title: A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding.
abstract: We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1(C1229W) catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity.
authors.affiliation: Department of Biology, Center for Integrated Protein Science Munich, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.
authors.forename: Carina
authors.initials: C
authors.lastname: Frauer
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
mesh_terms: D001667:Binding, Competitive; D002460:Cell Line; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000302:isolation & purification; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human