Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1.
We have developed the first economical and rapid nonradioactive assay method that is suitable for high-throughput screening of the important pharmacological target human DNA (cytosine-5)-methyltransferase 1 (DNMT1). The method combines three key innovations: the use of a truncated form of the enzyme that is highly active on a 26-bp hemimethylated DNA duplex substrate, the introduction of the methylation site into the recognition sequence of a restriction endonuclease, and the use of a fluorogenic read-out method. The extent of DNMT1 methylation is reflected in the protection of the DNA substrate from endonuclease cleavage that would otherwise result in a large fluorescence increase. The assay has been validated in a high-throughput format, and trivial changes in the substrate sequence and endonuclease allow adaptation of the method to any bacterial or human DNA methyltransferase.
Ye
Yu
Y
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Stivers
James T
JT
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human