PROBING THE PROTEIN-DNA INTERFACE OF THE ECORV MODIFICATION METHYLTRANSFERASE BOUND TO ITS RECOGNITION SEQUENCE, GATATC

The DNA contacts produced between the EcoRV modification methyltransfe rase and its recognition sequence, GATATC, have been determined. The e nzyme's general location in a methylase/DNA/sinefungin ternary complex was evaluated by protection from exonuclease III digestion. Important phosphate contacts were resolved using N-ethyl-N-nitrosourea ethylati on interference footprinting. Methylation protection and interference using dimethyl sulfate were employed to assess significant contacts to purinic bases. The protein-DNA interface was further probed using oli godeoxynucleotides containing base analogues within the GATATC sequenc e. Most of the experiments were carried out using hemimethylated seque nces, i.e., having 6-methyladenosine at the methylation site in one of the strands. The monomeric methylase was found to bind to the DNA in two different orientations for the methylation of each strand. The enz yme approaches the DNA, predominantly from one ''side'', and makes mos t of its contacts in the major groove. In either of the two binding ev ents contacts are made to the four phosphates NpNpNpGpA and the three bases GAT (where GAT represents the 5' half of the GATATC site) on bot h DNA strands. The phosphates and bases in the 3' ATC half are much le ss important. Although the contacts made to the equivalent locations o n each strand are similar, they display a slight but consistent change dependent on which strand contains the 6-methyldeoxyadenosine. This s trand variation shows completely reciprocal behavior, switching around exactly, depending entirely on the methylated deoxyadenosine location . It is this that provides evidence for the two binding modes. The res ults obtained are discussed in terms of possible models for the protei n-DNA interface.