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Nucleic Acids Research, 2002, Vol. 30, No. 17 3880-3885
© 2002 Oxford University Press

Promiscuous methylation of non-canonical DNA sites by HaeIII methyltransferase

Helen M. Cohen, Dan S. Tawfik2 and Andrew D. Griffiths*,1

MRC Centre for Protein Engineering and 1 MRC Laboratory for Molecular Biology, MRC Centre, Hills Road, Cambridge CB2 2QH, UK and 2 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76 100, Israel

*To whom correspondence should be addressed. Tel: +44 1223 402113; Fax: +44 1223 402140; Email: griff{at}mrc-lmb.cam.ac.uk

The cytosine C5 methyltransferase M.HaeIII recognises and methylates the central cytosine of its canonical site GGCC. Here we report that M.HaeIII can also, with lower efficiency, methylate cytosines located in a wide range of non-canonical sequences. Using bisulphite sequencing we mapped the methyl- cytosine residues in DNA methylated in vitro and in vivo by M.HaeIII. Methyl-cytosine residues were observed in multiple sequence contexts, most commonly, but not exclusively, at star sites (sites differing by a single base from the canonical sequence). The most frequently used star sites had changes at positions 1 and 4, but there is little or no methylation at star sites changed at position 2. The rate of methylation of non-canonical sites can be quite significant: a DNA substrate lacking a canonical site was methylated by M.HaeIII in vitro at a rate only an order of magnitude slower than an otherwise identical substrate containing the canonical site. In vivo methylation of non-canonical sites may therefore be significant and may have provided the starting point for the evolution of restriction–modification systems with novel sequence specificities.


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