| Abstract: | 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. |
