, USA).

    Techniques: BAC Assay, Sequencing, Activity Assay, Luciferase, Marker, Cell Culture, Transfection, Standard Deviation

    The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Journal: PLoS ONE

    Article Title: Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

    doi: 10.1371/journal.pone.0006445

    Figure Lengend Snippet: The EGFP is an effective selection marker for FACS. (A) 32D cells were mock transfected or transfected with I- Sce I-linearized BAC E11-IGR-β-catenin-ERα DNA by electroporation. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. The EGFP fluorescence was compared by flow cytometry. In the range of the indicated marker, there are 0.84, 7.97 and 67.76% of EGFP positive cells in mock, unsorted and sorted cell populations respectively. (B) HTB56 cells were mock transfected or transfected with supercoiled BAC E11-IGR-β-catenin-ERα DNA by lipofectamine reagent. The BAC-transfected cells were sorted by FACS 24 hours after transfection and the sorted cells were incubated at 37°C in 5% CO 2 for another 24 hours. Compared by flow cytometry, there are 0.16, 9.80 and 79.05% of EGFP positive cells in mock, unsorted and sorted cell populations respectively in the range of the indicated marker.

    Article Snippet: Before transfection into 32D cells, the supercoiled BAC DNA was linearized by digestion with I-Sce I restriction enzyme (New England Biolabs, Ipswich, MA, USA).

    Techniques: Selection, Marker, FACS, Transfection, BAC Assay, Electroporation, Incubation, Fluorescence, Flow Cytometry, Cytometry