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Nucleic Acids Research Advance Access published online on May 1, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn200
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© 2008 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

Chemical mapping of cytosines enzymatically flipped out of the DNA helix

Dalia Daujotyte, Zita Liutkeviciute, Gintautas Tamulaitis and Saulius Klimasauskas*

Institute of Biotechnology, Graiciuno 8, LT-02241 Vilnius, Lithuania

*To whom correspondence should be addressed. Tel: +370 5 2602114; Fax: +370 5 2602116; Email: klimasau{at}ibt.lt Present address: Dalia Daujotyt e, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK

Received February 27, 2008. Revised April 3, 2008. Accepted April 3, 2008.

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein–DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein–DNA complexes.


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