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

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.

Figure 1.

Break light cytosine-C5 methyltransferase activity assay. The fluorescence of the hemimethylated oligonucleotide 1 is quenched by the dabcyl group. It is a substrate for cytosine-C5 methyltransferases such as DNMT1 or M.SssI and upon methylation yields the fully methylated oligonucleotide 2, which is cleaved by GlaI, separating the fluorophore from the quencher and resulting in an increase in fluorescence, which is proportional to the concentration of oligonucleotide 3.

Figure 2.

Urea-PAGE analysis of partially and fully methylated oligonucleotides incubated with GlaI. Partially methylated oligonucelotide 1 or fully methylated oligonucleotide 2 (10 μM) were incubated with GlaI (8 U) for 1 h. (A) Fluorescence image. (B) White light image. Lanes: M, oligonucleotide ladder (bases); lane 1, oligonucleotide 1; lane 2, GlaI treated oligonucleotide 1; lane 3, oligonucleotide 2; lane 4, GlaI-treated oligonucleotide 2.

Figure 3.

Dependence of GlaI activity on oligonucleotide concentration. (A) Using partially methylated oligonucleotide 1 as the substrate and (B) using fully methylated oligonucleotide 2 as the substrate.

Figure 4.

Analysis of time courses for coupled and stepwise enzymatic reactions. (A) Comparison of time courses of product formation in stepwise (filled circles) and coupled (solid line) assays expressed as fractional turnover. (B) Gel analysis of coupled and stepwise enzymatic reactions. The fluorescence image is of a 20% urea-polyacrylamide gel of stepwise and coupled assays and was recorded before staining. Lanes were as follows: 1, coupled assay negative control (no AdoMet); 2, stepwise assay negative control (no AdoMet) incubated for 0 min; 3, stepwise assay negative control (no AdoMet) incubated for 64 min; 4-11, stepwise assay incubated for 0, 1, 2, 4, 8, 16, 32 and 64 min respectively; 12, coupled assay. (C) As in (B) but after staining with GelRed stain.

Figure 5.

Activity of DNMT1 with oligonucleotide 1. (A) Comparison of fluorescence changes in a full assay (solid black line) and a negative control assay lacking DNMT1 (dotted line). (B) Plot of change in the concentration of oligonucleotide 3 after background subtraction.

Figure 6.

Effect of increasing oligonucleotide 1 concentration on DNMT1 activity. (A) Real-time data averaged from duplicate assays. Assays contained oligonucleotide 1 at concentrations of 30 nM (black line), 60 nM (orange line), 120 nM (green line) and 240 nM (blue line) with associated negative controls (lacking DNMT1) shown as dotted lines. (B) Plot of steady-state rate against concentration of oligonucleotide 1.

Figure 7.

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

Figure 8.

Kinetic analysis of M.SssI activity. (A) Background-subtracted raw data showing the first 600 s for methylation of oligonucleotide 1 by M.SssI. The concentration of oligonucleotide 1 increases from lowest to highest as follows: 28, 47, 78, 130, 220, 360, 600 and 1000 nM. (B) Dependence of M.SssI activity on oligonucleotide concentration. (C) Dependence of M.SssI activity on AdoMet concentration.