CHEMICAL DISPLAY OF THYMINE RESIDUES FLIPPED OUT BY DNA METHYLTRANSFERASES

2The DNA cytosine-C5 methyltransferase M.Hhal flips its target base ou t of the DNA helix during interaction with the substrate sequence GCGC . Binary and ternary complexes between M.Hhal and hemimethylated DNA d uplexes were used to examine the suitability of four chemical methods to detect flipped-out bases in protein-DNA complexes. These methods pr obe the structural peculiarities of pyrimidine bases in DNA. We find t hat in cases when the target cytosine is replaced with thymine (G (T) under bar GC), KMnO4 proved an efficient probe for positive display of flipped-out thymines. The generality of this procedure was further ve rified by examining a DNA adenine-N6 methyltransferase, M.Taql, in whi ch case an enhanced reactivity of thymine replacing the target adenine (TCG (T) under bar) in the recognition sequence TCGA was also observe d. Our results support the proposed base-flipping mechanism for adenin e methyltransferases, and offer a convenient laboratory tool for detec tion of flipped-out thymines in protein-DNA complexes.