HIGH-RESOLUTION FOOTPRINTING OF A TYPE-I METHYLTRANSFERASE REVEALS A LARGE STRUCTURAL DISTORTION WITHIN THE DNA RECOGNITION SITE

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN(6)RTCG, transferring a methyl group fr om S-adenosyl methionine to a specific adenine on each DNA strand, We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusual ly large: the protein protects the DNA on both strands for at least tw o complete turns of the helix, indicating that the enzyme completely e ncloses the DNA in the complex, The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompass ing the recognition site, Within this region, however, there is a rema rkably hyper-reactive site on each strand. The two sites of enhanced c leavage are co-incident with the two adenines that are the target base s for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that t he distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in th e reaction pathway.