bcni  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bcni
    Bcni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bcni/product/Thermo Fisher
    Average 94 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    bcni - by Bioz Stars, 2022-10
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    Thermo Fisher bcni sites
    <t>DNA</t> cleavage by <t>BcnI.</t> The proposed DNA cleavage mechanism based on bulk kinetic studies of BcnI ( 7 ). It involves enzyme association (the lower limit for the bimolecular rate constant 3 × 10 7 M −1 s −1 ) at the asymmetric target site in either the ‘G’ orientation (the preferred binding mode, catalytic center close to the ‘G’ strand 5΄-CC G GG-3΄), or the ‘C’ orientation (catalytic center close to the ‘C’ strand 5΄-CC C GG-3΄), followed by rapid hydrolysis of the first DNA strand, slow cleavage of the second strand, and product release. The observed cleavage of the second strand [ k obs (C) and k obs (G)] starts with a slow switch in the BcnI orientation [rate constants k switch (G-nick) and k switch (C-nick)], which presumably involves sliding and hopping of enzyme on the DNA, and a rapid hydrolysis reaction [ k 2 (C) and k 2 (G)]. The major fraction of DNA (70–80%) is cleaved via the G-nick intermediate.
    Bcni Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bcni sites/product/Thermo Fisher
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    bcni sites - by Bioz Stars, 2022-10
    94/100 stars
      Buy from Supplier

    94
    Thermo Fisher bcni
    <t>DNA</t> cleavage by <t>BcnI.</t> The proposed DNA cleavage mechanism based on bulk kinetic studies of BcnI ( 7 ). It involves enzyme association (the lower limit for the bimolecular rate constant 3 × 10 7 M −1 s −1 ) at the asymmetric target site in either the ‘G’ orientation (the preferred binding mode, catalytic center close to the ‘G’ strand 5΄-CC G GG-3΄), or the ‘C’ orientation (catalytic center close to the ‘C’ strand 5΄-CC C GG-3΄), followed by rapid hydrolysis of the first DNA strand, slow cleavage of the second strand, and product release. The observed cleavage of the second strand [ k obs (C) and k obs (G)] starts with a slow switch in the BcnI orientation [rate constants k switch (G-nick) and k switch (C-nick)], which presumably involves sliding and hopping of enzyme on the DNA, and a rapid hydrolysis reaction [ k 2 (C) and k 2 (G)]. The major fraction of DNA (70–80%) is cleaved via the G-nick intermediate.
    Bcni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bcni/product/Thermo Fisher
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bcni - by Bioz Stars, 2022-10
    94/100 stars
      Buy from Supplier

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    DNA cleavage by BcnI. The proposed DNA cleavage mechanism based on bulk kinetic studies of BcnI ( 7 ). It involves enzyme association (the lower limit for the bimolecular rate constant 3 × 10 7 M −1 s −1 ) at the asymmetric target site in either the ‘G’ orientation (the preferred binding mode, catalytic center close to the ‘G’ strand 5΄-CC G GG-3΄), or the ‘C’ orientation (catalytic center close to the ‘C’ strand 5΄-CC C GG-3΄), followed by rapid hydrolysis of the first DNA strand, slow cleavage of the second strand, and product release. The observed cleavage of the second strand [ k obs (C) and k obs (G)] starts with a slow switch in the BcnI orientation [rate constants k switch (G-nick) and k switch (C-nick)], which presumably involves sliding and hopping of enzyme on the DNA, and a rapid hydrolysis reaction [ k 2 (C) and k 2 (G)]. The major fraction of DNA (70–80%) is cleaved via the G-nick intermediate.

    Journal: Nucleic Acids Research

    Article Title: The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

    doi: 10.1093/nar/gkx294

    Figure Lengend Snippet: DNA cleavage by BcnI. The proposed DNA cleavage mechanism based on bulk kinetic studies of BcnI ( 7 ). It involves enzyme association (the lower limit for the bimolecular rate constant 3 × 10 7 M −1 s −1 ) at the asymmetric target site in either the ‘G’ orientation (the preferred binding mode, catalytic center close to the ‘G’ strand 5΄-CC G GG-3΄), or the ‘C’ orientation (catalytic center close to the ‘C’ strand 5΄-CC C GG-3΄), followed by rapid hydrolysis of the first DNA strand, slow cleavage of the second strand, and product release. The observed cleavage of the second strand [ k obs (C) and k obs (G)] starts with a slow switch in the BcnI orientation [rate constants k switch (G-nick) and k switch (C-nick)], which presumably involves sliding and hopping of enzyme on the DNA, and a rapid hydrolysis reaction [ k 2 (C) and k 2 (G)]. The major fraction of DNA (70–80%) is cleaved via the G-nick intermediate.

    Article Snippet: The 22.8 kbp DNA construct devoid of BcnI sites ( ) was preincubated with streptavidin-coated 1 μm magnetic beads (MyOne, ThermoFisher Scientific) and immobilized on the antidigoxigenin-covered bottom surface of the flow cell consisting of a capillary with a quadratic cross section (VitroCom).

    Techniques: Binding Assay

    Sliding and jumping of BcnI on DNA. ( A ) Mean square displacements as a function of time for the motion of BcnI along DNA in the absence of divalent ions or in the presence of Ca 2+ and Mg 2+ . The diffusion constants D 1 were obtained from linear fits to the data at the shortest times (red lines). Number of analyzed traces was 102 for the experiment with Ca 2+ , 44 for the experiment with Mg 2+ , and 12 for the experiment with no divalent cations. ( B ) Jump size distribution of BcnI in the presence of Mg 2+ and Ca 2+ . Jumps in the absence of divalent ions were too rare to obtain statistically meaningful data.

    Journal: Nucleic Acids Research

    Article Title: The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

    doi: 10.1093/nar/gkx294

    Figure Lengend Snippet: Sliding and jumping of BcnI on DNA. ( A ) Mean square displacements as a function of time for the motion of BcnI along DNA in the absence of divalent ions or in the presence of Ca 2+ and Mg 2+ . The diffusion constants D 1 were obtained from linear fits to the data at the shortest times (red lines). Number of analyzed traces was 102 for the experiment with Ca 2+ , 44 for the experiment with Mg 2+ , and 12 for the experiment with no divalent cations. ( B ) Jump size distribution of BcnI in the presence of Mg 2+ and Ca 2+ . Jumps in the absence of divalent ions were too rare to obtain statistically meaningful data.

    Article Snippet: The 22.8 kbp DNA construct devoid of BcnI sites ( ) was preincubated with streptavidin-coated 1 μm magnetic beads (MyOne, ThermoFisher Scientific) and immobilized on the antidigoxigenin-covered bottom surface of the flow cell consisting of a capillary with a quadratic cross section (VitroCom).

    Techniques: Diffusion-based Assay

    Preferred binding orientation of BcnI. Single molecule FRET experiments were performed with Alexa488-labeled BcnI(N18C+C202S) and Alexa546-labeled DNA. ( A ) FRET efficiency histogram obtained for ‘C’ strand-labeled DNA. Based on the available structures of BcnI–DNA complexes (PDB IDs: 2ODI and 3IMB), the expected distance between the Alexa488 (green sphere) and Alexa546 (red sphere) dyes is 71.3 and 56.2 Å for the different orientations of the protein–DNA complexes. This is equivalent to FRET efficiencies of 0.27 and 0.61 for protein bound in the ‘C’ and ‘G’ orientations, respectively (see sketches at the top). A double-Gaussian fit to the FRET efficiency data (red line) is consistent with preferred binding of BcnI in the ‘G’ orientation. ( B ) FRET efficiency histogram obtained for ‘G’ strand-labeled DNA. The expected FRET efficiencies are 0.61 and 0.23 for protein bound in the ‘C’ and ‘G’ orientations, respectively (see sketches at the top). As with the ‘C’ strand-labeled DNA, the experimental data is consistent with the preferential binding in the ‘G’ orientation.

    Journal: Nucleic Acids Research

    Article Title: The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

    doi: 10.1093/nar/gkx294

    Figure Lengend Snippet: Preferred binding orientation of BcnI. Single molecule FRET experiments were performed with Alexa488-labeled BcnI(N18C+C202S) and Alexa546-labeled DNA. ( A ) FRET efficiency histogram obtained for ‘C’ strand-labeled DNA. Based on the available structures of BcnI–DNA complexes (PDB IDs: 2ODI and 3IMB), the expected distance between the Alexa488 (green sphere) and Alexa546 (red sphere) dyes is 71.3 and 56.2 Å for the different orientations of the protein–DNA complexes. This is equivalent to FRET efficiencies of 0.27 and 0.61 for protein bound in the ‘C’ and ‘G’ orientations, respectively (see sketches at the top). A double-Gaussian fit to the FRET efficiency data (red line) is consistent with preferred binding of BcnI in the ‘G’ orientation. ( B ) FRET efficiency histogram obtained for ‘G’ strand-labeled DNA. The expected FRET efficiencies are 0.61 and 0.23 for protein bound in the ‘C’ and ‘G’ orientations, respectively (see sketches at the top). As with the ‘C’ strand-labeled DNA, the experimental data is consistent with the preferential binding in the ‘G’ orientation.

    Article Snippet: The 22.8 kbp DNA construct devoid of BcnI sites ( ) was preincubated with streptavidin-coated 1 μm magnetic beads (MyOne, ThermoFisher Scientific) and immobilized on the antidigoxigenin-covered bottom surface of the flow cell consisting of a capillary with a quadratic cross section (VitroCom).

    Techniques: Binding Assay, Labeling

    Observation of sliding and jumping of BcnI on DNA. ( A ) Experimental configuration. A 22.8 kb DNA molecule lacking BcnI recognition sites (black) was attached to a magnetic bead (grey sphere) and to the surface of a flow cell. The DNA was stretched sidewards using a magnet placed next to the flow cell. An evanescent field (green) of a TIRF-microscope was illuminating quantum-dot labeled enzymes bound to the DNA (yellow). Most of the freely diffusing enzymes in solution (orange) were outside of the evanescent field. ( B ) Representative kymographs of the movement of quantum-dot labeled BcnI on the DNA in the presence of Mg 2+ ions (top) (straight horizontal line in the lower part of the kymograph depicts protein stuck on the surface of the flow cell), in the presence of Ca 2+ ions (middle), and without divalent metal ions (bottom). Protein association, jumps, and protein dissociation are marked with red, green and blue arrows, respectively. Positions of the magnetic bead and fluorescent BcnI proteins are marked on the right-hand side with magenta and black arrows, respectively.

    Journal: Nucleic Acids Research

    Article Title: The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

    doi: 10.1093/nar/gkx294

    Figure Lengend Snippet: Observation of sliding and jumping of BcnI on DNA. ( A ) Experimental configuration. A 22.8 kb DNA molecule lacking BcnI recognition sites (black) was attached to a magnetic bead (grey sphere) and to the surface of a flow cell. The DNA was stretched sidewards using a magnet placed next to the flow cell. An evanescent field (green) of a TIRF-microscope was illuminating quantum-dot labeled enzymes bound to the DNA (yellow). Most of the freely diffusing enzymes in solution (orange) were outside of the evanescent field. ( B ) Representative kymographs of the movement of quantum-dot labeled BcnI on the DNA in the presence of Mg 2+ ions (top) (straight horizontal line in the lower part of the kymograph depicts protein stuck on the surface of the flow cell), in the presence of Ca 2+ ions (middle), and without divalent metal ions (bottom). Protein association, jumps, and protein dissociation are marked with red, green and blue arrows, respectively. Positions of the magnetic bead and fluorescent BcnI proteins are marked on the right-hand side with magenta and black arrows, respectively.

    Article Snippet: The 22.8 kbp DNA construct devoid of BcnI sites ( ) was preincubated with streptavidin-coated 1 μm magnetic beads (MyOne, ThermoFisher Scientific) and immobilized on the antidigoxigenin-covered bottom surface of the flow cell consisting of a capillary with a quadratic cross section (VitroCom).

    Techniques: Flow Cytometry, Microscopy, Labeling

    BcnI conformational dynamics in the absence and in the presence of DNA. ( A ) Structures of apo-BcnI (PDB ID 2ODH) ( A ) and the BcnI–DNA complex (PDB ID: 2ODI). The green and red spheres indicate the Cβ atoms of N18 and V105 residues used for fluorescent labeling. The transition of BcnI from the apo- (‘open’) to the DNA-bound (‘closed’) conformation reduces the distance between the Cβ atoms from 5.5 to 4.2 nm, causing an increase in FRET efficiency. The central G and C nucleotides of the BcnI target site are depicted in magenta and cyan, respectively. Single molecule FRET experiments were performed with double-labeled BcnI(N18C+V105C+C202S)–Alexa488–Alexa546 using a confocal microscope. FRET efficiency histograms obtained for double-labeled BcnI(N18C+V105C+C202S)–Alexa488–Alexa546 in the absence or presence of Ca 2+ ions are shown for BcnI only ( B ), BcnI in the presence of non-specific DNA ( C ) and BcnI in the presence of specific DNA ( D ).

    Journal: Nucleic Acids Research

    Article Title: The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

    doi: 10.1093/nar/gkx294

    Figure Lengend Snippet: BcnI conformational dynamics in the absence and in the presence of DNA. ( A ) Structures of apo-BcnI (PDB ID 2ODH) ( A ) and the BcnI–DNA complex (PDB ID: 2ODI). The green and red spheres indicate the Cβ atoms of N18 and V105 residues used for fluorescent labeling. The transition of BcnI from the apo- (‘open’) to the DNA-bound (‘closed’) conformation reduces the distance between the Cβ atoms from 5.5 to 4.2 nm, causing an increase in FRET efficiency. The central G and C nucleotides of the BcnI target site are depicted in magenta and cyan, respectively. Single molecule FRET experiments were performed with double-labeled BcnI(N18C+V105C+C202S)–Alexa488–Alexa546 using a confocal microscope. FRET efficiency histograms obtained for double-labeled BcnI(N18C+V105C+C202S)–Alexa488–Alexa546 in the absence or presence of Ca 2+ ions are shown for BcnI only ( B ), BcnI in the presence of non-specific DNA ( C ) and BcnI in the presence of specific DNA ( D ).

    Article Snippet: The 22.8 kbp DNA construct devoid of BcnI sites ( ) was preincubated with streptavidin-coated 1 μm magnetic beads (MyOne, ThermoFisher Scientific) and immobilized on the antidigoxigenin-covered bottom surface of the flow cell consisting of a capillary with a quadratic cross section (VitroCom).

    Techniques: Labeling, Microscopy

    DNA cleavage by BcnI. ( A ) Scheme of the experiment. A 7.4 kb DNA fragment with a single BcnI recognition site was attached to the flow cell surface and a magnetic bead. The DNA was held stretched at 2 pN force and supercoiled with +25 turns. Subsequently, 0.2–1 nM BcnI was added. DNA nicking recovered the initial DNA length and cleavage of the second DNA strand released the bead. The time interval Δ t between these two events is the lifetime of the nicked intermediate, or the inverse of the second DNA strand cleavage rate. ( B ) Experimental results. The cumulative distribution of the measured Δ t values is shown in the inset. A double-exponential fit yielded k obs (1) = 0.3 ± 0.1 s −1 (30% events) and k obs (2) = 0.06 ± 0.01 s −1 (70% events).

    Journal: Nucleic Acids Research

    Article Title: The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

    doi: 10.1093/nar/gkx294

    Figure Lengend Snippet: DNA cleavage by BcnI. ( A ) Scheme of the experiment. A 7.4 kb DNA fragment with a single BcnI recognition site was attached to the flow cell surface and a magnetic bead. The DNA was held stretched at 2 pN force and supercoiled with +25 turns. Subsequently, 0.2–1 nM BcnI was added. DNA nicking recovered the initial DNA length and cleavage of the second DNA strand released the bead. The time interval Δ t between these two events is the lifetime of the nicked intermediate, or the inverse of the second DNA strand cleavage rate. ( B ) Experimental results. The cumulative distribution of the measured Δ t values is shown in the inset. A double-exponential fit yielded k obs (1) = 0.3 ± 0.1 s −1 (30% events) and k obs (2) = 0.06 ± 0.01 s −1 (70% events).

    Article Snippet: The 22.8 kbp DNA construct devoid of BcnI sites ( ) was preincubated with streptavidin-coated 1 μm magnetic beads (MyOne, ThermoFisher Scientific) and immobilized on the antidigoxigenin-covered bottom surface of the flow cell consisting of a capillary with a quadratic cross section (VitroCom).

    Techniques: Flow Cytometry

    Dynamics of BcnI binding to its target site. Single molecule TRIF-based FRET experiments using surface-tethered 36 bp long Cy5-labeled DNA and Cy3-labeled BcnI(N18C+C202S). ( A ) Time trajectory of donor and acceptor fluorescence measured on a single DNA in the presence of Ca 2+ . Arrows indicate the event duration times. ( B ) Cumulative distribution of the measured event durations. Double exponential fit to the data is shown as a solid line. Fit parameters are given next to the plot.

    Journal: Nucleic Acids Research

    Article Title: The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

    doi: 10.1093/nar/gkx294

    Figure Lengend Snippet: Dynamics of BcnI binding to its target site. Single molecule TRIF-based FRET experiments using surface-tethered 36 bp long Cy5-labeled DNA and Cy3-labeled BcnI(N18C+C202S). ( A ) Time trajectory of donor and acceptor fluorescence measured on a single DNA in the presence of Ca 2+ . Arrows indicate the event duration times. ( B ) Cumulative distribution of the measured event durations. Double exponential fit to the data is shown as a solid line. Fit parameters are given next to the plot.

    Article Snippet: The 22.8 kbp DNA construct devoid of BcnI sites ( ) was preincubated with streptavidin-coated 1 μm magnetic beads (MyOne, ThermoFisher Scientific) and immobilized on the antidigoxigenin-covered bottom surface of the flow cell consisting of a capillary with a quadratic cross section (VitroCom).

    Techniques: Binding Assay, Labeling, Fluorescence