Journal: Nucleic Acids Research

Article Title: Identification of recognition residues for ligation-based detection and quantitation of pseudouridine and N6-methyladenosine

doi: 10.1093/nar/gkm657

Figure Lengend Snippet: ( A ) Chemical structures of Ψ and m 6 A. ( B ) Scheme for T4 DNA ligase-catalyzed joining of two DNA substrates. In the ternary RNA/DNA complex, the black line corresponds to the 30-mer RNA template with the modified nucleotide (open circle) located at the 15th position. Blue lines correspond to the ligation substrates with the recognition residue shown as a filled blue circle.

Article Snippet: The optimized ligation reactions were carried out with 0.15 µM 30-mer RNA with or without Ψ or m6 A modifications, 0.5 µM floater and 0.38 µM of 5′-32 P-labeled anchor in 66 mM Tris–HCl, pH 7.6, 0.5 mM ZnCl2 , 10 mM DTT, 66 µM ATP, 15% DMSO and 0.25 U/µl T4 DNA ligase (USB Inc.).

Techniques: Modification, Ligation

Journal: PLoS ONE

Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

doi: 10.1371/journal.pone.0138774

Figure Lengend Snippet: Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

Techniques: DNA Ligation, Labeling, Incubation, Ligation, Electrophoresis

Journal: PLoS ONE

Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

doi: 10.1371/journal.pone.0138774

Figure Lengend Snippet: Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

Techniques: Labeling, Incubation, Titration, Ligation, Electrophoresis

Journal: PLoS ONE

Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

doi: 10.1371/journal.pone.0138774

Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

Techniques: Mutagenesis, Labeling, Incubation, Ligation, Electrophoresis

Journal: PLoS ONE

Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

doi: 10.1371/journal.pone.0138774

Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

Techniques: Mutagenesis, Labeling, Ligation, Electrophoresis

Journal: Biochemistry and Biophysics Reports

Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

doi: 10.1016/j.bbrep.2016.10.006

Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles in the absence of a magnetic field on the ambient temperature. The ordinate axis represents the ligation efficiency, which is normalized by that at 16 °C. The standard deviations are obtained from 6 independent experiments.

Article Snippet: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

Techniques: DNA Ligation, Ligation

Journal: Biochemistry and Biophysics Reports

Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

doi: 10.1016/j.bbrep.2016.10.006

Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The ambient temperature is 16 °C. The ordinate axis represents the ligation efficiency under an ac magnetic field, which is normalized by that in the absence of a magnetic field. The inset shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles, noting that the surface temperature increases with an increase in the field amplitude. The standard deviations are obtained from 6 independent experiments.

Article Snippet: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

Techniques: DNA Ligation, Ligation