Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase.

Abstract

Previously, the purification of DNA methyltransferase from murine P815 mastocytoma cells by immunoaffinity chromatography was described (Pfeifer, G.P., Grünwald, S., Palitti, F., Kaul, S., Boehm, T.L.J., Hirth, H.P. and Drahovsky, D. (1985) J. Biol. Chem. 260, 13787-13793). Proteins that stimulate the enzymatic activity of DNA methyltransferase have been purified from the same cells. These proteins, which partially coelute with DNA methyltransferase from DEAE-cellulose and heparin-agarose, are separated from the enzyme during the immunoaffinity purification step. A further purification of the stimulating proteins was achieved by butanol extraction, DEAE-cellulose chromatography and gel filtration on Superose 12. Two DNA methyltransferase-stimulating protein fractions were obtained. SDS-polyacrylamide gel electrophoresis of one fraction showed a single polypeptide with a molecular mass of 29 kDa. The second fraction consisted of 5 or 6 polypeptides with molecular masses 78-82 and 51-54 kDa. The proteins stimulate both de novo and maintenance activity of DNA methyltransferase about 3-fold. They enhance the methylation of any natural DNA and of poly[(dI-dC).(dI-dC)] but inhibit the methylation of poly[(dG-dC).(dG-dC)]. The purified proteins do not form a tight complex with DNA methyltransferase; however, they bind both to double-stranded and single-stranded DNA. The sequence specificity of DNA methyltransferase is obviously altered in presence of these proteins.

• Authors: A Tomassetti, P H Driever, G P Pfeifer, D Drahovsky
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 951 Issue 1 Pg. 201-12 (Nov 10 1988) ISSN: 0006-3002 [Print] Netherlands
• PMID: 3142521 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Butanols
• Proteins
• 1-Butanol
• DNA
• DNA (Cytosine-5-)-Methyltransferases

Topics

• 1-Butanol
• Animals
• Butanols
• Centrifugation, Density Gradient
• Chromatography, Affinity
• Chromatography, DEAE-Cellulose
• Chromatography, Gel
• DNA (metabolism)
• DNA (Cytosine-5-)-Methyltransferases (isolation & purification, metabolism)
• Electrophoresis, Polyacrylamide Gel
• Enzyme Activation (drug effects)
• Immunoassay
• Mast-Cell Sarcoma (analysis)
• Methylation
• Mice
• Molecular Weight
• Osmolar Concentration
• Proteins (isolation & purification, metabolism, pharmacology)
• Substrate Specificity
• Tumor Cells, Cultured