Nucleic Acids Research Advance Access originally published online on January 7, 2009
Nucleic Acids Research 2009 37(3):e22; doi:10.1093/nar/gkn1029
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Nucleic Acids Research, 2009, Vol. 37, No. 3 e22
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding
Department of Biology, Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany
* To whom correspondence should be addressed. Tel: +49 89 2180 74232; Fax: +49 89 2180 74236; Email: h.leonhardt{at}lmu.de
Received October 21, 2008. Revised December 3, 2008. Accepted December 10, 2008.
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 Dnmt1C1229W 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.