t7 exonuclease  (New England Biolabs)


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    Name:
    T7 Exonuclease
    Description:
    T7 Exonuclease 5 000 units
    Catalog Number:
    m0263l
    Price:
    261
    Size:
    5 000 units
    Category:
    Exonucleases
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    Structured Review

    New England Biolabs t7 exonuclease
    T7 Exonuclease
    T7 Exonuclease 5 000 units
    https://www.bioz.com/result/t7 exonuclease/product/New England Biolabs
    Average 97 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    t7 exonuclease - by Bioz Stars, 2020-07
    97/100 stars

    Images

    1) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

    2) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    3) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    4) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    5) Product Images from "Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *"

    Article Title: Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.067439

    Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π.  B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown.  C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as  error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.
    Figure Legend Snippet: Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π. B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown. C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.

    Techniques Used: Clone Assay, Plasmid Preparation, Construct, End Labeling, Incubation, Ligation, Binding Assay, Mutagenesis

    6) Product Images from "5?CAG and 5?CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs"

    Article Title: 5?CAG and 5?CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

    Journal: Molecular Biology International

    doi: 10.4061/2011/213824

    Preparation of leading and lagging strand templates, p-t junctions and miniforks. The DNA fragments in the 100 base pair (bp) ladder are 100, 200, 300, 400, 500/517, 600, 700, 800, 900, and 1000 pbs. (a) The agarose gels stained by ethidium bromide (EtBr) show the products of the PCR obtained with the four plasmids (p-Empty, p-5′GTT16, p-5′CTG17, and p-5′CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand. The name of the plasmids used for the PCR is indicated at the top of the gels. (b) The agarose gels stained by EtBr show the products of the T7 exonuclease digestion. The PCR products obtained with p-5′CTG17 (CTG17) and p-5′CAG23 (CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand were treated (+) or not (−) by T7 exonuclease. After treatment with T7 exonuclease, the appearance of a DNA band with a slower electrophoretic migration and a weaker intensity (indicated by a backward arrow) than the ds DNA is indicative of ss DNA production. (c) The ss leading strand templates containing either 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) are mixed with increasing amounts of radiolabelled p821 (p821*) to generate the p-t junctions. Species are resolved on a native gel. Free p821 migrates faster than the p-t junctions. (d) The p-t junctions containing 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) on their leading strand are mixed with increasing amounts of ss lagging strand template to assemble the miniforks. Species are resolved on a native gel. The miniforks migrate more slowly than the p-t junctions.
    Figure Legend Snippet: Preparation of leading and lagging strand templates, p-t junctions and miniforks. The DNA fragments in the 100 base pair (bp) ladder are 100, 200, 300, 400, 500/517, 600, 700, 800, 900, and 1000 pbs. (a) The agarose gels stained by ethidium bromide (EtBr) show the products of the PCR obtained with the four plasmids (p-Empty, p-5′GTT16, p-5′CTG17, and p-5′CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand. The name of the plasmids used for the PCR is indicated at the top of the gels. (b) The agarose gels stained by EtBr show the products of the T7 exonuclease digestion. The PCR products obtained with p-5′CTG17 (CTG17) and p-5′CAG23 (CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand were treated (+) or not (−) by T7 exonuclease. After treatment with T7 exonuclease, the appearance of a DNA band with a slower electrophoretic migration and a weaker intensity (indicated by a backward arrow) than the ds DNA is indicative of ss DNA production. (c) The ss leading strand templates containing either 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) are mixed with increasing amounts of radiolabelled p821 (p821*) to generate the p-t junctions. Species are resolved on a native gel. Free p821 migrates faster than the p-t junctions. (d) The p-t junctions containing 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) on their leading strand are mixed with increasing amounts of ss lagging strand template to assemble the miniforks. Species are resolved on a native gel. The miniforks migrate more slowly than the p-t junctions.

    Techniques Used: Staining, Polymerase Chain Reaction, Migration, CTG Assay

    Strategy of the preparation of model replication miniforks. (a) The left and right sides of the figure correspond to the strategy used to build the ss leading and lagging strand templates of the minifork, respectively. The ss leading and lagging strand templates are combined with the radiolabelled p821 primer to assemble the minifork by strand hybridization. Replication miniforks are prepared in three consecutive steps. The first step (Step 1: PCR) consists of a PCR using plasmids containing a random or a TNR sequence (shown as a black rectangle) and oligonucleotides that flank the random sequence or the TNR unit. For each PCR, one of the oligonucleotides (p1033/4ps (colored in blue) for the preparation of the ss leading strand template, and (50T/4ps)/p867 (colored in red) for the preparation of the ss lagging strand template) carries 4 phosphorothioate linkages (represented as filled blue and red spheres for p1033/4ps and (50T/4ps)/p867, resp.) at its 5′ end. After PCR, the ds PCR products are digested by the T7 exonuclease that specifically degrades the DNA strand (colored in green or orange for the preparation of the ss leading or lagging strand template, resp.) that does not contain the phosphorothioate linkages (Step 2: T7 exonuclease digestion). The minifork (shown in a rounded rectangle) is assembled by hybridization of the ss leading and lagging strand templates and the radiolabelled p821 primer (in green) (Step 3: Hybridization). A gap of 15 nts exists between the 3′ end of the p821 primer and the base of the ss tail of the lagging strand template to facilitate the assembly of the DNA polymerase at the p-t junction. (b) A minifork containing n repeats of 5′CTG is shown. The repeats are located on the leading strand template.
    Figure Legend Snippet: Strategy of the preparation of model replication miniforks. (a) The left and right sides of the figure correspond to the strategy used to build the ss leading and lagging strand templates of the minifork, respectively. The ss leading and lagging strand templates are combined with the radiolabelled p821 primer to assemble the minifork by strand hybridization. Replication miniforks are prepared in three consecutive steps. The first step (Step 1: PCR) consists of a PCR using plasmids containing a random or a TNR sequence (shown as a black rectangle) and oligonucleotides that flank the random sequence or the TNR unit. For each PCR, one of the oligonucleotides (p1033/4ps (colored in blue) for the preparation of the ss leading strand template, and (50T/4ps)/p867 (colored in red) for the preparation of the ss lagging strand template) carries 4 phosphorothioate linkages (represented as filled blue and red spheres for p1033/4ps and (50T/4ps)/p867, resp.) at its 5′ end. After PCR, the ds PCR products are digested by the T7 exonuclease that specifically degrades the DNA strand (colored in green or orange for the preparation of the ss leading or lagging strand template, resp.) that does not contain the phosphorothioate linkages (Step 2: T7 exonuclease digestion). The minifork (shown in a rounded rectangle) is assembled by hybridization of the ss leading and lagging strand templates and the radiolabelled p821 primer (in green) (Step 3: Hybridization). A gap of 15 nts exists between the 3′ end of the p821 primer and the base of the ss tail of the lagging strand template to facilitate the assembly of the DNA polymerase at the p-t junction. (b) A minifork containing n repeats of 5′CTG is shown. The repeats are located on the leading strand template.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Sequencing, CTG Assay

    7) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh124

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

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

    9) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    10) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    11) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

    12) Product Images from "Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *"

    Article Title: Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.067439

    Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π.  B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown.  C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as  error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.
    Figure Legend Snippet: Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π. B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown. C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.

    Techniques Used: Clone Assay, Plasmid Preparation, Construct, End Labeling, Incubation, Ligation, Binding Assay, Mutagenesis

    13) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh124

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

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

    15) Product Images from "Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection"

    Article Title: Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection

    Journal: Genes & Development

    doi: 10.1101/gad.308254.117

    Exo1 efficiently and specifically functions downstream from the MRX–Sae2 nuclease in the processing of dsDNA with blocked ends. ( A ) A representative experiment showing the processing of streptavidin-blocked dsDNA by MRX–Sae2 in the absence (lane 2 ) or presence (lane 3 ) of Exo1. ( B ) Representative DNA clipping reactions with MRX–Sae2 with various concentrations of Exo1, as indicated. ( C – F ) Exo1 degrades the MRX–Sae2 nuclease products in a specific manner. Representative kinetic nuclease assays with MRX–Sae2 and either no additional exonuclease ( C ), Exo1 ( D ), Exo1 D173A (nuclease-dead; E ), or T7 exonuclease ( F ). The DNA was either blocked by streptavidin ( left ) or bound by 30 nM Ku ( right ). ( G ) Quantitation of experiments such as those from C – F showing overall substrate utilization in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM. ( H ) Quantitation of experiments such as those from C – F showing the fraction of endonuclease products versus total DNA in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM.
    Figure Legend Snippet: Exo1 efficiently and specifically functions downstream from the MRX–Sae2 nuclease in the processing of dsDNA with blocked ends. ( A ) A representative experiment showing the processing of streptavidin-blocked dsDNA by MRX–Sae2 in the absence (lane 2 ) or presence (lane 3 ) of Exo1. ( B ) Representative DNA clipping reactions with MRX–Sae2 with various concentrations of Exo1, as indicated. ( C – F ) Exo1 degrades the MRX–Sae2 nuclease products in a specific manner. Representative kinetic nuclease assays with MRX–Sae2 and either no additional exonuclease ( C ), Exo1 ( D ), Exo1 D173A (nuclease-dead; E ), or T7 exonuclease ( F ). The DNA was either blocked by streptavidin ( left ) or bound by 30 nM Ku ( right ). ( G ) Quantitation of experiments such as those from C – F showing overall substrate utilization in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM. ( H ) Quantitation of experiments such as those from C – F showing the fraction of endonuclease products versus total DNA in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM.

    Techniques Used: Quantitation Assay

    16) Product Images from "BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *"

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.025825

    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.
    Figure Legend Snippet: BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Techniques Used: Hybridization, Labeling, Transfection, Plasmid Preparation, Western Blot, Over Expression

    17) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    18) Product Images from "Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes"

    Article Title: Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2009.06.009

    Backtracking of Trailing Polymerase Caused by Collision with Leading Polymerase (A) Experimental approach. A second G stop at position −27 relative to the first G stop was introduced. Drawn to scale. Numbers corresponding to each substrate also apply in (B) and (C). (B) Transcription products resolved by denaturing PAGE as in Figure 2 C. (C–E) T7 exonuclease mapping of elongation complexes and models, as in Figure 3 .
    Figure Legend Snippet: Backtracking of Trailing Polymerase Caused by Collision with Leading Polymerase (A) Experimental approach. A second G stop at position −27 relative to the first G stop was introduced. Drawn to scale. Numbers corresponding to each substrate also apply in (B) and (C). (B) Transcription products resolved by denaturing PAGE as in Figure 2 C. (C–E) T7 exonuclease mapping of elongation complexes and models, as in Figure 3 .

    Techniques Used: Polyacrylamide Gel Electrophoresis

    Exonuclease Mapping of Trailing Polymerase after Collision (A) (Box) Experimental approach. (Lower) Mono- and dielongation complexes with 3′ end-labeled, nontranscribed strand DNA were incubated with NTPs (or with NTPs and TFIIS), digested with T7 exonuclease, and analyzed by denaturing PAGE. DNA fragment sizes are indicated to the left. The position of the elongation complexes relative to the active site of the leading polymerase (−82, −79, etc.) is indicated on the right. Grey arrow indicates distance between the active site of the elongation complex at the G stop and the position of that same elongation complex as marked by T7 exonuclease. Double arrows (black) mark distances between elongation complexes, and thin lines between lanes indicate major exonuclease stops (see text for details). (B) (Box) Experimental design. (Right) Transcription products resolved by denaturing PAGE as in  Figure 2 C. (C) Relationship between lengths of transcripts and positions of trailing elongation complex as marked by exonuclease. Angled line indicates forward translocation toward the point of collision. Horizontal lines with arrows indicate backtracking, and gray, vertical lines with arrows indicate transcript cleavage and the return of RNAPII to forward translocation. (D) Summary of results, drawn to scale. Distances between elongation complexes are indicated.
    Figure Legend Snippet: Exonuclease Mapping of Trailing Polymerase after Collision (A) (Box) Experimental approach. (Lower) Mono- and dielongation complexes with 3′ end-labeled, nontranscribed strand DNA were incubated with NTPs (or with NTPs and TFIIS), digested with T7 exonuclease, and analyzed by denaturing PAGE. DNA fragment sizes are indicated to the left. The position of the elongation complexes relative to the active site of the leading polymerase (−82, −79, etc.) is indicated on the right. Grey arrow indicates distance between the active site of the elongation complex at the G stop and the position of that same elongation complex as marked by T7 exonuclease. Double arrows (black) mark distances between elongation complexes, and thin lines between lanes indicate major exonuclease stops (see text for details). (B) (Box) Experimental design. (Right) Transcription products resolved by denaturing PAGE as in Figure 2 C. (C) Relationship between lengths of transcripts and positions of trailing elongation complex as marked by exonuclease. Angled line indicates forward translocation toward the point of collision. Horizontal lines with arrows indicate backtracking, and gray, vertical lines with arrows indicate transcript cleavage and the return of RNAPII to forward translocation. (D) Summary of results, drawn to scale. Distances between elongation complexes are indicated.

    Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Translocation Assay

    TFIIS Induces Oscillation of Trailing Polymerase upon Collision with Leading Polymerase (A) (Box) Experimental design. (Right) Unlabeled elongation complexes were incubated with ATP/UTP/GTP and α- 32 P GTP. After 5 min, TFIIS was added (0.2 and 1 molar ratios to RNAPII). Products were analyzed by 20% denaturing PAGE. Length of transcripts generated by leading polymerase (L, on left side) and trailing polymerase (T, on the right), and the expected number of guanines in these transcripts (in brackets), are indicated. (B) Depiction of the situation with a 26 nt (upper, T26), and with a 5 nt (lower, T5) long cleaved nascent RNA, an alternative view to that shown in Figure 5 D. Numbers indicate positions relative to that of the leading polymerase's active site. Long arrows, position of cleavage induced by TFIIS. Short arrows, position of T7 exonuclease footprint.
    Figure Legend Snippet: TFIIS Induces Oscillation of Trailing Polymerase upon Collision with Leading Polymerase (A) (Box) Experimental design. (Right) Unlabeled elongation complexes were incubated with ATP/UTP/GTP and α- 32 P GTP. After 5 min, TFIIS was added (0.2 and 1 molar ratios to RNAPII). Products were analyzed by 20% denaturing PAGE. Length of transcripts generated by leading polymerase (L, on left side) and trailing polymerase (T, on the right), and the expected number of guanines in these transcripts (in brackets), are indicated. (B) Depiction of the situation with a 26 nt (upper, T26), and with a 5 nt (lower, T5) long cleaved nascent RNA, an alternative view to that shown in Figure 5 D. Numbers indicate positions relative to that of the leading polymerase's active site. Long arrows, position of cleavage induced by TFIIS. Short arrows, position of T7 exonuclease footprint.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Generated

    Elongation Complex Footprint and Mapping of the Transcription Bubble (A) (Upper) Summary of elongation complex preparation, drawn to scale. (Lower) Elongation complexes with 5′ end-labeled, 131-mer transcribed DNA were digested with increasing amounts of DNase I, as indicated. The footprint region where DNase I cleavage is repressed (bold line) and a hypersensitive site (sphere) are indicated on the right. M, thymine-specific cleavage marker. Elongation complex (oval) and RNA (bold line) are indicated on the left. Position of the active site is taken as +1. (B) (Upper) Schematic diagram of the locations of the RNA primer (bold text) and G stop (gray box) in the elongation complex. The 150-mer transcribed strand (TS), the corresponding end-labeled nontranscribed strand (NTS), and the direction of exonuclease digestion are indicated. (Lower) Elongation complexes incubated alone, or with ATP/UTP/GTP and TFIIS, were digested with T7 exonuclease to mark their position(s), modified with increasing amounts of KMnO 4 (2 and 8 mM), and analyzed by denaturing PAGE (lanes 1–4). Bands corresponding to the major elongation complexes (2 mM KMnO 4 experiment shown) were isolated, cleaved at modified thymines with piperidine, and resolved by denaturing PAGE (lanes 5 and 6). DNA sequence is shown on the left. Spheres, modified thymines. Arrowheads, positions of active sites. (C) Crystal structure of the elongation complex with modeled DNA paths (dashed lines). Space-filling model shows the DNA (color) engulfed in protein bulk. (D) Area protected by RNAPII (DNase I footprinting, solid line; crystallography model, dashed line) and their transcription bubbles. Only reconstituted elongation complex was mapped by DNase I footprinting. However, T7 exonuclease digests to the same distance from the edge of the two footprints (±1 base; positions indicated by vertical arrow) before and after transcription, showing that RNAPII moved forward upon NTP addition.
    Figure Legend Snippet: Elongation Complex Footprint and Mapping of the Transcription Bubble (A) (Upper) Summary of elongation complex preparation, drawn to scale. (Lower) Elongation complexes with 5′ end-labeled, 131-mer transcribed DNA were digested with increasing amounts of DNase I, as indicated. The footprint region where DNase I cleavage is repressed (bold line) and a hypersensitive site (sphere) are indicated on the right. M, thymine-specific cleavage marker. Elongation complex (oval) and RNA (bold line) are indicated on the left. Position of the active site is taken as +1. (B) (Upper) Schematic diagram of the locations of the RNA primer (bold text) and G stop (gray box) in the elongation complex. The 150-mer transcribed strand (TS), the corresponding end-labeled nontranscribed strand (NTS), and the direction of exonuclease digestion are indicated. (Lower) Elongation complexes incubated alone, or with ATP/UTP/GTP and TFIIS, were digested with T7 exonuclease to mark their position(s), modified with increasing amounts of KMnO 4 (2 and 8 mM), and analyzed by denaturing PAGE (lanes 1–4). Bands corresponding to the major elongation complexes (2 mM KMnO 4 experiment shown) were isolated, cleaved at modified thymines with piperidine, and resolved by denaturing PAGE (lanes 5 and 6). DNA sequence is shown on the left. Spheres, modified thymines. Arrowheads, positions of active sites. (C) Crystal structure of the elongation complex with modeled DNA paths (dashed lines). Space-filling model shows the DNA (color) engulfed in protein bulk. (D) Area protected by RNAPII (DNase I footprinting, solid line; crystallography model, dashed line) and their transcription bubbles. Only reconstituted elongation complex was mapped by DNase I footprinting. However, T7 exonuclease digests to the same distance from the edge of the two footprints (±1 base; positions indicated by vertical arrow) before and after transcription, showing that RNAPII moved forward upon NTP addition.

    Techniques Used: Labeling, Marker, Incubation, Modification, Polyacrylamide Gel Electrophoresis, Isolation, Sequencing, Footprinting

    Transcription Bubbles Remain Intact and Separated upon Collision (A) Experimental design. (B) Elongation complexes (end-labeled NTS) were incubated with NTPs and TFIIS, digested with T7 exonuclease, and modified with increasing amounts of KMnO 4  (12, 18, and 24 mM) as indicated and resolved by denaturing PAGE. Major T7 exonuclease-marked bands as seen in  Figure 3 , lane 17, line, were isolated, cleaved at modified thymines with piperidine, and analyzed by denaturing PAGE. The positions of isolated bands are indicated at the top. Thin lines between lanes on the autoradiograph indicate transcription bubbles. Naked NTS (6 mM KMnO 4  footprinting) is shown in lane 16. Important distances and positions are indicated on the right. Arrowheads indicate the respective active sites. (C) As in (B), but both in the absence and presence of TFIIS, modified with increasing amounts of permanganate (4, 8, and 18 mM). (D) Summary of results obtained in the absence (upper) and presence (lower) of TFIIS, drawn to scale.
    Figure Legend Snippet: Transcription Bubbles Remain Intact and Separated upon Collision (A) Experimental design. (B) Elongation complexes (end-labeled NTS) were incubated with NTPs and TFIIS, digested with T7 exonuclease, and modified with increasing amounts of KMnO 4 (12, 18, and 24 mM) as indicated and resolved by denaturing PAGE. Major T7 exonuclease-marked bands as seen in Figure 3 , lane 17, line, were isolated, cleaved at modified thymines with piperidine, and analyzed by denaturing PAGE. The positions of isolated bands are indicated at the top. Thin lines between lanes on the autoradiograph indicate transcription bubbles. Naked NTS (6 mM KMnO 4 footprinting) is shown in lane 16. Important distances and positions are indicated on the right. Arrowheads indicate the respective active sites. (C) As in (B), but both in the absence and presence of TFIIS, modified with increasing amounts of permanganate (4, 8, and 18 mM). (D) Summary of results obtained in the absence (upper) and presence (lower) of TFIIS, drawn to scale.

    Techniques Used: Labeling, Incubation, Modification, Polyacrylamide Gel Electrophoresis, Isolation, Autoradiography, Footprinting

    19) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    20) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    21) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

    Related Articles

    other:

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells
    Article Snippet: Together, these results support the notion that the presence of DNA ligase IV–XRCC4 protects DNA ends from end degradation by T7 exonuclease.

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *
    Article Snippet: T7 exonuclease and XhoI digestion suggested that the band that was unaffected following the addition of BCL2 with compatible ends was indeed circular ( C ).).

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *
    Article Snippet: Results showed that T7 exonuclease was able to digest the bands corresponding to the substrate and linear dimer DNA but not the one in between ( B , lanes 2 and 3 ) (data not shown), suggesting the circular nature of the products.

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells
    Article Snippet: We also show that DNA ligase IV–XRCC4 can contribute to the protection of ends from degradation by T7 exonuclease.

    Purification:

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *
    Article Snippet: .. T7 exonuclease digestion was performed by incubating purified EJ products with either increasing concentrations or 5 units of T7 exonuclease (New England Biolabs) at 25 °C for 2 h. In some cases, a fraction of EJ products was digested with XhoI (4 units) (37 °C for 4 h) prior to T7 exonuclease digestion. .. The EJ products of interest were cut out from PAGE, and DNA was eluted in a solution containing Tris (10 m m ), EDTA (1 m m ) and NaCl (0.5 m ).

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    New England Biolabs t7 exonuclease
    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against <t>T7</t> exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    T7 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Journal: Nucleic Acids Research

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    doi:

    Figure Lengend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Article Snippet: We also show that DNA ligase IV–XRCC4 can contribute to the protection of ends from degradation by T7 exonuclease.

    Techniques: Incubation, Labeling, Electrophoresis, Autoradiography

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Journal: The Journal of Biological Chemistry

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    doi: 10.1074/jbc.M110.140350

    Figure Lengend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Article Snippet: T7 exonuclease digestion was performed by incubating purified EJ products with either increasing concentrations or 5 units of T7 exonuclease (New England Biolabs) at 25 °C for 2 h. In some cases, a fraction of EJ products was digested with XhoI (4 units) (37 °C for 4 h) prior to T7 exonuclease digestion.

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis