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

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

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.

Mentions: The break light assay was used to determine (oligonucleotide 1) and for M.SssI. In the activity assays (Figure 8A), a slowing of the rate of fluorescence change were observed in the initial phase (up to ∼100 s), but this was followed by a stable steady-state phase (up to 600 s), which was used to determine the reaction rates. The source of the variation in rate during the initial phase is at present unknown: some of the possible factors might include temperature equilibration of the plate, mixing of the wells or rate-limiting product release, resulting in a burst phase. For both substrates, the dependence of M.SssI activity upon substrate concentration was fitted to the Michaelis–Menten equation. was found to be 234 ± 36 nM (Figure 8B), which compares well with the value of 180 nM reported previously (46) for a 30-bp hemimethylated oligonucleotide, although for an alternative sequence of the same length, M.SssI showed a significantly higher of 1.6 µM (47). Using our assay, for M.SssI was measured to be 675 ± 110 nM (Figure 8C). The literature values for are 50 nM (46) and 2.2 µM (48) and may depend on the precise assay conditions used; however, the value determined using our assay falls between these two. The range of values calculated for kcat, from data shown in Figures 7, 8B and C are 0.98 ± 0.02, 0.73 ± 0.03 and 0.49 ± 0.02 turnovers/min, respectively. These values are similar to the reported kcat for the protein of 0.16 turnovers/min (46).Figure 8.


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

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

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

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.
Mentions: The break light assay was used to determine (oligonucleotide 1) and for M.SssI. In the activity assays (Figure 8A), a slowing of the rate of fluorescence change were observed in the initial phase (up to ∼100 s), but this was followed by a stable steady-state phase (up to 600 s), which was used to determine the reaction rates. The source of the variation in rate during the initial phase is at present unknown: some of the possible factors might include temperature equilibration of the plate, mixing of the wells or rate-limiting product release, resulting in a burst phase. For both substrates, the dependence of M.SssI activity upon substrate concentration was fitted to the Michaelis–Menten equation. was found to be 234 ± 36 nM (Figure 8B), which compares well with the value of 180 nM reported previously (46) for a 30-bp hemimethylated oligonucleotide, although for an alternative sequence of the same length, M.SssI showed a significantly higher of 1.6 µM (47). Using our assay, for M.SssI was measured to be 675 ± 110 nM (Figure 8C). The literature values for are 50 nM (46) and 2.2 µM (48) and may depend on the precise assay conditions used; however, the value determined using our assay falls between these two. The range of values calculated for kcat, from data shown in Figures 7, 8B and C are 0.98 ± 0.02, 0.73 ± 0.03 and 0.49 ± 0.02 turnovers/min, respectively. These values are similar to the reported kcat for the protein of 0.16 turnovers/min (46).Figure 8.

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