dnase i footprinting  (New England Biolabs)


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    New England Biolabs dnase i footprinting
    ( A ) <t>DNase</t> I <t>footprinting</t> analysis of the binding of wild-type and N-terminally truncated derivatives of SimR to the simR–simX intergenic region. A DNA fragment containing the simR–simX intergenic region, 5′-end labelled on either the upper strand (left panel) or the lower strand (right panel), was exposed to DNase I in the presence of saturating concentrations of SimR protein (200 nM for wild-type SimR, SimRΔN10 and SimRΔN15; 400 nM for SimRΔN22 and SimRΔN25). The sequencing ladders were generated by subjecting the probes to Maxam-Gilbert G+A chemical sequencing. Regions protected from DNase I cleavage (operators O X and O R ) by wild-type SimR are indicated by solid vertical bars, and those protected by the N-terminally truncated SimR derivatives are indicated by open bars. Inverted repeats within the DNase I protected regions are indicated by convergent arrows. ( B ) Sequence of the simR–simX intergenic region summarizing the DNase I footprinting data. Regions protected by wild-type SimR are indicated by solid lines, and those protected by the N-terminally truncated SimR derivatives are indicated by dotted lines. Also indicated are the simRp and simXp transcription start points and putative −10 sequences, the simR and simX ribosome-binding sites (RBS), and the imperfect inverted repeats within the footprints.
    Dnase I Footprinting, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding"

    Article Title: The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr640

    ( A ) DNase I footprinting analysis of the binding of wild-type and N-terminally truncated derivatives of SimR to the simR–simX intergenic region. A DNA fragment containing the simR–simX intergenic region, 5′-end labelled on either the upper strand (left panel) or the lower strand (right panel), was exposed to DNase I in the presence of saturating concentrations of SimR protein (200 nM for wild-type SimR, SimRΔN10 and SimRΔN15; 400 nM for SimRΔN22 and SimRΔN25). The sequencing ladders were generated by subjecting the probes to Maxam-Gilbert G+A chemical sequencing. Regions protected from DNase I cleavage (operators O X and O R ) by wild-type SimR are indicated by solid vertical bars, and those protected by the N-terminally truncated SimR derivatives are indicated by open bars. Inverted repeats within the DNase I protected regions are indicated by convergent arrows. ( B ) Sequence of the simR–simX intergenic region summarizing the DNase I footprinting data. Regions protected by wild-type SimR are indicated by solid lines, and those protected by the N-terminally truncated SimR derivatives are indicated by dotted lines. Also indicated are the simRp and simXp transcription start points and putative −10 sequences, the simR and simX ribosome-binding sites (RBS), and the imperfect inverted repeats within the footprints.
    Figure Legend Snippet: ( A ) DNase I footprinting analysis of the binding of wild-type and N-terminally truncated derivatives of SimR to the simR–simX intergenic region. A DNA fragment containing the simR–simX intergenic region, 5′-end labelled on either the upper strand (left panel) or the lower strand (right panel), was exposed to DNase I in the presence of saturating concentrations of SimR protein (200 nM for wild-type SimR, SimRΔN10 and SimRΔN15; 400 nM for SimRΔN22 and SimRΔN25). The sequencing ladders were generated by subjecting the probes to Maxam-Gilbert G+A chemical sequencing. Regions protected from DNase I cleavage (operators O X and O R ) by wild-type SimR are indicated by solid vertical bars, and those protected by the N-terminally truncated SimR derivatives are indicated by open bars. Inverted repeats within the DNase I protected regions are indicated by convergent arrows. ( B ) Sequence of the simR–simX intergenic region summarizing the DNase I footprinting data. Regions protected by wild-type SimR are indicated by solid lines, and those protected by the N-terminally truncated SimR derivatives are indicated by dotted lines. Also indicated are the simRp and simXp transcription start points and putative −10 sequences, the simR and simX ribosome-binding sites (RBS), and the imperfect inverted repeats within the footprints.

    Techniques Used: Footprinting, Binding Assay, Sequencing, Generated

    2) Product Images from "Structural Basis for Eukaryotic Transcription-Coupled DNA Repair Initiation"

    Article Title: Structural Basis for Eukaryotic Transcription-Coupled DNA Repair Initiation

    Journal: Nature

    doi: 10.1038/nature24658

    Rad26 binds to the upstream DNA and bubble fork of Pol II EC and bends the upstream DNA a , Atomic model for the scaffold in the Pol II EC-Rad26 complex displayed inside the segmented cryo-EM density, obtained by subtracting Pol II and Rad26 from the Pol II EC-Rad26 map. Orange asterisk: Rad26 density not modeled. Top-right inset: scaffold density (in yellow) in the context of the full complex. b , Superposition of the scaffolds from the Pol II EC-Rad26 and Pol II EC structures, with the latter shown in darker colors. c , Close-up view of the interaction between Rad26 and the scaffold. d , DNase I footprinting assay of Pol II EC-Rad26. The experiment was repeated independently 3 times with similar results. e , Close-up of the Rad26 HD2-1 “wedge” (yellow arrow) that interacts with the upstream bubble fork. Same view as c except with Rad26 in a surface charge representation. Right inset: closer view of the interaction, looking from the transcription bubble towards the upstream DNA. g , Major interactions between Rad26 and the Wall and Protrusion regions of Pol II. The Pol II motifs that bind to Rad26 are shown as surface representations, with the corresponding residues listed. f , Effect of transcription bubble size in the affinity of Rad26 for Pol II EC. Mismatches were added to the upstream fork, downstream fork, or both. Data shown as mean and standard deviation (n = 3). P-values (two-tailed Student’s t test): not shown = not significant; * =
    Figure Legend Snippet: Rad26 binds to the upstream DNA and bubble fork of Pol II EC and bends the upstream DNA a , Atomic model for the scaffold in the Pol II EC-Rad26 complex displayed inside the segmented cryo-EM density, obtained by subtracting Pol II and Rad26 from the Pol II EC-Rad26 map. Orange asterisk: Rad26 density not modeled. Top-right inset: scaffold density (in yellow) in the context of the full complex. b , Superposition of the scaffolds from the Pol II EC-Rad26 and Pol II EC structures, with the latter shown in darker colors. c , Close-up view of the interaction between Rad26 and the scaffold. d , DNase I footprinting assay of Pol II EC-Rad26. The experiment was repeated independently 3 times with similar results. e , Close-up of the Rad26 HD2-1 “wedge” (yellow arrow) that interacts with the upstream bubble fork. Same view as c except with Rad26 in a surface charge representation. Right inset: closer view of the interaction, looking from the transcription bubble towards the upstream DNA. g , Major interactions between Rad26 and the Wall and Protrusion regions of Pol II. The Pol II motifs that bind to Rad26 are shown as surface representations, with the corresponding residues listed. f , Effect of transcription bubble size in the affinity of Rad26 for Pol II EC. Mismatches were added to the upstream fork, downstream fork, or both. Data shown as mean and standard deviation (n = 3). P-values (two-tailed Student’s t test): not shown = not significant; * =

    Techniques Used: Footprinting, Standard Deviation, Two Tailed Test

    3) Product Images from "Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF"

    Article Title: Functional Phosphorylation Sites in the C-Terminal Region of the Multivalent Multifunctional Transcriptional Factor CTCF

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.6.2221-2234.2001

    Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.
    Figure Legend Snippet: Mutating of the major C-terminal CKII sites does not affect interaction of CTCF with c- myc CTSs assayed by EMSAs (A), methylation interference (B), and DNase I footprinting (C). Lanes: 1, full-length (FL) wtCTCF; 2, CTCF/2-mut; 3, CTCF/4-mut; 4, 11ZF protein. See the text for more details. F, free probe; B, protein-bound probe.

    Techniques Used: Methylation, Footprinting

    4) Product Images from "One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator"

    Article Title: One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator

    Journal: mBio

    doi: 10.1128/mBio.01860-16

    VirF 21 autoregulates virF expression. (A) β-Galactosidase activity of virF-lacZ fusions in response to increased levels of VirF 21 or VirF 30 . Ectopic expression of VirF 21 or VirF 30 was obtained in E. coli p virF - lacZ strains carrying pAC-21 or pAC-30, respectively. pGIP7, empty vector. Values are averages of three experiments, and standard deviations indicated. (B) In vivo levels of lacZ mRNA were monitored in the same samples used in the β-galactosidase assay summarized in panel A. Triplicate samples were evaluated, and error bars indicate standard errors of the mean expression levels (RQ values). (C) In vivo levels of virB mRNA were monitored in the Δ virF S. flexneri strain (M90T Fd) carrying pF-M81L (VirF 30 ) or ectopically expressing VirF 21 under IPTG control (pAC-21). Triplicate samples were evaluated; error bars show standard errors of the mean expression levels (RQ values). (D) Western blot analysis of cell extracts of M90T F3xFT carrying pAC-21, with or without ectopic induction of VirF 21 . The level of expression of VirF 30 was monitored with an anti-FLAG antibody. VirF 21 induction was monitored with an anti-VirF antibody. Asterisks indicate unspecific cross-hybridization with an unknown protein in the extract. (E) Identification of the VirF 21 binding site on the virF promoter based on DNase I footprinting results. Plasmid pMYSH6504 DNA ( 41 ) was incubated with 0, 10 or 20 µl of in vitro -translated VirF 21 . The samples were DNase I treated and subsequently analyzed as described in Materials and Methods, using ML-U30 and ML-U29 as primers. Sequencing ladders were generated with the same 5′-end-labeled plus- or minus-strand-specific primers. The VirF 21 -protected site is indicated by vertical black lines and shown by shading on both strands of the virF promoter sequence.
    Figure Legend Snippet: VirF 21 autoregulates virF expression. (A) β-Galactosidase activity of virF-lacZ fusions in response to increased levels of VirF 21 or VirF 30 . Ectopic expression of VirF 21 or VirF 30 was obtained in E. coli p virF - lacZ strains carrying pAC-21 or pAC-30, respectively. pGIP7, empty vector. Values are averages of three experiments, and standard deviations indicated. (B) In vivo levels of lacZ mRNA were monitored in the same samples used in the β-galactosidase assay summarized in panel A. Triplicate samples were evaluated, and error bars indicate standard errors of the mean expression levels (RQ values). (C) In vivo levels of virB mRNA were monitored in the Δ virF S. flexneri strain (M90T Fd) carrying pF-M81L (VirF 30 ) or ectopically expressing VirF 21 under IPTG control (pAC-21). Triplicate samples were evaluated; error bars show standard errors of the mean expression levels (RQ values). (D) Western blot analysis of cell extracts of M90T F3xFT carrying pAC-21, with or without ectopic induction of VirF 21 . The level of expression of VirF 30 was monitored with an anti-FLAG antibody. VirF 21 induction was monitored with an anti-VirF antibody. Asterisks indicate unspecific cross-hybridization with an unknown protein in the extract. (E) Identification of the VirF 21 binding site on the virF promoter based on DNase I footprinting results. Plasmid pMYSH6504 DNA ( 41 ) was incubated with 0, 10 or 20 µl of in vitro -translated VirF 21 . The samples were DNase I treated and subsequently analyzed as described in Materials and Methods, using ML-U30 and ML-U29 as primers. Sequencing ladders were generated with the same 5′-end-labeled plus- or minus-strand-specific primers. The VirF 21 -protected site is indicated by vertical black lines and shown by shading on both strands of the virF promoter sequence.

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation, In Vivo, Western Blot, Hybridization, Binding Assay, Footprinting, Incubation, In Vitro, Sequencing, Generated, Labeling

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    New England Biolabs dnase i footprinting
    ( A ) <t>DNase</t> I <t>footprinting</t> analysis of the binding of wild-type and N-terminally truncated derivatives of SimR to the simR–simX intergenic region. A DNA fragment containing the simR–simX intergenic region, 5′-end labelled on either the upper strand (left panel) or the lower strand (right panel), was exposed to DNase I in the presence of saturating concentrations of SimR protein (200 nM for wild-type SimR, SimRΔN10 and SimRΔN15; 400 nM for SimRΔN22 and SimRΔN25). The sequencing ladders were generated by subjecting the probes to Maxam-Gilbert G+A chemical sequencing. Regions protected from DNase I cleavage (operators O X and O R ) by wild-type SimR are indicated by solid vertical bars, and those protected by the N-terminally truncated SimR derivatives are indicated by open bars. Inverted repeats within the DNase I protected regions are indicated by convergent arrows. ( B ) Sequence of the simR–simX intergenic region summarizing the DNase I footprinting data. Regions protected by wild-type SimR are indicated by solid lines, and those protected by the N-terminally truncated SimR derivatives are indicated by dotted lines. Also indicated are the simRp and simXp transcription start points and putative −10 sequences, the simR and simX ribosome-binding sites (RBS), and the imperfect inverted repeats within the footprints.
    Dnase I Footprinting, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i footprinting/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i footprinting - by Bioz Stars, 2022-08
    86/100 stars
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    99
    New England Biolabs dnase i rnase free
    <t>DNase</t> I footprinting of CTCF associated with 5caC-containing DNA. (A) Overview of CTCF DNase I footprinting with variably carboxylated radiolabeled DNA probes representing strong and weak CTCF motifs. (B) Gel analysis of CTCF DNase I footprinting performed with the CD45 exon 5 probe, ±CpG 5caC. The DNase I reaction contained 342.3-42.8 nM CTCF and 7.52 nM DNA probe. The location of carboxylated cytosine residues are indicated by C*. M signifies oligonucleotide marker and arrowheads indicate lanes used to generate histograms. (C) Lane histogram densitometry analysis of the indicated lanes from (B). The region protected by CTCF binding is shown.
    Dnase I Rnase Free, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    ( A ) DNase I footprinting analysis of the binding of wild-type and N-terminally truncated derivatives of SimR to the simR–simX intergenic region. A DNA fragment containing the simR–simX intergenic region, 5′-end labelled on either the upper strand (left panel) or the lower strand (right panel), was exposed to DNase I in the presence of saturating concentrations of SimR protein (200 nM for wild-type SimR, SimRΔN10 and SimRΔN15; 400 nM for SimRΔN22 and SimRΔN25). The sequencing ladders were generated by subjecting the probes to Maxam-Gilbert G+A chemical sequencing. Regions protected from DNase I cleavage (operators O X and O R ) by wild-type SimR are indicated by solid vertical bars, and those protected by the N-terminally truncated SimR derivatives are indicated by open bars. Inverted repeats within the DNase I protected regions are indicated by convergent arrows. ( B ) Sequence of the simR–simX intergenic region summarizing the DNase I footprinting data. Regions protected by wild-type SimR are indicated by solid lines, and those protected by the N-terminally truncated SimR derivatives are indicated by dotted lines. Also indicated are the simRp and simXp transcription start points and putative −10 sequences, the simR and simX ribosome-binding sites (RBS), and the imperfect inverted repeats within the footprints.

    Journal: Nucleic Acids Research

    Article Title: The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding

    doi: 10.1093/nar/gkr640

    Figure Lengend Snippet: ( A ) DNase I footprinting analysis of the binding of wild-type and N-terminally truncated derivatives of SimR to the simR–simX intergenic region. A DNA fragment containing the simR–simX intergenic region, 5′-end labelled on either the upper strand (left panel) or the lower strand (right panel), was exposed to DNase I in the presence of saturating concentrations of SimR protein (200 nM for wild-type SimR, SimRΔN10 and SimRΔN15; 400 nM for SimRΔN22 and SimRΔN25). The sequencing ladders were generated by subjecting the probes to Maxam-Gilbert G+A chemical sequencing. Regions protected from DNase I cleavage (operators O X and O R ) by wild-type SimR are indicated by solid vertical bars, and those protected by the N-terminally truncated SimR derivatives are indicated by open bars. Inverted repeats within the DNase I protected regions are indicated by convergent arrows. ( B ) Sequence of the simR–simX intergenic region summarizing the DNase I footprinting data. Regions protected by wild-type SimR are indicated by solid lines, and those protected by the N-terminally truncated SimR derivatives are indicated by dotted lines. Also indicated are the simRp and simXp transcription start points and putative −10 sequences, the simR and simX ribosome-binding sites (RBS), and the imperfect inverted repeats within the footprints.

    Article Snippet: DNase I footprinting Templates for DNase I footprinting were amplified by PCR using one unlabelled primer and one primer 5′-end labelled using [γ32 -P] ATP and T4 polynucleotide kinase (New England Biolabs).

    Techniques: Footprinting, Binding Assay, Sequencing, Generated

    Rad26 binds to the upstream DNA and bubble fork of Pol II EC and bends the upstream DNA a , Atomic model for the scaffold in the Pol II EC-Rad26 complex displayed inside the segmented cryo-EM density, obtained by subtracting Pol II and Rad26 from the Pol II EC-Rad26 map. Orange asterisk: Rad26 density not modeled. Top-right inset: scaffold density (in yellow) in the context of the full complex. b , Superposition of the scaffolds from the Pol II EC-Rad26 and Pol II EC structures, with the latter shown in darker colors. c , Close-up view of the interaction between Rad26 and the scaffold. d , DNase I footprinting assay of Pol II EC-Rad26. The experiment was repeated independently 3 times with similar results. e , Close-up of the Rad26 HD2-1 “wedge” (yellow arrow) that interacts with the upstream bubble fork. Same view as c except with Rad26 in a surface charge representation. Right inset: closer view of the interaction, looking from the transcription bubble towards the upstream DNA. g , Major interactions between Rad26 and the Wall and Protrusion regions of Pol II. The Pol II motifs that bind to Rad26 are shown as surface representations, with the corresponding residues listed. f , Effect of transcription bubble size in the affinity of Rad26 for Pol II EC. Mismatches were added to the upstream fork, downstream fork, or both. Data shown as mean and standard deviation (n = 3). P-values (two-tailed Student’s t test): not shown = not significant; * =

    Journal: Nature

    Article Title: Structural Basis for Eukaryotic Transcription-Coupled DNA Repair Initiation

    doi: 10.1038/nature24658

    Figure Lengend Snippet: Rad26 binds to the upstream DNA and bubble fork of Pol II EC and bends the upstream DNA a , Atomic model for the scaffold in the Pol II EC-Rad26 complex displayed inside the segmented cryo-EM density, obtained by subtracting Pol II and Rad26 from the Pol II EC-Rad26 map. Orange asterisk: Rad26 density not modeled. Top-right inset: scaffold density (in yellow) in the context of the full complex. b , Superposition of the scaffolds from the Pol II EC-Rad26 and Pol II EC structures, with the latter shown in darker colors. c , Close-up view of the interaction between Rad26 and the scaffold. d , DNase I footprinting assay of Pol II EC-Rad26. The experiment was repeated independently 3 times with similar results. e , Close-up of the Rad26 HD2-1 “wedge” (yellow arrow) that interacts with the upstream bubble fork. Same view as c except with Rad26 in a surface charge representation. Right inset: closer view of the interaction, looking from the transcription bubble towards the upstream DNA. g , Major interactions between Rad26 and the Wall and Protrusion regions of Pol II. The Pol II motifs that bind to Rad26 are shown as surface representations, with the corresponding residues listed. f , Effect of transcription bubble size in the affinity of Rad26 for Pol II EC. Mismatches were added to the upstream fork, downstream fork, or both. Data shown as mean and standard deviation (n = 3). P-values (two-tailed Student’s t test): not shown = not significant; * =

    Article Snippet: DNase I footprinting An aliquot of 20 nM Pol II EC (with 5´-P labeled template DNA strand) was incubated with 0-150 nM Rad26 in the binding buffer (see above) at 23 °C for 30 min. Then DNase I (NEB, USA) was added to a final concentration of 0.04 units/ml and the digestion was carried out for 1 minute (50 seconds if Rad26 was absent) at 23 °C.

    Techniques: Footprinting, Standard Deviation, Two Tailed Test

    DNase I footprinting of CTCF associated with 5caC-containing DNA. (A) Overview of CTCF DNase I footprinting with variably carboxylated radiolabeled DNA probes representing strong and weak CTCF motifs. (B) Gel analysis of CTCF DNase I footprinting performed with the CD45 exon 5 probe, ±CpG 5caC. The DNase I reaction contained 342.3-42.8 nM CTCF and 7.52 nM DNA probe. The location of carboxylated cytosine residues are indicated by C*. M signifies oligonucleotide marker and arrowheads indicate lanes used to generate histograms. (C) Lane histogram densitometry analysis of the indicated lanes from (B). The region protected by CTCF binding is shown.

    Journal: bioRxiv

    Article Title: TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

    doi: 10.1101/480525

    Figure Lengend Snippet: DNase I footprinting of CTCF associated with 5caC-containing DNA. (A) Overview of CTCF DNase I footprinting with variably carboxylated radiolabeled DNA probes representing strong and weak CTCF motifs. (B) Gel analysis of CTCF DNase I footprinting performed with the CD45 exon 5 probe, ±CpG 5caC. The DNase I reaction contained 342.3-42.8 nM CTCF and 7.52 nM DNA probe. The location of carboxylated cytosine residues are indicated by C*. M signifies oligonucleotide marker and arrowheads indicate lanes used to generate histograms. (C) Lane histogram densitometry analysis of the indicated lanes from (B). The region protected by CTCF binding is shown.

    Article Snippet: DNase I (1.25E-3U/ul; NEB, #M0303L) was added and samples were incubated at room temperature for the indicated time intervals.

    Techniques: Footprinting, Marker, Binding Assay

    DNase I Footprinting of Control DNA in Nucleosomes. (a) DNase I cleavage of bare and nucleosomal control DNA duplexes. The + symbol indicates a location of relatively increased cleavage in the nucleosomal DNA, while * indicates areas of relatively diminished cleavage. Note that the cleavage in the nucleosomal DNA is globally lower than in the bare DNA, both due to shielding by histones and to the presence of unlabeled chicken DNA; both nuclease concentration and visual contrast have been increased in the nucleosomal sample to compensate. (b,c) Changes in DNase I cleavage mapped to the model nucleosome upon which this 146 base pair sequence is loosely based (PDB ID 1AOI, reference 42 ). The histone octamer has been removed for clarity. Regions of strong cleavage are colored green, weak cleavage is colored yellow, and absent or drastically reduced cleavage is red. No data are available where the DNA is colored blue. The most robustly cleaved regions correlate with locations where the minor groove is open to the nuclease and the backbone points outward; regions of diminished or absent cleavage point inward toward the octamer.

    Journal: Biochemistry

    Article Title: Effect of the Spiroiminodihydantoin Lesion on Nucleosome Stability and Positioning

    doi: 10.1021/acs.biochem.6b00093

    Figure Lengend Snippet: DNase I Footprinting of Control DNA in Nucleosomes. (a) DNase I cleavage of bare and nucleosomal control DNA duplexes. The + symbol indicates a location of relatively increased cleavage in the nucleosomal DNA, while * indicates areas of relatively diminished cleavage. Note that the cleavage in the nucleosomal DNA is globally lower than in the bare DNA, both due to shielding by histones and to the presence of unlabeled chicken DNA; both nuclease concentration and visual contrast have been increased in the nucleosomal sample to compensate. (b,c) Changes in DNase I cleavage mapped to the model nucleosome upon which this 146 base pair sequence is loosely based (PDB ID 1AOI, reference 42 ). The histone octamer has been removed for clarity. Regions of strong cleavage are colored green, weak cleavage is colored yellow, and absent or drastically reduced cleavage is red. No data are available where the DNA is colored blue. The most robustly cleaved regions correlate with locations where the minor groove is open to the nuclease and the backbone points outward; regions of diminished or absent cleavage point inward toward the octamer.

    Article Snippet: Either concentrated histone exchange samples or bare DNA (as a control) were combined with bovine pancreatic DNase I (New England Biolabs) and incubated at 37 °C.

    Techniques: Footprinting, Concentration Assay, Sequencing