Visualization and measurement of DNA methyltransferase activity in living cells.
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.
Ludwig Maximilians University Munich, Department of Biology II, Martinsried, Germany.
Lothar
L
Schermelleh
Fabio
F
Spada
Heinrich
H
Leonhardt
D000818:Animals; D001973:Bromodeoxyuridine / Q000032:analysis / Q000378:metabolism; D002477:Cells / Q000201:enzymology*; D003584:Cytological Techniques; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000378:metabolism
D018809:Proliferating Cell Nuclear Antigen; D004248:DNA (Cytosine-5-)-Methyltransferases; D001973:Bromodeoxyuridine