DNA Methyltransferases (DNMT)
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DNA methylation is a major epigenetic modification in the genome of higher eukaryotes. This DNA covalent modification is catalyzed by DNA methyltransferase enzymes (DNMTs or DNA MTases) and consist in the addition of a methyl group from S-adenosyl-L-methionine (AdoMet) to the fifth carbon position of cytosine (cytosine-5 or C5), mostly within CpG dinucleotides. This methylation, together with histone modifications, plays an important role in modulating chromatin structure, thus controlling gene expression and many other chromatin-dependent processes.
DNA methylation is accomplished by three enzymes: DNMT1, DNMT3a and DNMT3b. These enzymes can be further classified as de novo methyltransferases, enzymes that are able to methylate previously unmethylated CpG sequences, or maintenance methyltransferases, which copy pre-existing methylation marks onto new DNA strands during replication1,2. DNMT1 is the most abundant DNA methyltransferase in mammalian cells and predominatly methylates hemimethylated CpG dinucleotides as a maintenance methyltransferase, but DNMT1 has also been shown to function as a de novo DNA methyltransferase3,4,5. DNMT3a methylated CpG dinucleotides at a slower rate than DNMT1, but a greater rate than DNMT3b.
In addition to DNMT1, DNMT3a and DNMT3b there are two non-canonical family members, DNMT2 and DNMT3L. DNMT2 is not a DNA methyltransferase, it methylates cytosine 38 in the anticodon loop of tRNA16 but does not methylate DNA. DNMT3L is closely related to DNMT3a and DNMT3b structurally, but is catalytically inactive as a DNA methyltransferase. DNMT3L is known to associate with both DNMT3a and DNMT3b and may be responsible for the recruitment of histone deacetylases to direct repression onto newly established imprints6.
Active Motif's DNMT Activity / Inhibition Assay is a time-saving, non-radioactive assay to measure DNA methyltransferase activity and/or inhibition from recombinant DNMT enzymes (DNMT1, DNMT3a & DNMT3b) or nuclear extract samples. This sensitive ELISA-based method uses the ability of methyl CpG binding domain (MBD) proteins to bind methylated DNA with high affinity. In the DNMT assay method, a universal CpG-enriched DNA substrate has been immobilized on a 96-stripwell plate. Purified DNMTs or DNMT activities from nuclear extracts will catalyze the transfer of methyl groups from the provided AdoMet reagent to the coated DNA substrate. The resulting methylated DNA will be recognized by the His-tagged recombinant MBD2b in an amount proportional to the enzyme activity. Addition of a polyHistidine antibody conjugated to horseradish peroxidase (HRP) provides a sensitive colorimetric readout that is easiy quantified by spectrophotometry. The assay includes a sample of purified DNMT1 enzyme as a positive control.
To see a list of available DNMT Activity / Inhibition Assay products, click Available Products.
- Non-radioactive – colorimetric assay is easily quantified by spectrophotometry on a microplate reader at 450 nm
- Sensitive – unique methyl CpG binding domain (MBD) protein approach enhances the sensitivity of detection from either purified DNMT proteins or nuclear extracts
- Fast – assay can be completed in less than 3 hours
- Less effort required – kit is compatible with multi-channel pipettors to streamline wash steps
- Flexible – stripwell plate allows screening in low or high throughput
- Okano M. et al. (1999) Cell, 99:247-257.
- Jeltsch, A. (2006) Curr. Top. Microbiol. Immunol. 301:203-225.
- Fatemi et al. (2002) Eur. J. Biochem. 269:4981-4984.
- Gowher, H. et al. (2005) Biochemistry 44:9899-9904.
- Liang et al. (2002) Mol. Cell Biol. 22:480-491.
- Deplus, R. et al. (2002) Nucleic Acids Res. 30:3831-3838.