Protein loops are major contributors to DNA strand separation and high-fidelity substrate recognition for DNA methyltransferase CcrM.

Strand separation is a newly described DNA recognition mechanism, and in the case of the cell cycle-regulated DNA methyltransferase (CcrM), it leads to an extraordinary level of substrate discrimination relative to other methyltransferases. The structural mechanisms underlying the process of DNA strand separation remain poorly understood. Two highly conserved loops in CcrM, loop-2B and loop-45, are inserted between the strand-separated DNA interface and are likely to generate and stabilize the strand-separated conformation. During strand separation, residues within loop-2B, loop-45, and loop-6E contact the DNA strand that undergoes methylation (target strand). Highly conserved loop residues R44 and F125 are positioned between the separated DNA strands and appear essential for maintaining the strand-separated intermediate. Replacement of F125 results in various perturbations of strand separation that are correlated to the size of the substituted residue. Global fitting of kinetic data shows that stabilization of DNA strand separation is perturbed by each mutation, leading to a reduced rate of methylation in some cases. These data support a functional role for these loops in generating and stabilizing the strand-separated intermediate. Insights into CcrM's mechanism of DNA strand separation are likely applicable to understand strand-separation mechanisms for other enzymes.