Amersham typhoon imager

The cleavage reaction was started by the addition of an oligonucleotide duplex (to a final concentration of 3.8 nM) to the activated Cas9-sgRNA complex. The reaction mixture was incubated at 37 °C in a buffer (20 mM HEPES pH 7.5, 125 mM KCl, 1 mM EDTA, 1 mM DTT, 6 mM MgCl₂, and 7% of glycerol). The volume of the reaction mixture was 60 μL. Aliquots of 10 µL were taken at different time points (from 10 to 420 min). The reaction was stopped by the addition of 10 μL of a solution containing 8 M urea, TBE (9 mM Tris-NH₂, 9 mM H₃BO₃, and 0.1 mM EDTA), 0.01% of bromophenol blue, and 0.01% of xylene cyanol with incubation for 5 min at 95 °C. The reaction products were analyzed by electrophoresis in a 15% polyacrylamide gel containing 8 M urea, followed by gel scanning on an Amersham Typhoon Imager (Cytiva, Marlborough, MA, USA).

The fluorescently labeled nucleosome (final concentration of 0.1 µm) was mixed with either 0, 0.4, 0.6, or 0.8 µm of the p53 protein, and incubated at 25°C for 30 min in buffer containing 20 mm Tris-HCl (pH 7.5 or pH 8.0), 100 mm NaCl, 0.4 mm ß-mercaptoethanol, 0.7 mm DTT, and 0.01% NP−40. The reaction products were then fractionated by 5% non-denaturing polyacrylamide gel electrophoresis in 0.5xTBE buffer, and visualized by ethidium bromide staining or Cy5 fluorescence signal detection, using an Amersham Typhoon imager (Cytiva).

An enzymatic hydroxylation of a single-strand m3C-containing DNA was studied using a chemical quenching technique recently described for the ALKBH2 dioxygenase in [7 (link)]. Briefly, a typical reaction mixture contained an equimolar amount (2 µM) of the protein and FAM-labeled DNA substrate (m3C_FAM) in a buffer consisting of 50 mM of HEPES-KOH (pH 7.8), 50 mM of KCl, 10 mM of MgCl₂, 1 mM of 2OG, 2 mM of sodium ascorbate, and 40 µM of (NH₄)₂Fe(SO₄)₂·6H₂O. An equal volume of 0.2 M NaOH was added to 5 µm of aliquots of the mixture to terminate the reaction at certain time points. After neutralization with HCl and desalting, each of the aliquots was supplemented by the complementary DNA strand and treated with the HpaII restriction endonuclease specific to the repaired sequence (CCGG) according to the manufacturer’s protocol. The resulting reaction product was separated from the substrate using denaturating PAGE and visualized by the Amersham Typhoon Imager (Cytiva, Uppsala, Sweden) (Supplementary Figure S1). Data for the wt, Y143F, and Y143A proteins were converted to mol/L units and then fitted to a single exponent (Equation (1)), where P, P₀, and P_max are amounts of product at any given time, at the zero-time point, and the end time point, respectively; k_obs is the observed rate constant.
$$P=P_0+P_{max}(1-\exp \left(-k_{obs}t\right))$$