bamhi ecori  (New England Biolabs)


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    Name:
    BamHI
    Description:
    BamHI 50 000 units
    Catalog Number:
    r0136l
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    249
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    Category:
    Restriction Enzymes
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    New England Biolabs bamhi ecori
    BamHI
    BamHI 50 000 units
    https://www.bioz.com/result/bamhi ecori/product/New England Biolabs
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    bamhi ecori - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein"

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.780239

    DNA maps of the three circular DNA constructs used in the experiments. Linear dotted lines indicate the region of the inserted construct. U stands for genomic DNA from the tyrT UAS sequence, and P stands for proV NRE sequence respectively. The full size of the constructs is 4 kb, and the inserts are 1.3 kb. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.
    Figure Legend Snippet: DNA maps of the three circular DNA constructs used in the experiments. Linear dotted lines indicate the region of the inserted construct. U stands for genomic DNA from the tyrT UAS sequence, and P stands for proV NRE sequence respectively. The full size of the constructs is 4 kb, and the inserts are 1.3 kb. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Techniques Used: Construct, Sequencing

    AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) deposited on APTES-treated mica for a H-NS:DNA molar ratio of 20:1. The lower panels show the respective distributions of maximal bridged filament length. Control DNA deposited on APTES surface is compact with many self-crossings. The addition of H-NS results in several bridged regions between DNA strands, stabilizing DNA plectonemes. In certain cases DNA is branched, with the branching probability highest for PUUP constructs (indicated with white arrows ). Similarly, as is the case in Fig. 2 , PUUP constructs formed the longest, whereas UPPU formed the shortest bridged filaments. Scale bar , 500 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.
    Figure Legend Snippet: AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) deposited on APTES-treated mica for a H-NS:DNA molar ratio of 20:1. The lower panels show the respective distributions of maximal bridged filament length. Control DNA deposited on APTES surface is compact with many self-crossings. The addition of H-NS results in several bridged regions between DNA strands, stabilizing DNA plectonemes. In certain cases DNA is branched, with the branching probability highest for PUUP constructs (indicated with white arrows ). Similarly, as is the case in Fig. 2 , PUUP constructs formed the longest, whereas UPPU formed the shortest bridged filaments. Scale bar , 500 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Techniques Used: Construct

    AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) and pBR322 DNA. The lower panels show the respective distributions of maximal bridged filament length. White continuous arrows indicate bridge-loop structures; dashed arrows indicate loop-bridge-loop structures. The H-NS:DNA molar ratio was 13:1. PUUP construct formed the longest filaments exclusively on bridge-loop structures; UPPU formed shorter filaments with exclusively loop-bridge-loop structures; pBR322 DNA, which did not contain any strong binding sites for H-NS formed shortest filaments. 500 nm scale bar. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.
    Figure Legend Snippet: AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) and pBR322 DNA. The lower panels show the respective distributions of maximal bridged filament length. White continuous arrows indicate bridge-loop structures; dashed arrows indicate loop-bridge-loop structures. The H-NS:DNA molar ratio was 13:1. PUUP construct formed the longest filaments exclusively on bridge-loop structures; UPPU formed shorter filaments with exclusively loop-bridge-loop structures; pBR322 DNA, which did not contain any strong binding sites for H-NS formed shortest filaments. 500 nm scale bar. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Techniques Used: Construct, Binding Assay

    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.
    Figure Legend Snippet: Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Techniques Used: Footprinting, Construct, Plasmid Preparation, Binding Assay, Sequencing

    Ultra high vacuum AFM. A , images of different constructs bound by H-NS for H-NS:DNA molar ratio 20:1. Scale bars , 100 nm. B , the close-up view of the PUUP- and UPPU-bridged filaments and the corresponding model with measured shape parameters. The bridging H-NS dimers are indicated by blue ellipsoids . In this model PUUP binds H-NS with high frequency, resulting in higher periodicity of the bridged filament. In contrast, cooperative binding of H-NS to consecutive high-affinity sites of tandem P elements in UPPU stabilizes a plectoneme with high pitch and lower height and periodicity compared with PUUP DNA. We note that the height difference between the PUUP- and UPPU-bridged filaments might be explained by different protein interaction sites involved in H-NS dimer formation ( 45 ). Scale bars , 50 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.
    Figure Legend Snippet: Ultra high vacuum AFM. A , images of different constructs bound by H-NS for H-NS:DNA molar ratio 20:1. Scale bars , 100 nm. B , the close-up view of the PUUP- and UPPU-bridged filaments and the corresponding model with measured shape parameters. The bridging H-NS dimers are indicated by blue ellipsoids . In this model PUUP binds H-NS with high frequency, resulting in higher periodicity of the bridged filament. In contrast, cooperative binding of H-NS to consecutive high-affinity sites of tandem P elements in UPPU stabilizes a plectoneme with high pitch and lower height and periodicity compared with PUUP DNA. We note that the height difference between the PUUP- and UPPU-bridged filaments might be explained by different protein interaction sites involved in H-NS dimer formation ( 45 ). Scale bars , 50 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Techniques Used: Construct, Binding Assay

    2) Product Images from "Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts"

    Article Title: Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018554

    Replication fork leading strands progress up to the psoralen crosslink. (A) Sequence of the plasmid around the psoralen ICL with restriction sites used in B and C. Double arrows indicate the size of the replicated strands (lagging or leading) spanning the ICL site for the control or repaired plasmid. Single arrows indicate the progression of the leading strand to the ICL for the crosslinked plasmid. The strand size is shown above each product. (B) Mapping of leading strand progression for pTUC and pTUC-PSO after 65 min of incubation in Xenopus egg extracts in the continuous presence of [α- 32 P]-dATP. Plasmids were digested by the indicated enzymes and analyzed on 10% polyacrylamide denaturing gel. Faint bands visible in lanes 3 and 4 likely result from a star activity of Sac II. The two 28 nt size indicators are at two different positions due to gel smiling. (C) Mapping of leading strand progression for pTUC and pTUC-PSO at 25, 35, 50, 65, 85, 95, 120 and 180 min. Plasmids were digested with EcoRI and BamHI and subjected to migration on a 10% polyacrylamide denaturing gel. 28 nt and 46 nt fragments correspond to the DNA size expected for the leading strand arriving at the psoralen from the EcoRI side or BamHI side, respectively.
    Figure Legend Snippet: Replication fork leading strands progress up to the psoralen crosslink. (A) Sequence of the plasmid around the psoralen ICL with restriction sites used in B and C. Double arrows indicate the size of the replicated strands (lagging or leading) spanning the ICL site for the control or repaired plasmid. Single arrows indicate the progression of the leading strand to the ICL for the crosslinked plasmid. The strand size is shown above each product. (B) Mapping of leading strand progression for pTUC and pTUC-PSO after 65 min of incubation in Xenopus egg extracts in the continuous presence of [α- 32 P]-dATP. Plasmids were digested by the indicated enzymes and analyzed on 10% polyacrylamide denaturing gel. Faint bands visible in lanes 3 and 4 likely result from a star activity of Sac II. The two 28 nt size indicators are at two different positions due to gel smiling. (C) Mapping of leading strand progression for pTUC and pTUC-PSO at 25, 35, 50, 65, 85, 95, 120 and 180 min. Plasmids were digested with EcoRI and BamHI and subjected to migration on a 10% polyacrylamide denaturing gel. 28 nt and 46 nt fragments correspond to the DNA size expected for the leading strand arriving at the psoralen from the EcoRI side or BamHI side, respectively.

    Techniques Used: Sequencing, Plasmid Preparation, Incubation, Activity Assay, Migration

    TFO and purification of monomodified plasmid. (A) Structure of the TFO linked in 3′ to 4, 5′, 8-trimethylpsoralen and in 5′ to biotin. (B) Localization of the TFO binding site and position of the psoralen ICL on pTUC plasmid. P and A are schematic representations of primers for q-PCR used in (D). P primers surround the psoralen ICL site and amplify a region of 113 nt, the A primers amplify an undamaged regions of 129 nt. (C) Analysis of the crosslinked plasmid after UV irradiation and before (input) or after (purified) DTT elution from a strepatvidin column. DNA was digested with BamHI + EcoRI and radioactively labelled before electrophoresis on a 10% polyacrylamide denaturing gel. Interpretative diagrams of the relevant molecular species are shown on lane sides. (D) Input and purified plasmids were subjected to q-PCR with primers P and A. Mean values of 3 q-PCRs for the input and 12 q-PCRs for the purified plasmid are presented. For calculation details see Material and Methods . (E) After purification pTUC-PSO was analyzed on a 0.8% agarose gel TBE 1x alongside with control plasmid and a molecular weight ladder.
    Figure Legend Snippet: TFO and purification of monomodified plasmid. (A) Structure of the TFO linked in 3′ to 4, 5′, 8-trimethylpsoralen and in 5′ to biotin. (B) Localization of the TFO binding site and position of the psoralen ICL on pTUC plasmid. P and A are schematic representations of primers for q-PCR used in (D). P primers surround the psoralen ICL site and amplify a region of 113 nt, the A primers amplify an undamaged regions of 129 nt. (C) Analysis of the crosslinked plasmid after UV irradiation and before (input) or after (purified) DTT elution from a strepatvidin column. DNA was digested with BamHI + EcoRI and radioactively labelled before electrophoresis on a 10% polyacrylamide denaturing gel. Interpretative diagrams of the relevant molecular species are shown on lane sides. (D) Input and purified plasmids were subjected to q-PCR with primers P and A. Mean values of 3 q-PCRs for the input and 12 q-PCRs for the purified plasmid are presented. For calculation details see Material and Methods . (E) After purification pTUC-PSO was analyzed on a 0.8% agarose gel TBE 1x alongside with control plasmid and a molecular weight ladder.

    Techniques Used: Purification, Plasmid Preparation, Binding Assay, Polymerase Chain Reaction, Irradiation, Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility
    Article Snippet: .. The PCR product was digested with HindIII and BamHI and ligated into pMAL-cR1 v. 2 digested with the same enzymes (New England Biolabs, Inc.). .. The construct was transformed into E. coli XL1 blue, and, for expression of the maltose-binding∷hydin fusion protein, into BL21.

    Clone Assay:

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). ..

    other:

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins
    Article Snippet: Escherichia coli topoisomerase I ( Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

    Plasmid Preparation:

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). ..

    Article Title: Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit
    Article Snippet: .. Kinase assays using plasmid DNA substrates were performed with pcDNA3.1 digested with either XhoI, BamHI, EcoRV, and KpnI or pCAG-GFP digested with XbaI or EcoRI (New England Biolabs). .. The specific sequences recognized by the restriction enzymes and DNA termini generated are shown in .

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae
    Article Snippet: .. The ura3 -deficient S. cerevisiae strain BJ5465 ( α ura3–52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL1 ) was obtained from LGCPromochem, the NucleoSpin Plasmid kit was purchased from Macherey-Nagel, and the restriction enzymes BamHI, NheI, SpeI, SacI, and NotI from New England Biolabs. ..

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    New England Biolabs bamhi ecori
    DNA maps of the three circular DNA constructs used in the experiments. Linear dotted lines indicate the region of the inserted construct. U stands for genomic DNA from the tyrT UAS sequence, and P stands for proV NRE sequence respectively. The full size of the constructs is 4 kb, and the inserts are 1.3 kb. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, <t>BamHI,</t> and <t>EcoRI</t> restriction endonucleases, respectively.
    Bamhi Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bamhi ecori/product/New England Biolabs
    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    bamhi ecori - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    85
    New England Biolabs bamh1 ecori
    DNA maps of the three circular DNA constructs used in the experiments. Linear dotted lines indicate the region of the inserted construct. U stands for genomic DNA from the tyrT UAS sequence, and P stands for proV NRE sequence respectively. The full size of the constructs is 4 kb, and the inserts are 1.3 kb. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, <t>BamHI,</t> and <t>EcoRI</t> restriction endonucleases, respectively.
    Bamh1 Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bamh1 ecori/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bamh1 ecori - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    Image Search Results


    DNA maps of the three circular DNA constructs used in the experiments. Linear dotted lines indicate the region of the inserted construct. U stands for genomic DNA from the tyrT UAS sequence, and P stands for proV NRE sequence respectively. The full size of the constructs is 4 kb, and the inserts are 1.3 kb. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    doi: 10.1074/jbc.M117.780239

    Figure Lengend Snippet: DNA maps of the three circular DNA constructs used in the experiments. Linear dotted lines indicate the region of the inserted construct. U stands for genomic DNA from the tyrT UAS sequence, and P stands for proV NRE sequence respectively. The full size of the constructs is 4 kb, and the inserts are 1.3 kb. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.

    Techniques: Construct, Sequencing

    AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) deposited on APTES-treated mica for a H-NS:DNA molar ratio of 20:1. The lower panels show the respective distributions of maximal bridged filament length. Control DNA deposited on APTES surface is compact with many self-crossings. The addition of H-NS results in several bridged regions between DNA strands, stabilizing DNA plectonemes. In certain cases DNA is branched, with the branching probability highest for PUUP constructs (indicated with white arrows ). Similarly, as is the case in Fig. 2 , PUUP constructs formed the longest, whereas UPPU formed the shortest bridged filaments. Scale bar , 500 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    doi: 10.1074/jbc.M117.780239

    Figure Lengend Snippet: AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) deposited on APTES-treated mica for a H-NS:DNA molar ratio of 20:1. The lower panels show the respective distributions of maximal bridged filament length. Control DNA deposited on APTES surface is compact with many self-crossings. The addition of H-NS results in several bridged regions between DNA strands, stabilizing DNA plectonemes. In certain cases DNA is branched, with the branching probability highest for PUUP constructs (indicated with white arrows ). Similarly, as is the case in Fig. 2 , PUUP constructs formed the longest, whereas UPPU formed the shortest bridged filaments. Scale bar , 500 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.

    Techniques: Construct

    AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) and pBR322 DNA. The lower panels show the respective distributions of maximal bridged filament length. White continuous arrows indicate bridge-loop structures; dashed arrows indicate loop-bridge-loop structures. The H-NS:DNA molar ratio was 13:1. PUUP construct formed the longest filaments exclusively on bridge-loop structures; UPPU formed shorter filaments with exclusively loop-bridge-loop structures; pBR322 DNA, which did not contain any strong binding sites for H-NS formed shortest filaments. 500 nm scale bar. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    doi: 10.1074/jbc.M117.780239

    Figure Lengend Snippet: AFM images of control and H-NS bound circular DNA constructs (PUUP, UPPU, and UPUP) and pBR322 DNA. The lower panels show the respective distributions of maximal bridged filament length. White continuous arrows indicate bridge-loop structures; dashed arrows indicate loop-bridge-loop structures. The H-NS:DNA molar ratio was 13:1. PUUP construct formed the longest filaments exclusively on bridge-loop structures; UPPU formed shorter filaments with exclusively loop-bridge-loop structures; pBR322 DNA, which did not contain any strong binding sites for H-NS formed shortest filaments. 500 nm scale bar. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.

    Techniques: Construct, Binding Assay

    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Journal: The Journal of Biological Chemistry

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    doi: 10.1074/jbc.M117.780239

    Figure Lengend Snippet: Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.

    Techniques: Footprinting, Construct, Plasmid Preparation, Binding Assay, Sequencing

    Ultra high vacuum AFM. A , images of different constructs bound by H-NS for H-NS:DNA molar ratio 20:1. Scale bars , 100 nm. B , the close-up view of the PUUP- and UPPU-bridged filaments and the corresponding model with measured shape parameters. The bridging H-NS dimers are indicated by blue ellipsoids . In this model PUUP binds H-NS with high frequency, resulting in higher periodicity of the bridged filament. In contrast, cooperative binding of H-NS to consecutive high-affinity sites of tandem P elements in UPPU stabilizes a plectoneme with high pitch and lower height and periodicity compared with PUUP DNA. We note that the height difference between the PUUP- and UPPU-bridged filaments might be explained by different protein interaction sites involved in H-NS dimer formation ( 45 ). Scale bars , 50 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Journal: The Journal of Biological Chemistry

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    doi: 10.1074/jbc.M117.780239

    Figure Lengend Snippet: Ultra high vacuum AFM. A , images of different constructs bound by H-NS for H-NS:DNA molar ratio 20:1. Scale bars , 100 nm. B , the close-up view of the PUUP- and UPPU-bridged filaments and the corresponding model with measured shape parameters. The bridging H-NS dimers are indicated by blue ellipsoids . In this model PUUP binds H-NS with high frequency, resulting in higher periodicity of the bridged filament. In contrast, cooperative binding of H-NS to consecutive high-affinity sites of tandem P elements in UPPU stabilizes a plectoneme with high pitch and lower height and periodicity compared with PUUP DNA. We note that the height difference between the PUUP- and UPPU-bridged filaments might be explained by different protein interaction sites involved in H-NS dimer formation ( 45 ). Scale bars , 50 nm. H , X , N , B , and E indicate the cleavage sites for HindIII, XbaI, NheI, BamHI, and EcoRI restriction endonucleases, respectively.

    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.

    Techniques: Construct, Binding Assay

    Replication fork leading strands progress up to the psoralen crosslink. (A) Sequence of the plasmid around the psoralen ICL with restriction sites used in B and C. Double arrows indicate the size of the replicated strands (lagging or leading) spanning the ICL site for the control or repaired plasmid. Single arrows indicate the progression of the leading strand to the ICL for the crosslinked plasmid. The strand size is shown above each product. (B) Mapping of leading strand progression for pTUC and pTUC-PSO after 65 min of incubation in Xenopus egg extracts in the continuous presence of [α- 32 P]-dATP. Plasmids were digested by the indicated enzymes and analyzed on 10% polyacrylamide denaturing gel. Faint bands visible in lanes 3 and 4 likely result from a star activity of Sac II. The two 28 nt size indicators are at two different positions due to gel smiling. (C) Mapping of leading strand progression for pTUC and pTUC-PSO at 25, 35, 50, 65, 85, 95, 120 and 180 min. Plasmids were digested with EcoRI and BamHI and subjected to migration on a 10% polyacrylamide denaturing gel. 28 nt and 46 nt fragments correspond to the DNA size expected for the leading strand arriving at the psoralen from the EcoRI side or BamHI side, respectively.

    Journal: PLoS ONE

    Article Title: Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

    doi: 10.1371/journal.pone.0018554

    Figure Lengend Snippet: Replication fork leading strands progress up to the psoralen crosslink. (A) Sequence of the plasmid around the psoralen ICL with restriction sites used in B and C. Double arrows indicate the size of the replicated strands (lagging or leading) spanning the ICL site for the control or repaired plasmid. Single arrows indicate the progression of the leading strand to the ICL for the crosslinked plasmid. The strand size is shown above each product. (B) Mapping of leading strand progression for pTUC and pTUC-PSO after 65 min of incubation in Xenopus egg extracts in the continuous presence of [α- 32 P]-dATP. Plasmids were digested by the indicated enzymes and analyzed on 10% polyacrylamide denaturing gel. Faint bands visible in lanes 3 and 4 likely result from a star activity of Sac II. The two 28 nt size indicators are at two different positions due to gel smiling. (C) Mapping of leading strand progression for pTUC and pTUC-PSO at 25, 35, 50, 65, 85, 95, 120 and 180 min. Plasmids were digested with EcoRI and BamHI and subjected to migration on a 10% polyacrylamide denaturing gel. 28 nt and 46 nt fragments correspond to the DNA size expected for the leading strand arriving at the psoralen from the EcoRI side or BamHI side, respectively.

    Article Snippet: Denaturing gel electrophoretic analysis of crosslinked products Crosslinked samples were digested with BamHI + EcoRI (New England Biolabs), filled with [α-32 P]-dATP using AMV reverse transcriptase as described by the manufacturer (Finnzyme).

    Techniques: Sequencing, Plasmid Preparation, Incubation, Activity Assay, Migration

    TFO and purification of monomodified plasmid. (A) Structure of the TFO linked in 3′ to 4, 5′, 8-trimethylpsoralen and in 5′ to biotin. (B) Localization of the TFO binding site and position of the psoralen ICL on pTUC plasmid. P and A are schematic representations of primers for q-PCR used in (D). P primers surround the psoralen ICL site and amplify a region of 113 nt, the A primers amplify an undamaged regions of 129 nt. (C) Analysis of the crosslinked plasmid after UV irradiation and before (input) or after (purified) DTT elution from a strepatvidin column. DNA was digested with BamHI + EcoRI and radioactively labelled before electrophoresis on a 10% polyacrylamide denaturing gel. Interpretative diagrams of the relevant molecular species are shown on lane sides. (D) Input and purified plasmids were subjected to q-PCR with primers P and A. Mean values of 3 q-PCRs for the input and 12 q-PCRs for the purified plasmid are presented. For calculation details see Material and Methods . (E) After purification pTUC-PSO was analyzed on a 0.8% agarose gel TBE 1x alongside with control plasmid and a molecular weight ladder.

    Journal: PLoS ONE

    Article Title: Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

    doi: 10.1371/journal.pone.0018554

    Figure Lengend Snippet: TFO and purification of monomodified plasmid. (A) Structure of the TFO linked in 3′ to 4, 5′, 8-trimethylpsoralen and in 5′ to biotin. (B) Localization of the TFO binding site and position of the psoralen ICL on pTUC plasmid. P and A are schematic representations of primers for q-PCR used in (D). P primers surround the psoralen ICL site and amplify a region of 113 nt, the A primers amplify an undamaged regions of 129 nt. (C) Analysis of the crosslinked plasmid after UV irradiation and before (input) or after (purified) DTT elution from a strepatvidin column. DNA was digested with BamHI + EcoRI and radioactively labelled before electrophoresis on a 10% polyacrylamide denaturing gel. Interpretative diagrams of the relevant molecular species are shown on lane sides. (D) Input and purified plasmids were subjected to q-PCR with primers P and A. Mean values of 3 q-PCRs for the input and 12 q-PCRs for the purified plasmid are presented. For calculation details see Material and Methods . (E) After purification pTUC-PSO was analyzed on a 0.8% agarose gel TBE 1x alongside with control plasmid and a molecular weight ladder.

    Article Snippet: Denaturing gel electrophoretic analysis of crosslinked products Crosslinked samples were digested with BamHI + EcoRI (New England Biolabs), filled with [α-32 P]-dATP using AMV reverse transcriptase as described by the manufacturer (Finnzyme).

    Techniques: Purification, Plasmid Preparation, Binding Assay, Polymerase Chain Reaction, Irradiation, Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight