The Mechanism of DNA Cytosine-5 Methylation

Abstract

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-L-methionine (AdoMet) and product S-adenosyl-L-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. ), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k chem . We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k chem ) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. Cytosine methylation in DNA is catalyzed by DNA methyltransferases (MTases) 1 that transfer methyl groups from the ubiquitous donor S-adenosyl-L-methionine (AdoMet) producing modified cytosines with a methyl group at either C-5 or N-4 (1). In higher organisms, where only 5-methylcytosine is found, DNA methylation is essential for controlling a number of cellular processes including transcription, genomic imprinting, developmental regulation, mutagenesis, DNA repair, and chromatin organization (2). Aberrations in cytosine-5 methylation correlate with human genetic disease, and therefore, the MTases are potent candidate targets for developing new therapies (3). In prokaryotes, MTases are usually but not exclusively found as components of restriction modification systems (1).Besides their important physiological role, the MTases are attractive models for the study of protein-DNA interactions, a central event in many biological processes. The major advantages of bacterial C5-MTases as model systems are as follows: (a) wide diversity of targets recognized (over 200 specificities known); (b) ability to promote covalent reactions within the DNA; (c) their relatively simple molecular organization; and (d) high level of sequence and structural homology with eukaryotic enzymes. It is not surprising that most evidence of the catalytic mechanism of cytosine-5 methylation has been obtained from the studies of prokaryotic MTases. A particular example is HhaI MTase, a component of a type II restriction-modification system from Haemophilus haemolyticus. M.HhaI recognizes the tetranucleotide sequence GCGC and methylates the inner cytosine residue (boldface) and is one of the smallest in the C5-MTase family. This enzyme has been extensively examined by employing a variety of methods. Interaction with the substrates was shown to lead to dramatic conformational changes in both the bound DNA and the enzyme itself. MTase-mediated rotation of the target nucleotide out of the DNA helix (baseflipping) serves to deliver the base into a concave catalytic site in the enzyme (4). Subsequent massive movement of t...

Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a

“…4), which are characteristic of a ternary complex mechanism. Consistent with this notion, there is previous structural and mechanistic data demonstrating that DNMTs form a ternary complex (40,47,58). To determine the order of substrate binding and product release, product inhibition studies were pursued.…”

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

“…There have been suggestions that steps after methyl transfer are rate-limiting (7) and that the M.HhaI molecule in the absence of DNA does not have the capacity to bind either AdoMet or AdoHcy (3). However, results to the contrary have been reported recently (7,16).…”

“…There have been suggestions that steps after methyl transfer are rate-limiting (7) and that the M.HhaI molecule in the absence of DNA does not have the capacity to bind either AdoMet or AdoHcy (3). However, results to the contrary have been reported recently (7,16). To gain further insight into the energetic basis of the recognition of AdoMet and AdoHcy by M.HhaI, we have pursued a thorough characterization of the thermodynamics of the interaction including the changes in the solvation.…”

“…Subsequent to its isolation from Hemophilus haemolyticus nearly a quarter century ago, HhaI DNA methyltransferase (M.HhaI) 1 has been subjected to incisive investigations yielding detailed insights into the reaction chemistry (3)(4)(5)(6)(7). M.HhaI is an S-adenosyl-Lmethionine (AdoMet) dependent DNA methyltransferase that catalyzes the covalent attachment of a methyl group at the C-5 position of the aromatic ring of the first cytosine in the specific sequence 5Ј-GCGC-3Ј.…”

MtmMII-mediated C-Methylation during Biosynthesis of the Antitumor Drug Mithramycin Is Essential for Biological Activity and DNA-Drug Interaction

“…The substrates bind in a compulsory order to the enzyme to form a ternary complex in which the exchange reaction takes place; the products are also released in a compulsory order. Ordered Bi-Bi mechanisms have been described for some AdoMet-dependent methyltransferases (33,34) even involving processive movement and rapid exchange of a macromolecular substrate, without dissociation (35); nevertheless, other enzymes operate by different mechanisms (36).…”