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A real-time assay for CpG-specific cytosine-C5 methyltransferase activity

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity
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 (R2) 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.
0305-1048
e107
Wood, Robert J.
0a258dd5-9409-4619-92bc-6ad02453a856
McKelvie, Jennifer C.
a2dccde2-8662-4375-a9b8-5db8c0064aa0
Maynard-Smith, Michael D.
30959fca-0bc7-4790-ae67-ed4c2fbe151b
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9
Wood, Robert J.
0a258dd5-9409-4619-92bc-6ad02453a856
McKelvie, Jennifer C.
a2dccde2-8662-4375-a9b8-5db8c0064aa0
Maynard-Smith, Michael D.
30959fca-0bc7-4790-ae67-ed4c2fbe151b
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9

Wood, Robert J., McKelvie, Jennifer C., Maynard-Smith, Michael D. and Roach, Peter L. (2010) A real-time assay for CpG-specific cytosine-C5 methyltransferase activity. Nucleic Acids Research, 38 (9), e107. (doi:10.1093/nar/gkq047).

Record type: Article

Abstract

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 (R2) 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.

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Published date: 5 February 2010

Identifiers

Local EPrints ID: 177027
URI: http://eprints.soton.ac.uk/id/eprint/177027
ISSN: 0305-1048
PURE UUID: 555566d4-6bd2-4e35-ac8b-5f9c8ea108bc
ORCID for Peter L. Roach: ORCID iD orcid.org/0000-0001-9880-2877

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Date deposited: 28 Mar 2011 14:14
Last modified: 03 Dec 2019 01:53

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