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Nucleic Acids Research Advance Access first published online on September 25, 2009
This version published online on October 8, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp772
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

A directed evolution design of a GCG-specific DNA hemimethylase

Ruta Gerasimaite, Giedrius Vilkaitis and Saulius Klimasauskas*

Laboratory of Biological DNA Modification, 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

Received July 30, 2009. Revised September 2, 2009. Accepted September 2, 2009.

DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases.


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