Cookies on this website
We use cookies to ensure that we give you the best experience on our website. If you click 'Continue' we'll assume that you are happy to receive all cookies and you won't see this message again. Click 'Find out more' for information on how to change your cookie settings.

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-dependentMTase immobilization by fluorescence recovery after photobleaching (FRAP). This trapping assay monitors kinetic properties and activity-dependent immobilization ofMTases 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. © 2008 by John Wiley & Sons, Inc.

Original publication

DOI

10.1002/0471143030.cb2212s39

Type

Journal article

Journal

Current Protocols in Cell Biology

Publication Date

17/12/2008