Nucleic Acids Research, Vol 26, Issue 15 3473-3479, Copyright © 1998 by Oxford University Press
S Serva, E Weinhold, RJ Roberts and S Klimasauskas
The DNA cytosine-C5 methyltransferase M. Hha I flips its target base out of
the DNA helix during interaction with the substrate sequence GCGC. Binary
and ternary complexes between M. Hha I and hemimethylated DNA duplexes were
used to examine the suitability of four chemical methods to detect
flipped-out bases in protein-DNA complexes. These methods probe the
structural peculiarities of pyrimidine bases in DNA. We find that in cases
when the target cytosine is replaced with thymine (GTGC), KMnO4proved an
efficient probe for positive display of flipped- out thymines. The
generality of this procedure was further verified by examining a DNA
adenine-N6 methyltransferase, M. Taq I, in which case an enhanced
reactivity of thymine replacing the target adenine (TCGT) in the
recognition sequence TCGA was also observed. Our results support the
proposed base-flipping mechanism for adenine methyltransferases, and offer
a convenient laboratory tool for detection of flipped-out thymines in
protein-DNA complexes.
ARTICLES
Chemical display of thymine residues flipped out by DNA methyltransferases
Institute of Biotechnology, Laboratory of Biological DNA Modification, Graiciuno 8, LT-2028 Vilnius, Lithuania.
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