Kinetic Mechanism of Cytosine DNA MethyltransferaseMspI

141

109

DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-L-methionine.

Section: Molecular Mechanism Of Dnmt3asupporting

confidence: 77%

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

Yokochi

Robertson

2002

141

109

“…cently demonstrated for M.HhaI (10) and for another C5-MTase, M.MspI (6). The parallel measurement of the rate of methylation k chem and product release k off in a single experiment (Fig.…”

Section: Dissection Of the Chemistrymentioning

confidence: 99%

The Mechanism of DNA Cytosine-5 Methylation

Vilkaitis¹,

Merkienė²,

Serva³

et al. 2001

110

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...

“…5). Kinetic analyses of the reactions catalyzed by DNA MTases are generally carried out either qualitatively or use a set of simplified kinetic equations (4,8,9,11,13). Such simplifications can result in the loss of kinetic information or its misrepresentation.…”

Section: A Special Role Of Adomet In the T4 Dam Methylationmentioning

confidence: 99%

“…Elucidating the kinetic mechanism of the reactions catalyzed by these enzymes still remains an important problem to investigate in the area of biological DNA methylation. Kinetic schemes have been proposed for HhaI (4 -7), MspI (8), human Dnmt1 (9), and murine Dnmt1 (10) [C 5 -cytosine]MTases, and for EcoRI (11,12), EcaI (13), TaqI (14), EcoRV (15), and Type III EcoP15I [N 6 -adenine]MTases (16). All of these enzymes exhibit a sequential bi-bi mechanism; however, they differ with respect to the order of substrate binding and ratelimiting step.…”

mentioning

confidence: 99%

Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

Evdokimov¹,

Zinoviev²,

Malygin³

et al. 2002

. After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.