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.
Copyright 2008 by John Wiley & Sons, Inc.