The modification of DNA by DNA methyltransferases is widespread and has a variety of biological functions (1
). In bacteria, DNA methylation is involved in host defense mechanisms and strand discrimination during mismatch repair. In eukaryotic cells, DNA methylation is part of a highly complex epigenetic network regulating genome structure and activity (2
). In contrast to the bacterial enzymes, eukaryotic DNA methyltransferases contain large regulatory domains that are involved in numerous intermolecular interactions and control enzyme activity through a largely unknown mechanism (4
). The biochemical and cell biological characterization of DNA methyltransferases is pivotal for the understanding of epigenetic network regulation.
The basic biochemistry of the 5-methyl cytosine (5mC) methylation reaction is by now well understood. In a postreplicative reaction, DNA methyltransferases catalyze the transfer of a methyl group from S
-methionine (AdoMet) to the C5 position of the nucleobase. During this multi-step reaction, the target cytosine is flipped out of the double helix (base flipping) and the recipient C5 position is activated by a transient, covalent complex formation with the enzyme at the C6 position (5
). After methyl group transfer, the enzyme is released by β-elimination together with the proton at the C5 position. This last and crucial step of the enzymatic reaction can be exploited for a specific and mechanism-based inhibition with DNA substrates containing nucleotide analogs like 5-aza-dC or zebularine that are missing the essential proton at the C5 position (7–9
). Although the catalytic mechanism of the 5mC DNA methyltransferases is known, the crucial question how eukaryotic enzymes recognize and discriminate target sites for methylation remains elusive.
Over the past decades, a variety of biochemical assays has been developed to determine the activity of DNA methyltransferases. The most commonly used methyltransferase activity assays measure the transfer of radioactively labeled methyl groups from the cofactor AdoMet to DNA substrates (10–14
). Alternatively, DNA methylation by active methyltransferases can be monitored as protection against nucleolytic cleavage by restriction enzymes. The amount of methylated DNA can be measured as release or retention of terminal affinity probes of DNA substrates (15
). Another indirect approach uses bisulfite treatment followed by incorporation and detection of hapten-labeled dCTPs at non-converted sites (17
). Also direct detection of methylated cytosine residues by MALDI-TOF mass spectrometry (18
) or monitoring of conversion of AdoMet to S
-adenosyl-homocysteine (AdoHcy) by liquid chromatography and mass spectroscopy has been used (19
). All these methods depend on either radioisotopes, expensive and demanding equipment, and/or multiple-step protocols.
Here, we present a simple, non-radioactive and versatile method to measure DNA methyltransferase activity. The assay measures methyltransferase activity as irreversible covalent complex formation with fluorescently labeled DNA substrates containing the mechanism-based inhibitor 5-aza-dC. The variation of DNA sequence and fluorescent label allows detection of DNA sequence specificity and discrimination of methyltransferase activity from DNA binding. We tested this assay using mammalian DNA methyltransferase 1 and mutants thereof.