Nucleic Acids Research, 2002, Vol. 30, No. 17 3818-3830
© 2002 Oxford University Press
Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships
Department of Molecular Medicine, Guy’s, King’s and St Thomas’ School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK
*To whom correspondence should be addressed. Tel: +44 207 848 5909; Fax: +44 207 733 3877; Email: kevin.ford{at}kcl.ac.uk
Present addresses:
Andrew R. McNamara, Sangamo BioScience Inc., Point Richmond Tech Center III, 501 Canal Boulevard, Suite A100, Richmond, CA 94804, USA
Paul J. Hurd, Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
DNA methylation is now seen as a primary signal in the cell for mediating transcriptional repression through chromatin formation. The construction and evaluation of enzymes capable of influencing this process in vivo is therefore of significant interest. We have fused the C5-cytosine DNA methyltransferases, M.HhaI and M.HpaII, which both methylate 4 bp sequences containing a CpG dinucleotide, to a three zinc finger protein recognising a 9 bp DNA sequence. DNA methylation analyses demonstrate specific DNA methylation by both enzymes at target sites comprising adjacent methyltransferase and zinc finger subsites, targeted M.HpaII being the most specific. Binding analysis of the targeted M.HpaII enzyme reveals an 8-fold preference for binding to its target site, compared to binding to a zinc finger site alone, and an 18-fold preference over binding to a methyltransferase site alone, thereby demonstrating enhanced binding by the fusion protein, compared to its component proteins. Both DNA binding and methylation are specific for the target site up to separations of 
40 bp between the zinc finger and methyltransferase subsites. Ex vivo plasmid methylation experiments are also described that demonstrate targeted methylation. These targeted enzymes, however, are shown to be not fully mono-functional, retaining a significant non-targeted activity most evident at elevated protein concentrations.
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