Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

Wang, Richard Y.-H.; Huang, Li Yen Mae; Ehrlich, Melanie

doi:10.1093/nar/12.8.3473

Cited by 37 publications

(24 citation statements)

References 55 publications

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“…The procedure used for isolating MDBP and DNA methyltransferase from human placental nuclei involved previously described methods with some modifications (14,15). Two full-term placenta minus the chorionic plate were minced and homogenized for 10 sec at full speed in a Waring Blender and then in a Potter-Elvejehm homogenizer with six strokes at 3/4 maximum speed using 3-4 volumes of 50 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 0.25 M sucrose, 0.1 mM phenylmethylsulfonyl fluoride, 1 ig/ml chymostatin (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…After a further 1:1 dilution with this buffer, the suspension was filtered through two layers of gauze, then through four layers, and lastly through a nylon membrane (HD3-70; Tetko, Depew, N.Y.). The filtrate was centrifuged and the crude nuclear pellet was rinsed, extracted with 0.3 M NaCl, and the extract centrifuged as previously described (14,15). The supernatant was diluted with 2.5 volumes of 25 mM potassium phosphate, pH 7.4, 0.1 mM EDTA, 0.2 mM dithiothreitol (DTT), 10% glycerol (buffer A) and applied at a flow rate of 80 ml/h to a phosphocellulose column (5 x 5 cm), which had been preequilibrated with buffer A plus 0.1 M KC1.…”

Section: Methodsmentioning

confidence: 99%

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A human DNA-binding protein is methylation-specific and sequence-specific

1986

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A nuclear protein isolated from human placenta, methylated DNA-binding protein (MDBP), binds selectively to DNA enriched in 5-methylcytosine. We now demonstrate that MDBP is a sequence-specific, as well as methylation-specific, DNA-binding protein. From ten restriction fragments of pBR322 DNA methylated with human DNA methyltransferase, one was bound to MDBP very much more strongly than any of the others. For this preferential binding to MDBP, the DNA had to be methylated. By a DNase I protection experiment (DNase I footprinting), a 22-base sequence within this methylated restriction fragment was shown to be specifically protected by MDBP. The sequence-specificity of MDBP coupled with its dependence on DNA methylation suggests that this is one of the proteins which modulates important functions of human DNA methylation in vivo.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A human DNA-binding protein is methylation-specific and sequence-specific

1986

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“…DNA reassociation experiments and subsequent restriction endonuclease analysis suggest that most CpG dinucleotides are symmetrically methylated (3). Purification of methylases from somatic nuclei and in vitro assays of their activities showed that in general, their preferred substrate was hemimethylated DNA (9,30). In addition, DNA-medi-ated gene transfer experiments showed that clonal inheritance of methylation patterns in vivo was restricted to the palindromic dinucleotide CpG (27).…”

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confidence: 99%

Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

et al. 1989

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We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.Postreplicative addition of methyl groups to DNA bases is a common modification of the DNAs of most organisms. Most higher eucaryotes contain exclusively 5-methylcytosine, whereas protozoans, some plants, and procaryotes contain N6-methyladenine either exclusively or in addition to 5-methylcytosine. It is not known whether cytosine and adenine methylations of DNA serve similar or different functions.Methylated bases are not distributed at random in the DNA but are found in stable, tissue-or cell-type specific patterns. In the case of the cytosine methylases, it is thought that methylation patterns are established with the differentiation of cell type and maintained by a methylase with limited de novo activity (for review, see reference 18). Because of the palindromic nature of many methylation sites in eucaryotes, a model based on the semiconservative nature of DNA replication has been proposed for the mechanism of activity of cytosine methylases (3,14,19). According to this model, DNA molecules of the parent cell, which are modified on both strands, yield hemimethylated sites after DNA replication. These sites become the target for a nondiscriminating maintenance methylase with a strong preference for a hemimethylated substrate. This enzyme methylates the base on the newly replicated daughter strand, thus faithfully maintaining the pattern through cell division. Support for this model has come from several lines of evidence. DNA reassociation experiments and subsequent restriction endonuclease analysis suggest that most CpG dinucleotides are symmetrically methylated (3). Purification of methylases from somatic nuclei and in vitro assays of their activities showed that in general, their preferred substrate was hemimethylated DNA (9, 30). In addition, DNA-medi-

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“…Eukaryotic DNA methyltransferase has been partially purified and characterized by several investigators (34,(43)(44)(45)(46). However, controversy still exists as to the existence of two distinct enzymes, since the de novo and maintenance methyltransferase activities do co-purify (26,44,46).…”

Section: Dna Methylation Patternsmentioning

confidence: 99%

DNA Methylation and Differentiation

Michalowsky¹,

Jones²

1989

Environmental Health Perspectives

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The methylation of specific cytosine residues in DNA has been implicated in regulating gene expression and facilitating functional specialization of cellular phenotypes. Generally, the demethylation of certain CpG sites correlates with transcriptional activation of genes. 5-Azacytidine is an inhibitor of DNA methylation and has been widely used as a potent activator of suppressed genetic information. Treatment of cells with 5-azacytidine results in profound phenotypic alterations. The drug-induced hypomethylation of DNA apparently perturbs DNA-protein interactions that may consequently alter transcriptional activity and cell determination. The inhibitory effect of cytosine methylation may be exerted via altered DNA-protein interactions specifically or may be transduced by a change in the conformation of chromatin.Recent studies have demonstrated that cytosine methylation also plays a central role in parental imprinting, which in turn determines the differential expression of maternal and paternal genomes during embryogenesis. In other words, methylation is the mechanism whereby the embryo retains memory of the gametic origin of each component of genetic information. A memory of this type would probably persist during DNA replication and cell division as methylation patterns are stable and heritable.

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Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

Cited by 37 publications

References 55 publications

A human DNA-binding protein is methylation-specific and sequence-specific

A human DNA-binding protein is methylation-specific and sequence-specific

Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

DNA Methylation and Differentiation

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