Fig. 5
From: A continuous kinetic assay for protein and DNA methyltransferase enzymatic activities
Monitoring DNA methyl transferase activity using the coupled kinetic assay. Steady-state kinetics of M.HaeIII methyl transfer activity with DNA fragment containing six methylation sites. a Scheme of DNA substrate for M.HaeIII containing six methylation sites (red) including one methylation site that is located at NotI recognition sequence. NotI cleavage at this site leads to two fragments of 550 and 1050 bp. b Kinetic traces of M.HaeIII at increased M.HaeIII concentrations (blue 1.5 μM, orange 3 μM and grey 6 μM) showing the increase in methylation rate. DNA concentration for all reactions was 400 nM. c DNA methylation by M.HaeIII prevents cleavage by NotI. Gel electrophoresis analysis of the DNA substrate cleavage by NotI prior and following methylation by M.HaeIII. Methylation of the DNA substrate by M.HaeIII was performed in the M.HaeIII optimal reaction buffer and in the coupled assay buffer to ensure that the later does not interfere with DNA methylation (reaction time 3 h, M.HaeIII concentration is 2 μM)