Mechanism of action of eukaryotic DNA methyltransferase: Use of 5-azacytosine-containing DNA

doi:10.1016/0022-2836(82)90395-3

Journal of Molecular Biology

Volume 162, Issue 3, 15 December 1982, Pages 679-692

https://doi.org/10.1016/0022-2836(82)90395-3 Get rights and content

Abstract

Hemimethylated DNA substrates prepared from cell cultures treated with 5-azacytidine are efficient acceptors of methyl groups from S-adenosylmethionine in the presence of a crude preparation of mouse spleen DNA methyltransferase. Partially purified methyltransferase was also capable of efficiently modifying single-stranded unmethylated DNA. The methylation of single-stranded DNA was less sensitive to inhibition by salt than duplex DNA. The presence of other DNA species in the reaction mix (duplex or single-stranded, methylated or unmethylated) inhibited the modification of the hemimethylated duplex DNA. The enzyme was specific for DNA, since the presence of RNA in reaction mixtures did not inhibit the methylation of DNA. DNA methyltransferase formed a tight-binding complex with hemimethylated duplex DNA containing high levels of 5-azacytosine, and this complex was not dissociated by high concentrations of salt. Treatment of cultured cells with biologically effective concentrations of 5-azacytidine and other cytidine analogs modified in the 5 position resulted in a loss of extractable active enzyme from the cells. The amount of extractable active enzyme recovered slowly with time after treatment. These results suggest that incorporation of 5-azacytidine into DNA inhibits the progress of DNA methyltransferase along the duplex, perhaps by the formation of a tight-binding complex. This complex formation might be irreversible, so that new enzyme synthesis might be required to reverse the block of DNA methylation.

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^☆: This work was supported by grant no. GM30892 from the National Institutes of Health.

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Journal of Molecular Biology

Mechanism of action of eukaryotic DNA methyltransferase: Use of 5-azacytosine-containing DNA☆

Abstract

Biochim. Biophys. Acta

Anal. Biochem

J. Mol. Biol

Trends Biochem. Sci

Cell

Devel. Biol

J. Biol. Chem

J. Mol. Biol

J. Mol. Biol

Cell

Anal. Biochem

Cell

Cell

Cell

Methods Enzymol

Eur. J. Biochem

Mol. Pharmacol