A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.
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.
School of Chemistry, University of Southampton, Southampton, Hampshire, SO17 1BJ, UK.
Robert J
RJ
Wood
Jennifer C
JC
McKelvie
Michael D
MD
Maynard-Smith
Peter L
PL
Roach
D002138:Calibration; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes; D015257:DNA-Cytosine Methylases / Q000032:analysis; D057075:Enzyme Assays / Q000379:methods* / Q000592:standards; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism
D009841:Oligonucleotides; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; D004262:DNA Restriction Enzymes