UNIT 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells
Published Online: 1 JUN 2008
Copyright © 2008 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Schermelleh, L., Spada, F. and Leonhardt, H. 2008. Visualization and Measurement of DNA Methyltransferase Activity in Living Cells. Current Protocols in Cell Biology. 39:22.12.1–22.12.16.
- Published Online: 1 JUN 2008
- Published Print: JUN 2008
In this unit, a live-cell assay to measure DNA (cytosine-5) methyltransferase (MTase) activity at the single-cell level is described. This method takes advantage of the irreversible binding of enzymatically active MTases to genomic DNA substituted with the mechanism-based inhibitor 5-aza-2¢-deoxycytidine (5-aza-dC). The procedure comprises incorporation of this nucleoside analog into DNA during replication and quantification of the time-dependent MTase immobilization by fluorescence recovery after photobleaching (FRAP). This trapping assay monitors kinetic properties and activity-dependent immobilization of MTases in their native environment and enables direct comparison of mutations and inhibitors that affect MTase regulation and catalytic activity in single living cells. In addition, a simplified protocol to obtain qualitative information on the activity of either endogenously or exogenously expressed MTases is provided. Curr. Protoc. Cell Biol. 39:22.12.1-22.12.16. © 2008 by John Wiley & Sons, Inc.
Keywords: DNA methylation; DNA (cytosine-5) methyltransferase; Dnmt1; trapping assay; fluorescence recovery after photobleaching; 5-aza-2¢-deoxycytidine; mechanism-based inhibitor