Figure 1.

Relative activity and kinetic mechanism of DNMT3A-C WT and Arg882 variants. (A) Methylation activity of 1 μM DNMT3A-C WT and Arg882 variants was measured for 5 and 10 min using ³[H] labelled S-Adenosylmethionine (AdoMet). The transfer of radiolabeled –CH₃ group to DNA was measured as counts per minute (CPM) using the MicroBeta scintillation counter. (B) Methylation activity of DNMT3A-C WT and Arg882 variants was measured for 10 min using 100 ng of pUC19 plasmid as a substrate, at concentrations of enzymes varying from 0.25 to 1 μM. The enzymes were pre-incubated with DNA for 10 min at room temperature and the reaction was initiated by addition of AdoMet. Each data point is an average and standard error of the mean (n≥ 3 independent experiments).The data shows reduced activity and loss of cooperativity for all the variant enzymes.