DNA-methylase from regenerating rat liver: purification and characterisation

Simon, Dietrich; Grunert, F.; Acken, U.v.; Dörining, H. P.; Kröger, Hans

doi:10.1093/nar/5.6.2153

Cited by 99 publications

(43 citation statements)

References 32 publications

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“…1). The substrate efficiency of DNA from a variety of organisms was also tested (data not shown) and varied widely, as reported by others (20,(27)(28)(29)(30). In general, G+C-rich DNAs were better substrates than G+C-poor DNAs.…”

Section: Methodsmentioning

confidence: 66%

“…The enzyme was extracted with 0.8 M KCl from purified rat liver and spleen nuclei as described (20,21). The crude nuclear extract in 20 mM NaCi in buffer A (50 mM Tris HCl, pH 7.8/1 mM dithiothreitol) was loaded onto a S-adenosyl-L-homocysteine agarose column (3 ml; Bethesda Research Laboratories); the column was washed with 30 ml of 60 mM KCI/buffer A/1 mM EDTA and 15 ml of the same buffer with 20 ,uM S-adenosylmethionine (AdoMet).…”

Section: Methodsmentioning

confidence: 99%

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Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

Farrance¹,

Ivarie²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Ethylation of poly(dC-dG) poly(dC-dG) with ethyl methanesulfonate (EtMes), a known carcinogen, at increasing molar ratios of EtMes/C-G base pairs progressively stimulated the methyl-accepting ability of the DNA during in vitro methylation by partially purified rat DNA (cytosine-5)-methyltransferase (EC 2.1.1.37). Maximum stimulation was 2-fold over mock-treated DNA when 2.7% of the guanines were modified at the N-7 position, the major site of ethylation by EtMes in DNA. If a CpG site "hemiethylated" at guanine N-7 mimics a hemimethylated CpG site, we calculate that the enzyme has a relative affinity for hemiethylated CpG 18-fold above unmodified CpG. If ethylation of a dioxyphosphate oxygen of the phosphodiester bond is responsible for stimulation, the relative affinity could be much higher, up to 370-fold.DNA methylation at cytosine C-5 in the rare CpG dinucleotide appears to play an important role in regulating the expression of many mammalian genes (reviewed in refs. 1-7) and is being extensively studied as a potential target for chemical and radiation-induced carcinogenesis. A large body of evidence now exists showing that chemical carcinogens and UV irradiation, both of which chemically modify DNA, significantly decrease enzymatic methylation of DNA in vivo and in vitro (8)(9)(10)(11)(12)(13)(14). Demethylation may, therefore, play a role in activation of the cancer cell phenotype as proposed by Holliday (15).By contrast, however, we recently found that one widely used chemical carcinogen, ethyl methanesulfonate (EtMes), inactivated specific gene expression (16) apparently by promoting an increase in enzymatic methylation of cultured cell DNA (17). Methyl methanesulfonate has also been reported to increase the level of 5-methylcytidine in DNA in regenerating rat liver (18). Because EtMes introduces ethyl groups primarily at guanine N-7 and secondarily at the phosphodiester bond (19), it was proposed that EtMes might promote enzymatic methylation (17) by creating a "fraudulent" hemimethylated site by rotation of an ethyl group at guanine N-7 or the phosphodiester in close proximity to cytosine C-5 in CpG sequences in B-DNA. This ethylated site might be recognized as hemimethylated by DNA (cytosine-5)-methyltransferase (MeTase; EC 2.1.1.37) and subsequently undergo methylation. We have tested a prediction of the model that unmethylated CpG sites modified by EtMes will be more efficient substrates for DNA methyltransferase in vitro than unmodified sites, and we report the results here. MATERIALS AND METHODSDNA MeTase. The enzyme was extracted with 0.8 M KCl from purified rat liver and spleen nuclei as described (20,21). The crude nuclear extract in 20 mM NaCi in buffer A (50 mM Tris HCl, pH 7.8/1 mM dithiothreitol) was loaded onto a S-adenosyl-L-homocysteine agarose column (3 ml; Bethesda Research Laboratories); the column was washed with 30 ml of 60 mM KCI/buffer A/1 mM EDTA and 15 ml of the same buffer with 20 ,uM S-adenosylmethionine (AdoMet). Enzyme was eluted with 0.3 M KCI in buffer A/1 mM EDTA a...

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Section: Methodsmentioning

confidence: 66%

Section: Methodsmentioning

confidence: 99%

Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

Farrance¹,

Ivarie²

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Bird has estimated that there may be as many as 30,000 CpG islands in the haploid mouse genome (9). It is also predicted that CpG islands will be found associated with the vast majority of genes, especially those that are widely expressed (32 (34). These enzymes catalyze the transfer ofthe methyl group from Sadenosylmethionine (SAM) to the 5-position of speciflc cytosine residues in DNA.…”

Section: Dna Methylation Patternsmentioning

confidence: 99%

“…These enzymes catalyze the transfer ofthe methyl group from Sadenosylmethionine (SAM) to the 5-position of speciflc cytosine residues in DNA. Methyltransferase enzymes recognize hemimethylated DNA (34) and maintain methylation patterns with high fidelity (35). Following replication, the maintenance methylases use the the methylation pattern on the template strand to restore symmetry and direct the precise methylation of the newly synthesized DNA strand (15).…”

Section: Dna Methylation Patternsmentioning

confidence: 99%

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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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CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication.

1992

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The physical parameters controlling the accessibility of antigen receptor loci to the V(D)J recombination activity are unknown. We have used minichromosome substrates to study the role that CpG methylation might play in controlling V(D)J recombination site accessibility. We find that CpG methylation decreases the V(D)J recombination of these substrates more than 100‐fold. The decrease correlates with a considerable increase in resistance to endonuclease digestion of the methylated minichromosome DNA. The minichromosomes acquire resistance to both the intracellular V(D)J recombinase and exogenous endonuclease only after DNA replication. Therefore, CpG methylation specifies a chromatin structure that, upon DNA replication, is resistant to eukaryotic site‐specific recombination. These findings are important to V(D)J recombination as well as to the chromatin assembly of methylated DNA during replication.

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DNA-methylase from regenerating rat liver: purification and characterisation

Cited by 99 publications

References 32 publications

Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

Ethylation of poly(dC-dG).poly(dC-dG) by ethyl methanesulfonate stimulates the activity of mammalian DNA methyltransferase in vitro.

DNA Methylation and Differentiation

CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication.

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