Enzymatic Hydroxylation of m3C-DNA

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.
$$P=P_0+P_{max}(1-\exp \left(-k_{obs}t\right))$$