DNA-cytosine-5-methyltransferase from P815 mouse mastocytoma cells: “Maintenance” and “de novo” activities are carried out by the same enzyme molecule

Grünwald, Stefan; Drahovský, D.

doi:10.1016/0020-711x(84)90147-2

Cited by 17 publications

(8 citation statements)

References 26 publications

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“…Methylation studies in vitro do not reveal such distinction of functions. In fact, whereas kinetic determinations show that Dnmt1 utilizes hemimethylated DNA 5-10-fold better than unmethylated DNA (15)(16)(17)(18)(19), they also show that this enzyme methylates unmethylated DNA more efficiently than Dnmt3A and Dnmt3B (4). Therefore, it is unclear whether the de novo activity of Dnmt1 is repressed in vivo.…”

Section: From the Institute Of Biosciences And Technology Center Formentioning

confidence: 96%

Recombinant Human DNA (Cytosine-5) Methyltransferase

Bacolla

Pradhan

Larson

et al. 2001

Journal of Biological Chemistry

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Steady-state kinetic analyses revealed that the methylation reaction of the human DNA (cytosine-5) methyltransferase 1 (DNMT1) is repressed by the N-terminal domain comprising the first 501 amino acids, and that repression is relieved when methylated DNA binds to this region. DNMT1 lacking the first 501 amino acids retains its preference for hemimethylated DNA. The methylation reaction proceeds by a sequential mechanism, and either substrate (S-adenosyl-L-methionine and unmethylated DNA) may be the first to bind to the active site. However, initial binding of S-adenosyl-L-methionine is preferred. The binding affinities of DNA for both the regulatory and the catalytic sites increase in the presence of methylated CpG dinucleotides and vary considerably (more than one hundred times) according to DNA sequence. DNA topology strongly influences the reaction rates, which increased with increasing negative superhelical tension. These kinetic data are consistent with the role of DNMT1 in maintaining the methylation patterns throughout development and suggest that the enzyme may be involved in the etiology of fragile X, a syndrome characterized by de novo methylation of a greatly expanded CGG⅐CCG triplet repeat sequence.The mammalian genome is epigenetically modified by the transfer of methyl groups from S-adenosyl-L-methionine (AdoMet) 1 to acceptor bases in double-stranded DNA, mostly at the carbon 5 of cytosine when the base is part of a CG dinucleotide (1). Several DNA methyltransferases (Dnmt) have been identified, including Dnmt1 (2), Dnmt2 (3), Dnmt3A, Dnmt3B (4), a splice variant of Dnmt1 (Dnmt1b) (5), and an oocytespecific isoform of Dnmt1 that lacks the first 118 N-terminal amino acids (6). Targeted mutations of Dnmt1 (7), Dnmt3A, and Dnmt3B genes are recessive lethals (8) in mice attesting to their essential role in development. In humans, mutations in the DNMT3B gene have been associated with the (immunodeficiency, centromere instability, and facial abnormalities syndrome (ICF syndrome) (9), a recessive disorder characterized biochemically by hypomethylation of satellite 2 and 3 DNA from the juxtacentromeric heterochromatin of chromosomes 1 and 16 (10 -12) as well as other common non-satellite repeats (13).Genetic studies suggest that Dnmt1 and Dnmt3 carry out two types of modifications as follows: both Dnmt3A and -B establish patterns of methylation early in embryogenesis by de novo methylation of cytosine residues, whereas Dnmt1 maintains such patterns throughout life by copying them onto newly synthesized DNA (4,14). Methylation studies in vitro do not reveal such distinction of functions. In fact, whereas kinetic determinations show that Dnmt1 utilizes hemimethylated DNA 5-10-fold better than unmethylated DNA (15-19), they also show that this enzyme methylates unmethylated DNA more efficiently than Dnmt3A and Dnmt3B (4). Therefore, it is unclear whether the de novo activity of Dnmt1 is repressed in vivo.Most cases of fragile X syndrome, a relatively frequent (1: 4000 births) hereditary neurological disea...

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Section: From the Institute Of Biosciences And Technology Center Formentioning

confidence: 96%

Recombinant Human DNA (Cytosine-5) Methyltransferase

Bacolla

Pradhan

Larson

et al. 2001

Journal of Biological Chemistry

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“…Complexes of the immunoaffinity-purified © iRL Press Limited, Oxford, England. heparin-agarose as previously described (Grunwald and Drahovsky, 1984). The enzymatically active fractions after heparinagarose chromatography were then applied onto a column of M2B1O antibody immobilized on Affi-Gel 10.…”

Section: Resultsmentioning

confidence: 99%

“…Nuclei were extracted in 50 mM Tris-HCI (pH 7.8), 0.8 M KCI, I mM EDTA, I mM dithioerythritol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 /Lg/ml chymostatin, 0.5% (w/v) Trison X-100, 10% (v/v) glycerol, and the nuclear extract was further purified by chromatographies on DEAE-cellulose and heparinagarose (Grunwald and Drahovsky, 1984). 0.2 mM PMSF was included in all buffers.…”

Section: Methodsmentioning

confidence: 99%

“…Immunoaffinity purification of DNA methyltransferase DNA methyltransferase from mouse P815 mastocytoma cells was isolated essentially as previously described (Grunwald and Drahovsky, 1984). Nuclei were extracted in 50 mM Tris-HCI (pH 7.8), 0.8 M KCI, I mM EDTA, I mM dithioerythritol, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 1 /Lg/ml chymostatin, 0.5% (w/v) Trison X-100, 10% (v/v) glycerol, and the nuclear extract was further purified by chromatographies on DEAE-cellulose and heparinagarose (Grunwald and Drahovsky, 1984).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore it appears that at least part of the DNA methyltransferase molecules bind directly to the terminal single-stranded segments and enter the double-stranded part of the DNA from there. The other possibility that the DNA methyltransferase may be arrested at the single-stranded termini after initial binding to the central double-stranded parts of the DNA molecules and moving processively along the DNA strand Morris, 1971a, 1971b;Pfeifer et al, 1983;Bestor and Ingram, 1983;Grunwald and Drahovsky, 1984) may be a less frequent event.…”

Section: Sequence Specificitymentioning

confidence: 99%

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Mouse DNA-cytosine-5-methyltransferase: sequence specificity of the methylation reaction and electron microscopy of enzyme-DNA complexes.

Pfeifer¹,

Spieß²,

Grünwald³

et al. 1985

The EMBO Journal

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Monoclonal antibodies prepared against DNA methyltransferase from human placenta undergo immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells. One of these monoclonal antibodies, M2B10, was used for the immunoaffinity purification of this enzyme. Complexes of the imnmunoaffinity-purified mouse DNA methyltransferase with DNA were visualized by electron microscopy. DNA methyltransferase was found to be distributed along linearized plasmid DNA with a higher incidence of enzyme molecules at the terminal segments. This binding to strand ends was significantly increased after dG-or dGdCtailing of the DNA, which is compatible with a preferred binding of the enzyme to single-stranded DNA. Sequence specificity analysis using methyl-sensitive restriction enzymes showed that the mouse DNA methyltransferase transferred methyl groups to the internal cytosines in 5'CCGG and 5'GCGC sequences, however, the external cytosine in 5'CCGG sequences was also methylated. Key words: DNA methyltransferase/DNA methylation/sequence specificity/electron microscopy/immunoaffinity purification mouse DNA methyltransferase with DNA were visualized by electron microscopy and the sequence specificity of the methylation reaction was analyzed by restriction enzyme analysis of in vitro methylated plasmid DNA. Results and DiscussionBinding of mouse and human DNA methyltransferase into immune complexes Monoclonal anti-human DNA methyltransferase antibodies (Kaul et al., 1984) cross-react with DNA methyltransferase isolated from mouse P815 mastocytoma cells. Figure 1 shows the interaction of monoclonal antibody M2B1O with the mouse and human DNA methyltransferase at different immunoglobin concentrations. Both enzymes are bound into immune complexes which can be removed from the solution by addition of formaline-fixed Staphylococcus aureus cells. When the immunoglobulins were added directly to the DNA methyltransferase incubation mixtures, without removing the immune complexes from the solution, no inhibitory effect was observed. This indicates that the monoclonal antibody M2BlO has a strong binding, but no neutralizing activity on both human and mouse DNA methyltransferase.Immunoaffinity purification of mouse DNA methyltransferase The cross-reactivity of the monoclonal antibody M2BlO allowed us to purify the DNA methyltransferase from mouse P815 cells by immunoaffinity chromatography. Prior to this step, the mouse DNA methyltransferase was pre-purified from high salt nuclear extracts by chromatographies on DEAE-cellulose and

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Purification of human DNA (cytosine‐5‐)‐methyltransferase

Zucker¹,

Riggs²,

Smith³

1985

J of Cellular Biochemistry

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We have developed a facile procedure for the purification of DNA methyltransferase activity from human placenta. The procedure avoids the isolation of nuclei and the dialysis and chromatography of large volumes. A purification of 38,000-fold from the whole cell extract has been achieved. The procedure employs ion exchange, affinity, and hydrophobic interaction chromatography coupled with preparative glycerol gradient centrifugation. A protein of 126,000 daltons was found to copurify with the activity and was the major band seen in the most highly purified material after SDS gel electrophoresis. This observation, coupled with an observed sedimentation coefficient of 6.3S, suggests that the enzyme is composed of a single polypeptide chain of this molecular weight. Hemimethylated DNA was found to be the preferred substrate for the enzyme at each stage in the purification. The ratio of the activity of the purified product on hemimethylated to that on unmethylated M13 duplex DNA was about 12 to 1. Thus, the purified activity has the properties postulated for a maintenance methyltransferase. The availability of highly purified human DNA methyltransferase should facilitate many studies on the structure, function, and expression of these activities in both normal and transformed cells.

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DNA-cytosine-5-methyltransferase from P815 mouse mastocytoma cells: “Maintenance” and “de novo” activities are carried out by the same enzyme molecule

Cited by 17 publications

References 26 publications

Recombinant Human DNA (Cytosine-5) Methyltransferase

Recombinant Human DNA (Cytosine-5) Methyltransferase

Mouse DNA-cytosine-5-methyltransferase: sequence specificity of the methylation reaction and electron microscopy of enzyme-DNA complexes.

Purification of human DNA (cytosine‐5‐)‐methyltransferase

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