Interaction of the type I methyltransferase M.EcoR124I with modified DNA substrates: sequence discrimination and base flipping.
We have analysed the DNA-protein contacts made between the type I DNA methyltransferase M.EcoR124I and its recognition sequence. The effects of base modifications have been probed by measuring the affinity of M.EcoR124I 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.
Mernagh
D R
DR
Division of Molecular and Cell Biology, School of Biological Sciences, University of Portsmouth, Portsmouth PO1 2DT, U.K.
Taylor
I A
IA
Kneale
G G
GG
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D019175:DNA Methylation; D004926:Escherichia coli; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013379:Substrate Specificity; D013872:Thionucleosides / Q000378:metabolism; D014498:Uracil / Q000378:metabolism
D009841:Oligonucleotides; D013872:Thionucleosides; D006151:Guanosine; D014498:Uracil; D004247:DNA; C005783:6-thioguanosine; C072994:EcoR124 modification methylase type I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)