exonuclease deficient dna polymerase  (New England Biolabs)


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    Name:
    DNA Polymerase I Klenow Fragment
    Description:
    DNA Polymerase I Klenow Fragment 1 000 units
    Catalog Number:
    M0210L
    Price:
    248
    Category:
    DNA Polymerases
    Size:
    1 000 units
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    Structured Review

    New England Biolabs exonuclease deficient dna polymerase
    DNA Polymerase I Klenow Fragment
    DNA Polymerase I Klenow Fragment 1 000 units
    https://www.bioz.com/result/exonuclease deficient dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exonuclease deficient dna polymerase - by Bioz Stars, 2021-09
    99/100 stars

    Images

    1) Product Images from "Single-stranded nucleic acid sensing and coacervation by linker histone H1"

    Article Title: Single-stranded nucleic acid sensing and coacervation by linker histone H1

    Journal: bioRxiv

    doi: 10.1101/2021.03.17.435841

    H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A biotinylated λ-DNA molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.
    Figure Legend Snippet: H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A biotinylated λ-DNA molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.

    Techniques Used: Fluorescence, Microscopy, Binding Assay, Imaging, Recombinase Polymerase Amplification

    2) Product Images from "Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase"

    Article Title: Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase

    Journal: Cell

    doi: 10.1016/j.cell.2019.06.032

    CMG Undergoes Directional Translocation on ssDNA (A) Schematic of the experimental setup. Individual DNA tethers were formed in channels 1–3 separated by laminar flow containing streptavidin-coated beads, biotinylated λ-DNA, and buffer, respectively. They were subsequently moved to orthogonal channels 4 and 5 for protein loading and imaging. The illustration in the zoom-in box was not drawn to scale. (B) (Left) Cartoon and 2D scan of a tethered ssDNA loaded with multiple Cy3-CMGs. (Right) Representative kymographs of CMG movement in the presence of 1 mM ATP and 10 nM Mcm10 under 5 pN of tension. (C) Representative kymographs of Cy3-CMG (green) on ssDNA in the presence of Mcm10 but without ATP. (D) Distribution of CMG translocation rates on ssDNA in the absence (red) and presence (blue) of ATP ( n = 40 and 62, respectively). .
    Figure Legend Snippet: CMG Undergoes Directional Translocation on ssDNA (A) Schematic of the experimental setup. Individual DNA tethers were formed in channels 1–3 separated by laminar flow containing streptavidin-coated beads, biotinylated λ-DNA, and buffer, respectively. They were subsequently moved to orthogonal channels 4 and 5 for protein loading and imaging. The illustration in the zoom-in box was not drawn to scale. (B) (Left) Cartoon and 2D scan of a tethered ssDNA loaded with multiple Cy3-CMGs. (Right) Representative kymographs of CMG movement in the presence of 1 mM ATP and 10 nM Mcm10 under 5 pN of tension. (C) Representative kymographs of Cy3-CMG (green) on ssDNA in the presence of Mcm10 but without ATP. (D) Distribution of CMG translocation rates on ssDNA in the absence (red) and presence (blue) of ATP ( n = 40 and 62, respectively). .

    Techniques Used: Translocation Assay, Imaging

    Related Articles

    Incubation:

    Article Title: Enhancer-promoter interactions and transcription are maintained upon acute loss of CTCF, cohesin, WAPL, and YY1
    Article Snippet: .. First, the pellet was resuspended in the end-repair buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 100 µg/mL BSA, 2 mM ATP, 5 mM DTT) and the 5’-ends were phosphorylated with 25 units of T4 Polynucleotide Kinase (NEB #M0201) for 15 min at 37 °C while shaking in a thermomixer at 1000 rpm for an interval of 15 sec every 3 min. Second, to convert the mixed types of nucleosomal ends to cohesive ends, we added 25 units of DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210) directly to the reaction and incubated the tube for an additional 15 min at 37 °C while shaking in a thermomixer at 1000 rpm for an interval of 15 sec every 3 min. Third, to repair the nucleosomal DNA ends to the blunt and ligatable ends, we supplemented 66 µM of dNTPs (dTTP, dGTP (NEB #N0446), biotin-dATP (Jena Bioscience #NU-835-BIO14), biotin-dCTP (Jena Bioscience #NU-809-BIOX), and 1X T4 DNA ligase reaction buffer (NEB #B0202) directly into the reaction and incubated for 45 min at 25 °C while shaking in a thermomixer at 1000 rpm for an interval of 15 sec every 3 min. ..

    Article Title: SARS-CoV-2 Restructures the Host Chromatin Architecture
    Article Snippet: .. To fill-in the DNA overhangs and add biotin, 35U DNA polymerase I (Klenow, NEB, M0210) together with 10 μl of 1mM biotin-dATP (Jeana Bioscience), 1μl 10mM dCTP/dGTP/dTTP were added and incubated at 37°C for 1 hour with rotation. ..

    Article Title: Nucleosome positioning on large tandem DNA repeats of the ‘601’ sequence engineered in Saccharomyces cerevisiae
    Article Snippet: .. The reaction was incubated for 30 minutes at 37°C, purified using the QIAquick PCR Purification Kit (Qiagen) and eluted in 80 µL H2 O. Nucleosomal DNA was 5’ phosphorylated by adding 333 µM dNTPs, 50 units of T4 polynucleotide kinase (NEB), 15 units of T4 DNA polymerase (NEB) and 5 units of Klenow DNA polymerase (NEB) to 80 µ L of repaired mononucleosomal DNA to a final reaction volume of 120 µ L. The reaction was incubated 30 minutes at room temperature, purified using the QIAquick PCR Purification Kit and eluted in 30 µ L H2O. ..

    Labeling:

    Article Title: Pax7 pioneer factor action requires both paired and homeo DNA binding domains
    Article Snippet: .. Labeling was done at room temperature for 20 min in the following buffer: 50 mM Tris pH 7,5, 10 mM MgCl2 , 1 mM DTT and 0.5 mg/ml BSA supplemented with 0.25 μM dGTP, dTTP, 0,2 μM α-32 P-dCTP (3,000 Ci/mmol, 10 mCi/mL, Perkin Elmer) and 1 unit of DNA polymerase I Large (Klenow) fragment from E. coli (NEB, catalog # M0210M). ..

    Purification:

    Article Title: Nucleosome positioning on large tandem DNA repeats of the ‘601’ sequence engineered in Saccharomyces cerevisiae
    Article Snippet: .. The reaction was incubated for 30 minutes at 37°C, purified using the QIAquick PCR Purification Kit (Qiagen) and eluted in 80 µL H2 O. Nucleosomal DNA was 5’ phosphorylated by adding 333 µM dNTPs, 50 units of T4 polynucleotide kinase (NEB), 15 units of T4 DNA polymerase (NEB) and 5 units of Klenow DNA polymerase (NEB) to 80 µ L of repaired mononucleosomal DNA to a final reaction volume of 120 µ L. The reaction was incubated 30 minutes at room temperature, purified using the QIAquick PCR Purification Kit and eluted in 30 µ L H2O. ..

    Polymerase Chain Reaction:

    Article Title: Nucleosome positioning on large tandem DNA repeats of the ‘601’ sequence engineered in Saccharomyces cerevisiae
    Article Snippet: .. The reaction was incubated for 30 minutes at 37°C, purified using the QIAquick PCR Purification Kit (Qiagen) and eluted in 80 µL H2 O. Nucleosomal DNA was 5’ phosphorylated by adding 333 µM dNTPs, 50 units of T4 polynucleotide kinase (NEB), 15 units of T4 DNA polymerase (NEB) and 5 units of Klenow DNA polymerase (NEB) to 80 µ L of repaired mononucleosomal DNA to a final reaction volume of 120 µ L. The reaction was incubated 30 minutes at room temperature, purified using the QIAquick PCR Purification Kit and eluted in 30 µ L H2O. ..

    Sonication:

    Article Title: Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations
    Article Snippet: .. Genomic DNA was extracted and approximately 10 μg of total genomic DNA was extracted and sonicated to an average size of 500 bp, on a Covaris S220 (LGC Genomics) in microtubes AFA (6 × 16 mm) using the following setup: peak incident power: 105 W, duty factor: 5%, 200 cycles, 80 s. DNA ends were subsequently repaired with T4 DNA polymerase (15 units, NEBiolabs) and Klenow DNA polymerase (5 units, NEBiolabs) and phosphorylated with T4 DNA kinase (50 units, NEBiolabs). ..

    Synthesized:

    Article Title: Viral Metagenomics Reveals Diverse Viruses in the Feces Samples of Raccoon Dogs
    Article Snippet: .. The cDNA of viral RNA was synthesized by using reverse transcription with six-base random primers, and then Klenow Fragment DNA polymerase (New England Biolabs, MA, USA) was used to generate the complementary chain of cDNA. ..

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  • 99
    New England Biolabs exonuclease deficient klenow fragment kf
    Translesion DNA synthesis by <t>Klenow</t> fragment (KF − ) and DNA polymerase η (pol η) on the 12-mer/24-mer primer-template duplex non-modified or containing a single, monofunctional adduct of AMD in the presence of all four <t>dNTPs</t> (“running start” DNA synthesis) for various time intervals (time points of 5–60 min are shown above the gels). The sequence of the primer–template sequence is shown in ( A ) and the position of the platinated G residue is indicated. Representative images of the DNA polymerase reaction products resolved on 15% polyacrylamide (PAA) gel are shown for DNA synthesis ( B ) by KF − in the presence of 25 μM of each of the four dNTPs and ( C ) by pol η in the presence of 100 μM of each of the four dNTPs. Lanes in Figure 2 B: 1, 3, 5, 7, 9, undamaged template; 2, 4, 6, 8, 10, the template containing the single, site-specific monofunctional adduct of AMD. Lanes in ( C ): 1–5, noPt, DNA synthesis using an undamaged template; 6–10, AMD, DNA synthesis using the template containing the single, monofunctional adduct of AMD. The pause sites (the product lengths) are shown on the right or left side of the gels.
    Exonuclease Deficient Klenow Fragment Kf, 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
    https://www.bioz.com/result/exonuclease deficient klenow fragment kf/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exonuclease deficient klenow fragment kf - by Bioz Stars, 2021-09
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    99
    New England Biolabs exonuclease deficient klenow fragment
    Survey of selectivity of enzymatic nucleotide incorporation opposite yDNA bases. A and C show yDNA template bases; B and D show natural bases as controls. Enzymes are Thermococcus litoralis <t>DNA</t> polymerase (Vent exo−) and <t>Klenow</t> fragment of DNA
    Exonuclease Deficient Klenow Fragment, 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
    https://www.bioz.com/result/exonuclease deficient klenow fragment/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exonuclease deficient klenow fragment - by Bioz Stars, 2021-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs exonuclease deficient dna polymerase
    H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A <t>biotinylated</t> <t>λ-DNA</t> molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.
    Exonuclease Deficient Dna Polymerase, 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
    https://www.bioz.com/result/exonuclease deficient dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exonuclease deficient dna polymerase - by Bioz Stars, 2021-09
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    Image Search Results


    Translesion DNA synthesis by Klenow fragment (KF − ) and DNA polymerase η (pol η) on the 12-mer/24-mer primer-template duplex non-modified or containing a single, monofunctional adduct of AMD in the presence of all four dNTPs (“running start” DNA synthesis) for various time intervals (time points of 5–60 min are shown above the gels). The sequence of the primer–template sequence is shown in ( A ) and the position of the platinated G residue is indicated. Representative images of the DNA polymerase reaction products resolved on 15% polyacrylamide (PAA) gel are shown for DNA synthesis ( B ) by KF − in the presence of 25 μM of each of the four dNTPs and ( C ) by pol η in the presence of 100 μM of each of the four dNTPs. Lanes in Figure 2 B: 1, 3, 5, 7, 9, undamaged template; 2, 4, 6, 8, 10, the template containing the single, site-specific monofunctional adduct of AMD. Lanes in ( C ): 1–5, noPt, DNA synthesis using an undamaged template; 6–10, AMD, DNA synthesis using the template containing the single, monofunctional adduct of AMD. The pause sites (the product lengths) are shown on the right or left side of the gels.

    Journal: International Journal of Molecular Sciences

    Article Title: Thermodynamic Insights by Microscale Thermophoresis into Translesion DNA Synthesis Catalyzed by DNA Polymerases Across a Lesion of Antitumor Platinum–Acridine Complex

    doi: 10.3390/ijms21207806

    Figure Lengend Snippet: Translesion DNA synthesis by Klenow fragment (KF − ) and DNA polymerase η (pol η) on the 12-mer/24-mer primer-template duplex non-modified or containing a single, monofunctional adduct of AMD in the presence of all four dNTPs (“running start” DNA synthesis) for various time intervals (time points of 5–60 min are shown above the gels). The sequence of the primer–template sequence is shown in ( A ) and the position of the platinated G residue is indicated. Representative images of the DNA polymerase reaction products resolved on 15% polyacrylamide (PAA) gel are shown for DNA synthesis ( B ) by KF − in the presence of 25 μM of each of the four dNTPs and ( C ) by pol η in the presence of 100 μM of each of the four dNTPs. Lanes in Figure 2 B: 1, 3, 5, 7, 9, undamaged template; 2, 4, 6, 8, 10, the template containing the single, site-specific monofunctional adduct of AMD. Lanes in ( C ): 1–5, noPt, DNA synthesis using an undamaged template; 6–10, AMD, DNA synthesis using the template containing the single, monofunctional adduct of AMD. The pause sites (the product lengths) are shown on the right or left side of the gels.

    Article Snippet: The exonuclease deficient Klenow fragment (KF−), T4 polynucleotide kinase, and dNTPs were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: DNA Synthesis, Modification, Sequencing

    Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Journal: PLoS ONE

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

    doi: 10.1371/journal.pone.0007029

    Figure Lengend Snippet: Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Article Snippet: Step 5 The resulting oligonucleotide was purified using urea polyacrylamide gel electrophoresis and used as a template to generate double stranded DNA using exonuclease deficient Klenow fragment (New England Biolabs) and the primer, dsHP ( AGCUATAGTTTAGCGGCCGCTTATACTACTCAAAAAAAAAAAAAAAAAA ) according the manufacturer's instructions.

    Techniques: shRNA, Activated Clotting Time Assay, Blocking Assay, Purification, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing

    Survey of selectivity of enzymatic nucleotide incorporation opposite yDNA bases. A and C show yDNA template bases; B and D show natural bases as controls. Enzymes are Thermococcus litoralis DNA polymerase (Vent exo−) and Klenow fragment of DNA

    Journal: Chembiochem : a European journal of chemical biology

    Article Title: Polymerase Amplification, Cloning, and Gene Expression of Benzo-homologous "yDNA" Base Pairs

    doi: 10.1002/cbic.200800339

    Figure Lengend Snippet: Survey of selectivity of enzymatic nucleotide incorporation opposite yDNA bases. A and C show yDNA template bases; B and D show natural bases as controls. Enzymes are Thermococcus litoralis DNA polymerase (Vent exo−) and Klenow fragment of DNA

    Article Snippet: The enzymes used were the exonuclease-deficient Klenow fragment of DNA polymerase I (Cat. No. M0212L from New England Biolabs) and the exonuclease-deficient Vent DNA polymerase from Thermococcus litoralis (Cat. No. M0257S from New England Biolabs.

    Techniques:

    H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A biotinylated λ-DNA molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.

    Journal: bioRxiv

    Article Title: Single-stranded nucleic acid sensing and coacervation by linker histone H1

    doi: 10.1101/2021.03.17.435841

    Figure Lengend Snippet: H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A biotinylated λ-DNA molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.

    Article Snippet: DNA preparationTo create a terminally biotinylated dsDNA template, the 12-base 5’ overhang on each end of genomic DNA from bacteriophage λ (48,502 bp; Roche) was filled in with a mixture of natural and biotinylated nucleotides by the exonuclease-deficient DNA polymerase I Klenow fragment (New England BioLabs).

    Techniques: Fluorescence, Microscopy, Binding Assay, Imaging, Recombinase Polymerase Amplification