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

Abstract

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.

CpG underrepresentation and the bacterial CpG-specific DNA methyltransferase M.MpeI

“…A similar distance (2.6-2.8 Å) is seen in complexes of other C5 methyltransferases with DNA in the presence of SAH. This has been interpreted as evidence for the formation of a partial covalent bond (23,24), which should be accompanied by a slight loss of substrate base planarity. However, our resolution is insufficient to detect such detail.…”

Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

“…27,28 During the process of methylation of C to 5mC in the presence of SAM, methyltransferases initiate a flipping of C out of the DNA helix, followed by the formation of a covalent bond between a cysteine in the methyltransferase and the C6 carbon of the flipped C. 29 This covalent interaction with the C6 carbon forms the activated cytosine which is then converted into an intermediate, namely 5,6-dihydrocytidine. [28][29][30] Dependent on the context, the cytosine intermediate can follow one of 2 possible routes: a) in the presence of SAM, it will accept the methyl group to form 5mC, and b) in the absence of SAM, the cytosine intermediate would be deaminated into U. 28 In line with bacterial methyltransferases, also mammalian DNMT-3A and -3B could deaminate C to U and importantly, 5mC to thymine (T).…”

Mechanisms of Superoxide Signaling in Epigenetic Processes: Relation to Aging and Cancer

“…These reversible, or more correctly, pseudo-reversible epigenetic processes of DNA and histone modification are probably the unique examples of chemical processes in which the forward and reversed reactions proceed by completely different mechanisms. Reactions of DNA methylation and histone acetylation go on by similar mechanisms with participation of the intermediates S-adenosyl-L-methionine (SAM) and Accoenzyme A (AcCoA) [8][9][10][11][12]. It is usually believed that these reactions are nucleophilic substitution processes, although they do not exactly correspond to the original name given by ED Hughes and C Ingold (1935).…”