Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase

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...

Section: Discussionmentioning

confidence: 99%

The Mechanism of DNA Cytosine-5 Methylation

Vilkaitis¹,

Merkienė²,

Serva³

et al. 2001

110

“…Our results, therefore, indicate that the interpretation of the catalytic competence of the M.HhaI-AdoMet complex based on isotope partitioning experiments should be viewed with caution. Despite the limitation of the isotope partitioning technique, it has been used successfully in a number of systems, EcoRI DNA methyltransferase (46), MspI DNA methyltransferase (47), the murine DNA (C-5 cytosine) methyltransferase (48), and RsrI DNA methyltransferase (49), to decipher the order of substrate binding. The requirement of DNA binding for the formation of competent complex was found in the case of murine DNA (C-5 cytosine) methyltransferase (48), whereas for the EcoRI DNA methyltransferase (46) and RsrI DNA methyltransferase (49) the enzyme-AdoMet complex was found to be catalytically active.…”

Section: Energetics Of Adometϫmhhai and Adohcyϫmhhai Recognition Rementioning

confidence: 99%

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Swaminathan¹,

Sankpal²,

Rao³

et al. 2002

Methyltransferase-catalyzed modification of substrate DNA, RNA, or protein serves as a key signal in the regulation and maintenance of diverse biological processes in a range of organisms from prokaryotes to eukaryotes (1, 2). 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 (3-7). 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. Its ability to perform the methyl transfer within the B-DNA helix was rationalized by a localized "DNA backbone rotation" by nearly 180°(base flipping) without significantly bending or kinking of the rest of the duplex (8 -11). Base flipping serves to deliver the base into a concave catalytic site in the enzyme (8). The Gln 287 side chain fills up the gap generated concomitantly in the DNA bound with M.HhaI, leading to a stable and specific interruption of the aromatic -stacked base pairs, a feature recently utilized to evaluate long range electron migration through DNA (12). M.HhaI methylates all of the cytosines in poly(dG-dC) DNA (3), with hemimethylated DNA being the preferred substrate (13). Both the reaction product S-adenosyl-L-homocysteine (AdoHcy) (14) and, by implication, the cofactor AdoMet (7), appear to share the ability to drive M.HhaI to trap the target cytosine in the catalytic site to form a productive complex. These results portray AdoMet and AdoHcy as important modulators of the multiple binding modes of the DNA-bound enzyme.Despite a wealth of information on the structural, dynamic, and kinetic aspects of the M.HhaI mechanism, little attention has been paid to the energetics of AdoMet and AdoHcy recognition reactions. No molecule other than ATP serves as a cofactor in more diverse reactions than does AdoMet (15). It was, therefore, of interest to understand the thermodynamic basis of recognition of the ubiquitous cofactor AdoMet by methyltransferases, a feature shared among structurally related enzymes that catalyze reactions with diverse substrate specificities (16 -18). For example, a snapshot of a step that follows methyl transfer has been elucidated by crystallographic analyses of the DNA intermediate covalently bound to M.HhaI (8) and M.HaeIII (19). Despite its absence in the original crystallization mix of the ternary complex, it was AdoHcy that was found, fortuitously, to reside within the cofactor binding pocket (8) shared by AdoMet (16). This illustrates that the transfer of the methyl group from AdoMet to the target cytosine results in the formation not only of the modified (i.e. methylated) DNA but also the transformation of AdoMet into AdoHcy. On the other * This work was supported by grants from the Department of Biotechnology, Government of India (to D. N. R. and A. S.) and the Department of Scien...

“…where v 20 (the partial specific volume of CcrM at 20°C) is 0.7374 ml/g, v 25 is 0.7396 ml/g, 20w (the density of pure water at 20°C) is 0.9982 g/ml, 25,b (the density of buffer b at 25°C) is 1.01132 g/ml for ultracentrifugation buffer, 20 Chemical Cross-linking-Chemical cross-linking of CcrM was achieved by using the bifunctional amine cross-linking agent, dimethyl suberimidate (DMS). A DMS stock solution of 50 mM was prepared in cross-linking buffer (50 mM potassium phosphate, pH 8.5, 150 mM potassium chloride, 3 mM EDTA, 5 mM ␤-ME, 10% glycerol) immediately prior to use.…”

Section: Fig 1 Synthetic Dna Substratesmentioning

confidence: 99%

“…An N 6 -45/45(sp)-mer with one methylation site containing a 3Ј-biotin label on the methylated strand and an abasic substitution for the target adenine on the nascent strand to facilitate tight binding (25,(35)(36)(37) was used. The biotinylated DNA was immobilized to a ligand load density of 187.2 response units on a streptavidin chip (BIACORE).…”

Section: Fig 1 Synthetic Dna Substratesmentioning

confidence: 99%

See 1 more Smart Citation

Identification of the Active Oligomeric State of an Essential Adenine DNA Methyltransferase from Caulobacter crescentus

Shier

Hancey

Benkovic

2001

Caulobacter crescentus contains one of the two known prokaryotic DNA methyltransferases that lacks a cognate endonuclease. This endogenous cell cycle regulated adenine DNA methyltransferase (CcrM) is essential for C. crescentus cellular viability. DNA methylation catalyzed by CcrM provides an obligatory signal for the proper progression through the cell cycle. To further our understanding of the regulatory role played by CcrM, we sought to investigate its biophysical properties. In this paper we employed equilibrium ultracentrifugation, velocity ultracentrifugation, and chemical cross-linking to show that CcrM is dimeric at physiological concentrations. However, surface plasmon resonance experiments in the presence of S-adenosyl-homocysteine evince that CcrM binds as a monomer to a defined hemi-methylated DNA substrate containing the canonical methylation sequence, GANTC. Initial velocity experiments demonstrate that dimerization of CcrM does not affect DNA methylation. Collectively, these findings suggest that CcrM is active as a monomer and provides a possible in vivo role for dimerization as a means to stabilize CcrM from premature catabolism.