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

Wood RJ, McKelvie JC, Maynard-Smith MD, Roach PL - Nucleic Acids Res. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Southampton, Southampton, Hampshire, SO17 1BJ, UK.

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 (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.

Show MeSH

Related in: MedlinePlus

Effect of GlaI concentration on the observed activity of M.SssI using oligonucleotide 1 as a substrate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875032&req=5

Figure 7: Effect of GlaI concentration on the observed activity of M.SssI using oligonucleotide 1 as a substrate.

Mentions: M.SssI was utilized as a model methyltransferase to test the application of the break light assay for kinetic analysis of cytosine-C5 methylation. To ensure that the rate of fluorescence increase was not limited by GlaI concentration at high (1 µM) DNA concentrations, a set of assays were prepared in which GlaI concentration was varied (0–3.2 U/well) and oligonucleotide 1 (1 µM), M.SssI (10 nM) and AdoMet (50 µM) concentrations were fixed. The concentration of DNA was over twice that required to ensure M.SssI was saturated and therefore functioning at Vmax. The observed rate of methylation was then plotted against GlaI concentration (Figure 7). Increasing concentrations of GlaI resulted in an increase in the observed initial rate of product formation up to a concentration of 1.6 U/well. Further increases in GlaI concentration did not result in a significant increase in the reaction rate and a concentration of 2.4 U/well GlaI was selected for the kinetic analysis of M.SssI activity, to ensure that the observed rate of fluorescence increase reflects the rate of methylation.Figure 7.


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

Wood RJ, McKelvie JC, Maynard-Smith MD, Roach PL - Nucleic Acids Res. (2010)

Effect of GlaI concentration on the observed activity of M.SssI using oligonucleotide 1 as a substrate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875032&req=5

Figure 7: Effect of GlaI concentration on the observed activity of M.SssI using oligonucleotide 1 as a substrate.
Mentions: M.SssI was utilized as a model methyltransferase to test the application of the break light assay for kinetic analysis of cytosine-C5 methylation. To ensure that the rate of fluorescence increase was not limited by GlaI concentration at high (1 µM) DNA concentrations, a set of assays were prepared in which GlaI concentration was varied (0–3.2 U/well) and oligonucleotide 1 (1 µM), M.SssI (10 nM) and AdoMet (50 µM) concentrations were fixed. The concentration of DNA was over twice that required to ensure M.SssI was saturated and therefore functioning at Vmax. The observed rate of methylation was then plotted against GlaI concentration (Figure 7). Increasing concentrations of GlaI resulted in an increase in the observed initial rate of product formation up to a concentration of 1.6 U/well. Further increases in GlaI concentration did not result in a significant increase in the reaction rate and a concentration of 2.4 U/well GlaI was selected for the kinetic analysis of M.SssI activity, to ensure that the observed rate of fluorescence increase reflects the rate of methylation.Figure 7.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry, University of Southampton, Southampton, Hampshire, SO17 1BJ, UK.

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 (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.

Show MeSH
Related in: MedlinePlus