A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has 
been developed. The assay applies a break light oligonucleotide in which the 
methylation of an unmethylated 5'-CG-3' site is enzymatically coupled to the 
development of a fluorescent signal. This sensitive assay can measure rates of 
DNA methylation down to 0.34 +/- 0.06 fmol/s. The assay is reproducible, with a 
coefficient of variation over six independent measurements of 4.5%. Product 
concentration was accurately measured from fluorescence signals using a linear 
calibration curve, which achieved a goodness of fit (R(2)) above 0.98. The 
oligonucleotide substrate contains three C5-methylated cytosine residues and one 
unmethylated 5'-CG-3' site. Methylation yields an oligonucleotide containing the 
optimal substrate for the restriction enzyme GlaI. Cleavage of the fully 
methylated oligonucleotide leads to separation of fluorophore from quencher, 
giving a proportional increase in fluorescence. This method has been used to 
assay activity of DNMT1, the principle maintenance methyltransferase in human 
cells, and for the kinetic characterization of the bacterial cytosine-C5 
methyltransferase M.SssI. The assay has been shown to be suitable for the 
real-time monitoring of DNMT1 activity in a high-throughput format, with low 
background signal and the ability to obtain linear rates of methylation over 
long periods, making this a promising method of high-throughput screening for 
inhibitors.