Trapped in action

Direct visualization of DNA methyltransferase activity in living cells

Lothar Schermelleh, Fabio Spada, Hariharan Easwaran, Kourosh Zolghadr, Jean B. Margot, M. Cristina Cardoso, Heinrich Leonhardt

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)751-756
Number of pages6
JournalNature Methods
Volume2
Issue number10
DOIs
StatePublished - Oct 2005
Externally publishedYes

Fingerprint

Methyltransferases
DNA
Catalytic DNA
decitabine
Immobilization
Azacitidine
Mutation
DNA Methylation
Green Fluorescent Proteins
Epigenomics
Nucleotides
Fluorescence
Gene Expression

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Biology
  • Cell Biology

Cite this

Schermelleh, L., Spada, F., Easwaran, H., Zolghadr, K., Margot, J. B., Cardoso, M. C., & Leonhardt, H. (2005). Trapped in action: Direct visualization of DNA methyltransferase activity in living cells. Nature Methods, 2(10), 751-756. https://doi.org/10.1038/nmeth794

Trapped in action : Direct visualization of DNA methyltransferase activity in living cells. / Schermelleh, Lothar; Spada, Fabio; Easwaran, Hariharan; Zolghadr, Kourosh; Margot, Jean B.; Cardoso, M. Cristina; Leonhardt, Heinrich.

In: Nature Methods, Vol. 2, No. 10, 10.2005, p. 751-756.

Research output: Contribution to journalArticle

Schermelleh, L, Spada, F, Easwaran, H, Zolghadr, K, Margot, JB, Cardoso, MC & Leonhardt, H 2005, 'Trapped in action: Direct visualization of DNA methyltransferase activity in living cells', Nature Methods, vol. 2, no. 10, pp. 751-756. https://doi.org/10.1038/nmeth794
Schermelleh, Lothar ; Spada, Fabio ; Easwaran, Hariharan ; Zolghadr, Kourosh ; Margot, Jean B. ; Cardoso, M. Cristina ; Leonhardt, Heinrich. / Trapped in action : Direct visualization of DNA methyltransferase activity in living cells. In: Nature Methods. 2005 ; Vol. 2, No. 10. pp. 751-756.
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