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Nucleic Acids Research 2005 33(10):3235-3244; doi:10.1093/nar/gki618
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Published online 7 June 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

Tsueu-Ju Su1, Mark R. Tock1, Stefan U. Egelhaaf1,2, Wilson C. K. Poon2 and David T. F. Dryden1,*

1School of Chemistry The King's Buildings The University of Edinburgh Edinburgh EH9 3JJ, UK 2School of Physics The King's Buildings The University of Edinburgh Mayfield Road, Edinburgh EH9 3JZ, UK

*To whom correspondence should be addressed. Tel: +44 131 650 4735; Fax: +44 131 650 6453; Email: David.Dryden{at}ed.ac.uk

Received March 21, 2005. Revised April 22, 2005. Accepted May 10, 2005.

The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5'-AACNNNNNNGTGC-3', where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequence-specific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a double-stranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response.

Present address: Stefan U. Egelhaaf, Institut fur Physik der kondensierten Materie, Heinrich-Heine Universitat, 4025-Dusseldorf, Germany


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