Nucleic Acids Research, Vol 24, Issue 24 4853-4858, Copyright © 1996 by Oxford University Press
DR Mernagh and GG Kneale
The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that
binds to the sequence GAAN6RTCG, transferring a methyl group from
S-adenosyl methionine to a specific adenine on each DNA strand. We have
investigated the protein-DNA interactions in the complex by DNase I and
hydroxyl radical footprinting. The DNase I footprint is unusually large:
the protein protects the DNA on both strands for at least two complete
turns of the helix, indicating that the enzyme completely encloses the DNA
in the complex. The higher resolution hydroxyl radical probe shows a
smaller, but still extensive, 18 bp footprint encompassing the recognition
site. Within this region, however, there is a remarkably hyper-reactive
site on each strand. The two sites of enhanced cleavage are co-incident
with the two adenines that are the target bases for methylation, showing
that the DNA is both accessible and highly distorted at these sites. The
hydroxyl radical footprint is unaffected by the presence of the cofactor
S-adenosyl methionine, showing that the distorted DNA structure induced by
M.EcoR124I is formed during the initial DNA binding reaction and not as a
transient intermediate in the reaction pathway.
ARTICLES
High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site
Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Hants, UK.
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