TU Darmstadt / ULB / TUbiblio

Trapped in action: direct visualization of DNA methyltransferase activity in living cells.

Schermelleh, Lothar and Spada, Fabio and Easwaran, Hariharan P. and Zolghadr, Kourosh and Margot, Jean B. and Cardoso, M Cristina and Leonhardt, Heinrich (2005):
Trapped in action: direct visualization of DNA methyltransferase activity in living cells.
In: Nature methods, pp. 751-6, 2, (10), ISSN 1548-7091,
[Online-Edition: http://www.cardoso-lab.org/publications/Schermelleh_et_al_20...],
[Article]

Abstract

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.

Item Type: Article
Erschienen: 2005
Creators: Schermelleh, Lothar and Spada, Fabio and Easwaran, Hariharan P. and Zolghadr, Kourosh and Margot, Jean B. and Cardoso, M Cristina and Leonhardt, Heinrich
Title: Trapped in action: direct visualization of DNA methyltransferase activity in living cells.
Language: German
Abstract:

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.

Journal or Publication Title: Nature methods
Volume: 2
Number: 10
Divisions: 10 Department of Biology > Cell Biology and Epigenetics
?? fb10_zoologie ??
10 Department of Biology
Date Deposited: 06 Mar 2010 07:42
Official URL: http://www.cardoso-lab.org/publications/Schermelleh_et_al_20...
Export:
Suche nach Titel in: TUfind oder in Google

Optionen (nur für Redakteure)

View Item View Item