Interaction of the type I methyltransferase M.EcoR124I with modified DNA substrates: sequence discrimination and base flipping

摘要

pWe have analysed the DNA–protein contacts made between the type I DNA methyltransferase M.iEco/iR124I and its recognition sequence. The effects of base modifications have been probed by measuring the affinity of M.iEco/iR124I for the modified sequences relative to that for the wild-type sequence by using gel-retardation competition assays. These results, along with those from methylation interference footprinting and photo-affinity cross-linking have identified the location of potential DNA contacts within the DNA recognition site. Substitution of 6-thioguanosine for each of the three specific guanines in the recognition sequence leads to a large (10–20-fold) decrease in the strength of DNA binding, indicating the importance of hydrogen-bonding interactions in the major groove of DNA. In contrast, replacement of either (or both) of the adenines at the target site for methylation by the enzyme, to produce either a base pair mismatch or loss of the base, leads to a marked increase in DNA-binding affinity. The results strongly support the proposal that type I methyltransferases employ a base-flipping mechanism to methylate their target base./p