The antibiotic target EcoDam (E.coli DNA adenine methyltransferase) plays critical roles in multiple biological pathways such as gene regulation, mismatch repair, and DNA replication by methylating the adenine in the DNA sequence 5'-GATC-3'. We demonstrate that EcoDam is regulated by the sequences immediately flanking biologically-derived GATC sites. A-tracts adjacent to GATC sites decrease methylation kinetics; however preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule unless both sites are surrounded by large amounts of non-specific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher-order specificity for EcoDam.

EcoDam carries out intra-site processive catalysis on short oligonucleotides whereby the enzyme:DNA complex methylates both strands of an unmethylated GATC prior to dissociation from the DNA. EcoDam dimerization on short, synthetic DNA results in enhanced catalysis and is not observed with large genomic DNA where the potential for inter-site processive methylation precludes any dimerization-dependent activation. High concentrations of short oligonucleotides results in enzyme activation that is inherent on genomic DNA where either multiple GATCs are available for methylation or the partitioning of the enzyme onto non-specific DNA is favored. These results are consistent with kinetic modeling which invokes substrate activation and dimerization and provides insights into regulatory checkpoints for an enzyme involved in multiple biological pathways.

The structural basis for EcoDam preferential methylation and processivity was elucidated using site directed mutagenesis of the conserved phosphate interactions involving residues R95, N126, N132, R116, and K139 that contact the DNA flanking the GATC site. Alanine substitutions at conserved positions do not impact k_cat/K_m^DNA nor DNA affinity, but cause large preferences for one GATC site over another when considering the pre-steady state efficiency constant k_chem/K_D ^DNA. These changes occur at the level of k_chem which results in significant decreases in processive catalysis. R95, N126, N132, and R116 repress the modulation of the enzyme's response to flanking sequence effects and do not show substrate-induced dimerization. This offers insight into the structural means by which an enzyme that does not completely enclose its substrate achieves processive catalysis and how interactions with DNA flanking the recognition site alter processivity.