<i>dam</i> methylase from <i>E. coli</i>

Kriebardis, Anastasios G.; Guschlbauer, Wilhelm

doi:10.1016/0014-5793(87)81509-0

Cited by 8 publications

(4 citation statements)

References 15 publications

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“…Because there is no high resolution structure for DAM, we used secondary structure predictions based on CD analysis to determine whether the structure of the protein fits that of previously characterized DNA methyltransferases and to confirm that the His-tagged enzyme is folded properly. Our secondary structure predictions for both DAM and hDAM are very similar, are within the range of observed values determined previously for DAM (32), and are from the inspection of the five related adenine methyltransferase crystal structures T4DAM, M.TaqI, M.DpnM, M.RsrI, and M.PvuII.…”

Section: Discussionsupporting

confidence: 59%

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Functional Characterization of Escherichia coli DNA Adenine Methyltransferase, a Novel Target for Antibiotics

Mashhoon

Carroll

Pruss

et al. 2004

Journal of Biological Chemistry

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We have characterized Escherichia coli DNA adenine methyltransferase, a critical regulator of bacterial virulence. Steady-state kinetics, product inhibition, and isotope exchange studies are consistent with a kinetic mechanism in which the cofactor S-adenosylmethionine binds first, followed by sequence-specific DNA binding and catalysis. The enzyme has a fast methyl transfer step followed by slower product release steps, and we directly demonstrate the competence of the enzyme cofactor complex. Methylation of adjacent GATC sites is distributive with DNA derived from a genetic element that controls the transcription of the adjacent genes. This indicates that the first methylation event is followed by enzyme release. The affinity of the enzyme for both DNA and S-adenosylmethionine was determined. Our studies provide a basis for further structural and functional analysis of this important enzyme and for the identification of inhibitors for potential therapeutic applications.Bacterial DNA methyltransferases generate N 4 -methylcytosine, C 5 -methylcytosine, and N 6 -methyladenosine in an S-adenosylmethionine-dependent reaction (1). Bacterial DNA methylation plays critical roles, including DNA repair, phage protection, gene regulation, and DNA replication, in diverse biological pathways. The majority of DNA methyltransferases form one-half of a restriction-modification system that protects the host bacteria against bacteriophage infection. Together with cognate restriction endonucleases, which generally cleave a short palindromic sequence, these restriction-modification systems provide the foundation for many recombinant DNA manipulations; the endonucleases and methyltransferases have provided many structural and mechanistic insights into the process of sequence-specific DNA recognition and modification.Not all DNA methyltransferases have an endonuclease partner or at least one which is known. Thus, DNA adenine methyltransferase (DAM, 1 methylates the adenine in GATC) in ␥-proteobacteria (2, 3), and the cell cycle-regulated methyltransferase (CcrM, methylates the adenine in GANTC) in ␣-proteobacteria (3, 4) are involved in post-replicative mismatch repair, DNA replication timing, cell cycle regulation, and the control of gene expression. DAM and CcrM have been identified as new targets for antibiotic development (5) because some pathogenic bacteria are either avirulent or not viable when the corresponding genes are removed. DNA adenine methylation regulates the pili formation genes in Escherichia coli and Salmonella, providing one of the first and clearest examples of epigenetic gene regulation (2). This DNA-mediated gene regulation involves differentially methylated GATC sites, which represent a small minority of the ϳ5,000 -20,000 GATC sites found in a typical bacterial genome.E. coli DAM is a functional monomer of 278 amino acids (6). Our present understanding of how this enzyme functions is based largely on a small number of reports (6 -10). Herman and Modrich (6) first characterized the enzyme with plasmid DNA, ...

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Section: Discussionsupporting

confidence: 59%

“…Concentrations were determined using calculated extinction coefficients. For gel mobility shift assays, DNA substrates were radiolabeled using [␥- 32 P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs). Unincorporated label was removed with Bio-Gel P-6 spin columns (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Escherichia coli DNA Adenine Methyltransferase, a Novel Target for Antibiotics

Mashhoon

Carroll

Pruss

et al. 2004

Journal of Biological Chemistry

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“…Second, the presence of AdoMet may serve to induce or stabilize a conformation of the enzyme that is more amenable to crystallization. Studies with adenine methyltransferases, M.£coRI and dam, suggest that AdoMet binding is associated with a conformational change in the methylase (Bergerat et al, 1991;Reich & Mashhoon, 1991; Kriebardis & Guschlbauer, 1987). It was recently reported that the binding of AdoMet and an inhibitor appears to be required for the growth of large crystals of rat catechol O-methyltransferase (Vidgren et al, 1991).…”

Section: Resultsmentioning

confidence: 99%

Purification, crystallization, and preliminary x-ray diffraction analysis of an M.HhaI-AdoMet complex

Kumar¹,

Cheng²,

Pflugrath³

et al. 1992

Biochemistry

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The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.

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“…Purity of Ado-Met was checked by HPLC ( 1). Homogenous Dam methylase was prepared from E. coli strain GM2290/pTP166, obtained from Prof. M. Marinus (University of Massachussetts), as described previously (12). Proteinase K was purchased from Boehringer Mannheim.…”

Section: Methodsmentioning

confidence: 99%

The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli

Bergerat

Guschlbauer²

1990

Nucleic Acids Research

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The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.

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dam methylase from E. coli

Cited by 8 publications

References 15 publications

Functional Characterization of Escherichia coli DNA Adenine Methyltransferase, a Novel Target for Antibiotics

Functional Characterization of Escherichia coli DNA Adenine Methyltransferase, a Novel Target for Antibiotics

Purification, crystallization, and preliminary x-ray diffraction analysis of an M.HhaI-AdoMet complex

The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli

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