![[feed]](/style/images/feed-icon-14x14.png){kind=icon}
Tools
Tools
| Item Type: | Article |
|---|---|
| Title: | Visualization and measurement of DNA methyltransferase activity in living cells |
| Creators Name: | Schermelleh, L., 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 |
| 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 |
Repository Staff Only: item control page
