Determinants of cofactor binding to DNA methyltransferases: insights from a systematic series of structural variants of S-adenosylhomocysteine.

S-Adenosylmethionine (AdoMet) is a commonly used cofactor, second only to ATP in the variety of reactions in which it participates. It is the methyl donor in the majority of methyl transfer reactions, including methylation of DNA, RNA, proteins and small molecules. Almost all structurally characterised methyltransferases share a conserved AdoMet-dependent methyltransferase fold, in which AdoMet is bound in the same orientation. Although potential interactions between the cofactor and methyltransferases have been inferred from crystal structures, there has not been a systematic study of the contributions of each functional group to binding. To explore the binding interaction we synthesised a series of seven analogues of the methyltransferase inhibitor S-adenosylhomocysteine (AdoHcy), each containing a single modification, and tested them for the ability to inhibit methylation by HhaI and HaeIII DNA methyltransferase. Comparison of the Ki values highlights the structural determinants for cofactor binding, and indicates which nucleoside and amino acid functional groups contribute significantly to AdoMet binding. An understanding of the binding of AdoHyc to methyltransferases will greatly assist the design of AdoMet inhibitors.

Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization.

Engineering the specificity of DNA-modifying enzymes has proven extremely challenging, as sequence recognition by these enzymes is poorly understood. Here we used directed evolution to generate a variant of HaeIII methyltransferase that efficiently methylates a novel target site. M.HaeIII methylates the internal cytosine of the canonical sequence GGCC, but there is promiscuous methylation of a variety of non-canonical sites, notably AGCC, at a reduced rate. Using in vitro compartmentalization (IVC), libraries of M.HaeIII genes were selected for the ability to efficiently methylate AGCC. A two-step mutagenesis strategy, involving initial randomization of DNA-contacting residues followed by randomization of the loop that lies behind these residues, yielded a mutant with a 670-fold improvement in catalytic efficiency (k(cat)/K(m)(DNA)) using AGCC and a preference for AGCC over GGCC. The mutant methylates three sites efficiently (AGCC, CGCC and GGCC). Indeed, it methylates CGCC slightly more efficiently than AGCC. However, the mutant discriminates against other non-canonical sites, including TGCC, as effectively as the wild-type enzyme. This study provides a rare example of a laboratory-evolved enzyme whose catalytic efficiency surpasses that of the wild-type enzyme with the principal substrate.

Promiscuous methylation of non-canonical DNA sites by HaeIII methyltransferase.

The cytosine C5 methyltransferase M.HaeIII recognises and methylates the central cytosine of its canonical site GGCC. Here we report that M.HaeIII can also, with lower efficiency, methylate cytosines located in a wide range of non-canonical sequences. Using bisulphite sequencing we mapped the methyl- cytosine residues in DNA methylated in vitro and in vivo by M.HaeIII. Methyl-cytosine residues were observed in multiple sequence contexts, most commonly, but not exclusively, at star sites (sites differing by a single base from the canonical sequence). The most frequently used star sites had changes at positions 1 and 4, but there is little or no methylation at star sites changed at position 2. The rate of methylation of non-canonical sites can be quite significant: a DNA substrate lacking a canonical site was methylated by M.HaeIII in vitro at a rate only an order of magnitude slower than an otherwise identical substrate containing the canonical site. In vivo methylation of non-canonical sites may therefore be significant and may have provided the starting point for the evolution of restriction-modification systems with novel sequence specificities.