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    New England Biolabs bsphi
    Bsphi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsphi  (New England Biolabs)


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    New England Biolabs bsphi
    Bsphi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsph1  (New England Biolabs)


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    New England Biolabs bsph1
    Bsph1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsp hi  (New England Biolabs)


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    New England Biolabs bsp hi
    ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after <t>Bsp</t> HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI <t>at</t> <t>37°C</t> for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.
    Bsp Hi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors"

    Article Title: Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

    Journal: bioRxiv

    doi: 10.1101/2023.08.10.552596

    ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.
    Figure Legend Snippet: ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.

    Techniques Used: Activity Assay, Fluorescence, Plasmid Preparation, Incubation, Marker

    ( a ) Mapping strategy for the D2 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D2 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify the cleavage site in the invading molecule after D2 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D2 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( d ) Graphical representation of the incisions mapped on plasmid-based D2 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primer are depicted on the right. Note that only chromatograms of the region of interest are shown. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( e ) Same as ( d ) but employing a D2.2 oligo for D-loop formation. Note that the second branched point is different from the previous one (from position 2012 to 2007 of pBKS).
    Figure Legend Snippet: ( a ) Mapping strategy for the D2 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D2 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify the cleavage site in the invading molecule after D2 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D2 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( d ) Graphical representation of the incisions mapped on plasmid-based D2 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primer are depicted on the right. Note that only chromatograms of the region of interest are shown. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( e ) Same as ( d ) but employing a D2.2 oligo for D-loop formation. Note that the second branched point is different from the previous one (from position 2012 to 2007 of pBKS).

    Techniques Used: Activity Assay, Fluorescence, Plasmid Preparation, Incubation, Marker, Sequencing, Derivative Assay, Mutagenesis

    ( a ) Mapping strategy for the D1 D-loop structure. The invading oligo is homologous to positions 1932 to 2022 of pBSK. After D1 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, after D1 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 16% denaturing PAGE and scanned a. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D1 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. The arrow indicates product of expected size. ( d ) and ( e ) Same as ( b ) and ( c ), but using Mus81. ( f ) Graphical representation of the incisions mapped on plasmid-based D1 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primers are shown. Note that only chromatograms of the region of interest are shown. Left: reactions with Mus81. Green arrows indicate Mus81 cleavage sites. Adenine incorporation after the cleavage point is indicated with a green dotted box. Nuclease-dead mutant controls are shown (ND). Right: Reactions with Yen1. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( g ) Same as ( f ), but employing a derivative D1.2 invading oligo. Note that the first branched point is different from the previous one (from position 1932 to 1924 nt of pBSK).
    Figure Legend Snippet: ( a ) Mapping strategy for the D1 D-loop structure. The invading oligo is homologous to positions 1932 to 2022 of pBSK. After D1 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, after D1 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 16% denaturing PAGE and scanned a. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D1 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. The arrow indicates product of expected size. ( d ) and ( e ) Same as ( b ) and ( c ), but using Mus81. ( f ) Graphical representation of the incisions mapped on plasmid-based D1 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primers are shown. Note that only chromatograms of the region of interest are shown. Left: reactions with Mus81. Green arrows indicate Mus81 cleavage sites. Adenine incorporation after the cleavage point is indicated with a green dotted box. Nuclease-dead mutant controls are shown (ND). Right: Reactions with Yen1. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( g ) Same as ( f ), but employing a derivative D1.2 invading oligo. Note that the first branched point is different from the previous one (from position 1932 to 1924 nt of pBSK).

    Techniques Used: Activity Assay, Fluorescence, Plasmid Preparation, Incubation, Marker, Sequencing, Derivative Assay, Mutagenesis

    restriction enzymes bsphi  (New England Biolabs)


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    New England Biolabs restriction enzymes bsphi
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    bsph1  (New England Biolabs)


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    restriction enzymes bsphi  (New England Biolabs)


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    r0517  (New England Biolabs)


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    bsph  (New England Biolabs)


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    New England Biolabs bsp hi
    ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after <t>Bsp</t> HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI <t>at</t> <t>37°C</t> for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.
    Bsp Hi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after <t>Bsp</t> HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI <t>at</t> <t>37°C</t> for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.
    Restriction Enzymes Bsphi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs r0517
    ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after <t>Bsp</t> HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI <t>at</t> <t>37°C</t> for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.
    R0517, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs bsph
    ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after <t>Bsp</t> HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI <t>at</t> <t>37°C</t> for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.
    Bsph, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bsph - by Bioz Stars, 2024-05
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    ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.

    Journal: bioRxiv

    Article Title: Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

    doi: 10.1101/2023.08.10.552596

    Figure Lengend Snippet: ( a ) Mapping strategy for the D3 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D3 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction after D3 D-loop formation and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) Same as ( b ), but using increasing concentrations of Mus81 (50, 100, 200, 400 nM). ( d ) To identify incisions in the plasmid after D3 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( e ) Same as ( d ) but using 400 nM Mus81. ( f ) Schematic representation of Yen1 (purple) and Mus81 (green) incisions on the plasmid-based D3 D-loop. ( g ) Same as ( f ) but for D2 D-loop substrate. ( h ) Same as ( f ) but for D1 D-loop substrate.

    Article Snippet: To analyse the incision in the donor plasmid molecule (pBSK), Rad51/Rad54-mediated D-loop formation (20 µL final volume) and cleavage with the indicated nuclease (as described), was followed by cleavage with 10 U Bsp HI (New England Biolabs, #R0517S) and incubated at 37°C for 20 min followed by dephosphorylation with 1 U Shrimp Alkaline Phosphatase (rSAP, New England Biolabs, #M0371S) and incubation for another 20 min. To verify D-loop formation and nuclease activity, half of each reaction was deproteinised and analysed on agarose gels as described above.

    Techniques: Activity Assay, Fluorescence, Plasmid Preparation, Incubation, Marker

    ( a ) Mapping strategy for the D2 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D2 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify the cleavage site in the invading molecule after D2 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D2 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( d ) Graphical representation of the incisions mapped on plasmid-based D2 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primer are depicted on the right. Note that only chromatograms of the region of interest are shown. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( e ) Same as ( d ) but employing a D2.2 oligo for D-loop formation. Note that the second branched point is different from the previous one (from position 2012 to 2007 of pBKS).

    Journal: bioRxiv

    Article Title: Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

    doi: 10.1101/2023.08.10.552596

    Figure Lengend Snippet: ( a ) Mapping strategy for the D2 D-loop structure. The invading oligo is homologous to positions 1932 to 2012 of pBSK. After D2 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify the cleavage site in the invading molecule after D2 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 12% denaturing PAGE and scanned. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D2 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. Arrows indicate products of expected sizes. ( d ) Graphical representation of the incisions mapped on plasmid-based D2 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primer are depicted on the right. Note that only chromatograms of the region of interest are shown. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( e ) Same as ( d ) but employing a D2.2 oligo for D-loop formation. Note that the second branched point is different from the previous one (from position 2012 to 2007 of pBKS).

    Article Snippet: To analyse the incision in the donor plasmid molecule (pBSK), Rad51/Rad54-mediated D-loop formation (20 µL final volume) and cleavage with the indicated nuclease (as described), was followed by cleavage with 10 U Bsp HI (New England Biolabs, #R0517S) and incubated at 37°C for 20 min followed by dephosphorylation with 1 U Shrimp Alkaline Phosphatase (rSAP, New England Biolabs, #M0371S) and incubation for another 20 min. To verify D-loop formation and nuclease activity, half of each reaction was deproteinised and analysed on agarose gels as described above.

    Techniques: Activity Assay, Fluorescence, Plasmid Preparation, Incubation, Marker, Sequencing, Derivative Assay, Mutagenesis

    ( a ) Mapping strategy for the D1 D-loop structure. The invading oligo is homologous to positions 1932 to 2022 of pBSK. After D1 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, after D1 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 16% denaturing PAGE and scanned a. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D1 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. The arrow indicates product of expected size. ( d ) and ( e ) Same as ( b ) and ( c ), but using Mus81. ( f ) Graphical representation of the incisions mapped on plasmid-based D1 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primers are shown. Note that only chromatograms of the region of interest are shown. Left: reactions with Mus81. Green arrows indicate Mus81 cleavage sites. Adenine incorporation after the cleavage point is indicated with a green dotted box. Nuclease-dead mutant controls are shown (ND). Right: Reactions with Yen1. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( g ) Same as ( f ), but employing a derivative D1.2 invading oligo. Note that the first branched point is different from the previous one (from position 1932 to 1924 nt of pBSK).

    Journal: bioRxiv

    Article Title: Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors

    doi: 10.1101/2023.08.10.552596

    Figure Lengend Snippet: ( a ) Mapping strategy for the D1 D-loop structure. The invading oligo is homologous to positions 1932 to 2022 of pBSK. After D1 D-loop formation, SSE activity on the invading molecule was analysed by fluorescence scanning after denaturing PAGE; SSE activity on the plasmid was analysed after Bsp HI treatment, radioactive labelling, and analysis on denaturing PAGE by phosphorimaging. ( b ) To identify cuts in the invading molecule, after D1 D-loop formation, increasing concentrations of Yen1 (50, 100, 200, 400 nM) or 400 nM Yen1 ND (ND) were added to the reaction and incubated for 1 h at 30°C. (-) indicates no enzyme. Reaction products were analysed by 16% denaturing PAGE and scanned a. A mixture of 5’-6FAM-end-labelled oligos of defined length was used as marker. ( c ) To identify incisions within the plasmid, after D1 D-loop formation, 400 nM Yen1 was added to the reaction and incubated for 1 h at 30°C. Then, pBSK was digested with 10 U Bsp HI at 37°C for 20 min, followed by treatment with rSAP for another 20 min at 37°C and heat inactivation at 99°C for 5 min. Reaction products were labelled using T4 PNK and 32 P-γ-ATP and analysed by 6% denaturing PAGE followed by phosphorimaging. A mixture of DNA fragments was radioactively labelled and used as marker. The arrow indicates product of expected size. ( d ) and ( e ) Same as ( b ) and ( c ), but using Mus81. ( f ) Graphical representation of the incisions mapped on plasmid-based D1 D-loops. The nucleotide sequence near the branch point is shown. Sequencing reactions were carried out as in . Chromatograms derived from sequencing using the reverse primers are shown. Note that only chromatograms of the region of interest are shown. Left: reactions with Mus81. Green arrows indicate Mus81 cleavage sites. Adenine incorporation after the cleavage point is indicated with a green dotted box. Nuclease-dead mutant controls are shown (ND). Right: Reactions with Yen1. Purple arrows indicate Yen1 cleavage sites and adenine incorporation is pointed out with a purple dotted box. Nuclease-dead mutant controls are shown (ND). ( g ) Same as ( f ), but employing a derivative D1.2 invading oligo. Note that the first branched point is different from the previous one (from position 1932 to 1924 nt of pBSK).

    Article Snippet: To analyse the incision in the donor plasmid molecule (pBSK), Rad51/Rad54-mediated D-loop formation (20 µL final volume) and cleavage with the indicated nuclease (as described), was followed by cleavage with 10 U Bsp HI (New England Biolabs, #R0517S) and incubated at 37°C for 20 min followed by dephosphorylation with 1 U Shrimp Alkaline Phosphatase (rSAP, New England Biolabs, #M0371S) and incubation for another 20 min. To verify D-loop formation and nuclease activity, half of each reaction was deproteinised and analysed on agarose gels as described above.

    Techniques: Activity Assay, Fluorescence, Plasmid Preparation, Incubation, Marker, Sequencing, Derivative Assay, Mutagenesis