DNA methylation plays an important role in epigenetic signalling, having an impact on gene regulation, chromatin structure, development and disease. Here, we review the structures and functions of the mammalian DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, including their domain structures, catalytic mechanisms, localisation, regulation, post-translational modifications and interaction with chromatin and other proteins, summarising data obtained in genetic, cell biology and enzymatic studies. We focus on the question of how the molecular and enzymatic properties of these enzymes are connected to the dynamics of DNA methylation patterns and to the roles the enzymes play in the processes of de novo and maintenance DNA methylation. Recent enzymatic and genome-wide methylome data have led to a new model of genomic DNA methylation patterns based on the preservation of average levels of DNA methylation in certain regions, rather than the methylation states of individual CG sites.

Dnmt3a-C, the catalytic domain of the Dnmt3a DNA-(cytosine-C5)-methyltransferase, is active in an isolated form but, like the full-length Dnmt3a, shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a-C methylated its own expression plasmid in E. coli, and protected it from cleavage by methylation-sensitive restriction enzymes. However, despite screening about 400 clones that were selected in three rounds from a random mutagenesis library of 60 000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids that had lost the restriction sites. To improve the catalytic activity of Dnmt3a-C by optimization of the sequence of the DNA substrate, we analyzed its flanking-sequence preference in detail by bisulfite DNA-methylation analysis and sequencing of individual clones. Based on the enrichment and depletion of certain bases in the positions flanking >1300 methylated CpG sites, we were able to define a sequence-preference profile for Dnmt3a-C from the −6 to the +6 position of the flanking sequence. This revealed preferences for T over a purine at position −2, A over G at −1, a pyrimidine at +1, and A and T over G at +3. We designed one “good” substrate optimized for methylation and one “bad” substrate designed not to be efficiently methylated, and showed that the optimized substrate is methylated >20 times more rapidly at its central CpG site. The optimized Dnmt3a-C substrate can be applied in enzymatic high-throughput assays with Dnmt3a-C (e.g., for inhibitor screening), because the increased activity provides an improved dynamic range and better signal/noise ratio.

The EcoDam and T4Dam DNA-(adenine N6)-methyltransferases both methylate the adenine residue in GATC sites. These enzymes are highly related in amino acid sequence, but they deviate in their contact to the first base pair of the target sequence. EcoDam contacts Gua1 with K9 (which corresponds to T4Dam A6), while T4Dam contacts Gua1 with R130 (which corresponds to EcoDam Y138). We have “transplanted” the T4Dam DNA recognition into EcoDam and show that the EcoDam K9A/Y138R double mutant is highly active and specific. We also studied the intermediates of this transition: The EcoDam K9A variant showed low activity and loss of recognition of Gua1 [Horton, et al., J. Mol. Biol. 2006, 358, 559–570]. In contrast, the EcoDam Y138R variant, which carries both Gua1 recognition elements (K9 from EcoDam and R138 corresponding to R130 from T4Dam), is fully active and specific. This result indicates that a smooth evolutionary pathway exists for changing the EcoDam DNA recognition mode to T4Dam without loss of activity and without generation of evolutionary intermediates with reduced activity. We consistently observed increased activity of EcoDam variants containing Y138R; this suggests that the transition from EcoDam (Gua1 recognition through K9) to T4Dam (Gua1 recognition through R130) was driven by selective pressure towards increased catalytic activity.

Employing an in vitro reconstitution approach, McGinty et al. studied the mechanism of stimulation of the Dot1-catalysed histone H3 methylation at Lys79 by histone H2B ubiquitylation at Lys120. To generate nucleosome particles that carry the ubiquitylation at Lys120, they chemically connected three polypeptides—the main parts of histone H3 and ubiquitin expressed in bacteria and a branched synthetic peptide. Using the semisynthetically produced nucleosome substrates and purified Dot1 enzyme, they showed that Dot1 is directly stimulated by the ubiquitylation, thus ruling out the need for further protein factors to mediate the effect.