A directed evolution design of a GCG-specific DNA hemimethylase

DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases

Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase

Methylation of the five position of cytosine in DNA plays important roles in epigenetic regulation in diverse organisms including humans. The transfer of methyl groups from the cofactor S-adenosyl-l-methionine is carried out by methyltransferase enzymes. Using the paradigm bacterial methyltransferase M.HhaI we demonstrate, in a chemically unperturbed system, the first direct real-time analysis of the key mechanistic events—the flipping of the target cytosine base and its covalent activation; these changes were followed by monitoring the hyperchromicity in the DNA and the loss of the cytosine chromophore in the target nucleotide, respectively. Combined with studies of M.HhaI variants containing redesigned tryptophan fluorophores, we find that the target base flipping and the closure of the mobile catalytic loop occur simultaneously, and the rate of this concerted motion inversely correlates with the stability of the target base pair. Subsequently, the covalent activation of the target cytosine is closely followed by but is not coincident with the methyl group transfer from the bound cofactor. These findings provide new insights into the temporal mechanism of this physiologically important reaction and pave the way to in-depth studies of other base-flipping systems

Structure and mechanism of the HhaI DNA methyltransferase

Summary in English, Latvian, LithuanianAvailable from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLELVLatvi

Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases

Christian Dalhoff, Gražvydas Lukinavičius, Saulius Klimas̆auskas & Elmar Weinhol