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Visualization and measurement of DNA methyltransferase activity in living cells

Item Type:Article
Title:Visualization and measurement of DNA methyltransferase activity in living cells
Creators Name:Schermelleh, L. and Spada, F. and Leonhardt, H.
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.
Keywords:DNA Methylation, DNA (Cytosine-5) Methyltransferase, Dnmt1, Trapping Assay, Fluorescence Recovery After Photobleaching, 5-aza-2′-deoxycytidine, Mechanism-Based Inhibitor, Animals
Source:Current Protocols in Cell Biology
ISSN:1934-2500
Publisher:Wiley (U.S.A.)
Volume:Chapter 22
Page Range:Unit 22.12
Date:June 2008
Official Publication:https://doi.org/10.1002/0471143030.cb2212s39
PubMed:View item in PubMed

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