How does a DNA interacting enzyme change its specificity during molecular evolution? A site-directed mutagenesis study at the DNA binding site of theDNA-(adenine-N-6)-methyltransferase EcoRV

The EcoRV DNA-(adenine-N6)-methyltransferase (MTase) recognizes GATATC sequ ences and modifies the first adenine residue within this site. Parts of its DNA interface show high sequence homology to DNA MTases of the dam family which recognize and modify GATC sequences. A phylogenetic analysis of M.Eco RV and dam-MTases suggests that EcoRV arose in evolution from a primordial dam-MTase in agreement to the finding that M.EcoRV also methylates GATC sit es albeit at a strongly reduced rate. GATCTC sites that deviate in only one position from the EcoRV sequence are preferred over general dam sites. We have investigated by site-directed mutagenesis the function of 17 conserved and nonconserved residues within three loops flanking the DNA binding clef t of M.EcoRV. M.EcoRV contacts the GATATC sequence with two highly cooperat ive recognition modules. The contacts to the GAT-part of the recognition se quence are formed by residues conserved between dam MTases and M.EcoRV. Mut ations at these positions lead to an increase in the discrimination between GATATC and GATC substrates. Our data show that the change in sequence spec ificity from dam (GATC) to EcoRV (GATATC) was accompanied by the generation of a second recognition module that contacts the second half of the target sequence. The new DNA contacts are formed by residues from all three loops that are not conserved between M.EcoRV and dam MTases. Mutagenesis at impo rtant residues within this module leads to variants that show a decreased a bility to recognize the TC-part of the GATATC sequence.