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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.

Original publication




Journal article


Curr Protoc Cell Biol

Publication Date



Chapter 22


Unit - 22.12


Animals, Bromodeoxyuridine, Cells, Cytological Techniques, DNA (Cytosine-5-)-Methyltransferases, Fluorescence Recovery After Photobleaching, Humans, Proliferating Cell Nuclear Antigen