Unit

UNIT 22.12 Visualization and Measurement of DNA Methyltransferase Activity in Living Cells

  1. Lothar Schermelleh,
  2. Fabio Spada,
  3. Heinrich Leonhardt

Published Online: 1 JUN 2008

DOI: 10.1002/0471143030.cb2212s39

Current Protocols in Cell Biology

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. 39:22.12:22.12.1–22.12.16.

Author Information

  1. Ludwig Maximilians University Munich (LMU), Department of Biology II, Martinsried, Germany

Publication History

  1. Published Online: 1 JUN 2008
  2. Published Print: JUN 2008

Abstract

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