Modification of DNA is a process fundamental to all life that requires the precise balancing of specificity and efficiency. Methylation of cytosine bases within a genome is essential to the stability of archeal genomes, pathogen protection in bacteria, and multi-cellularity in eukaryotes and the enzymes that generate 5- methylcytosine are highly conserved from an ancient ancestor. Simultaneously, the cytosine methyltransferases have diversified towards thousands of different sequence specificities with high levels of discrimination for cytosines only within the context of the cognate sequence. The bacterial M.HhaI is a model for cytosine methyltransferases as well as the process of enzyme-promoted base flipping and modification. We sought to understand the chemical details of sequence specificity, conformational dynamics, base flipping, and the architectural strategy that underlies translating substrate recognition with catalysis in this important enzyme.

First, the role of individual enzyme:substrate contacts were probed for their role in binding, catalysis, and the induced-fit closure of the catalytic loop via nucleobase analogs lacking bonding heteroatoms. Monitoring the rate of loop closure with a substrate lacking the base of the target cytosine demonstrated that base flipping preceeds loop closure and suggested base flipping as the thermodynamic barrier to Michaelis complex assembly. A massive difference in the contribution of individual enzyme:DNA contacts towards assembly of the ternary complex is observed. Similar disparities are observed in the contributions of individual hydrogen bonds toward catalysis, suggesting a segregation of the roles of the substrate contacts into discriminatory and base flipping contacts. These hypotheses are then demonstrated to be correct, as the individual contacts are shown to have profound differences in their ability to provide the energy to effect base flipping.

Mutagenesis is used to probe the role of individual residues in coordinating base flipping and loop closure. Modeling and analysis of the transient state kinetic data is used to confirm that base flipping precedes loop closure and that the loop does not actively eject the base from the helix. It is shown that the conformational dynamics leading to base flipping and catalysis are co-ordinated across numerous residues, with individual components having modest contributions to each step.

Finally, NMR spectroscopy, fluorescence lifetime studies, and kinetic studies are used to develop a more detailed picture of sequence-specific substrate recognition and the first observation of an intermediate along the flipping trajectory. It is observed that the enzyme utilizes discriminatory contacts to begin substrate recognition and disrupt the target base pair to initiate base flipping. Overall, these findings present the most detailed picture of sequence-specific cytosine methylation ever described.