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Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase
A Tomassetti1, P H Driever, G P Pfeifer
1Zentrum der Biologischen Chemie, Universität Frankfurt, F.R.G.
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.
Insights
Researchers purified proteins that stimulate DNA methyltransferase activity. These proteins alter DNA methylation sequence specificity and bind to DNA, but do not form a tight complex with the enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Epigenetics
Background:
- DNA methyltransferase (DNMT) regulates gene expression through DNA methylation.
- Previous studies purified DNMT from P815 mastocytoma cells using immunoaffinity chromatography.
Purpose of the Study:
- To purify and characterize proteins that modulate DNA methyltransferase activity.
- To investigate the interaction of these proteins with DNMT and DNA.
Main Methods:
- Immunoaffinity chromatography, DEAE-cellulose chromatography, heparin-agarose chromatography, butanol extraction, and gel filtration (Superose 12) were used for protein purification.
- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to determine the molecular masses of the purified proteins.
- Enzymatic assays were performed to assess the effect of purified proteins on DNMT activity using various DNA substrates.
Main Results:
- Two distinct fractions of DNMT-stimulating proteins were purified.
- One fraction contained a 29 kDa polypeptide, while the second fraction comprised polypeptides of 78-82 kDa and 51-54 kDa.
- These proteins stimulated both de novo and maintenance DNMT activity by approximately 3-fold.
- The proteins enhanced methylation of natural DNA and poly[(dI-dC).(dI-dC)] but inhibited methylation of poly[(dG-dC).(dG-dC)].
- The purified proteins bind to both double-stranded and single-stranded DNA, altering DNMT sequence specificity without forming a tight complex with the enzyme.
Conclusions:
- Proteins that stimulate DNA methyltransferase activity have been identified and purified.
- These stimulatory proteins interact with DNA and modify the sequence specificity of DNA methylation.
- The findings suggest a regulatory mechanism for DNA methylation involving accessory proteins.