title: Trapped in action: direct visualization of DNA methyltransferase activity in living cells.
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.
authors.affiliation: Ludwig Maximilians University Munich, Department of Biology II, Planegg-Martinsried, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Schermelleh
authors.forename: Fabio
authors.initials: F
authors.lastname: Spada
authors.forename: Hariharan P
authors.initials: HP
authors.lastname: Easwaran
authors.forename: Kourosh
authors.initials: K
authors.lastname: Zolghadr
authors.forename: Jean B
authors.initials: JB
authors.lastname: Margot
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Cardoso
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D019175:DNA Methylation; D004261:DNA Replication; D000077209:Decitabine; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D051379:Mice; D010777:Photochemistry / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000032:analysis
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; D001374:Azacitidine