Abstract
Purification of an enzymatic activity requires a simple and relatively expeditious assay of its activity. However, the nature of the demethylase reaction has been elusive for decades. Although a large body of evidence supported the hypothesis that active demethylation takes place during development and differentiation (1), the nature of the reaction was unknown. The main problem with understanding demethylaion of DNA is that true demethylation of DNA would involve cleavage of a stable carbon-carbon bond, which had been considered highly unlikely. Different laboratories have suggested that demethylation of DNA is accomplished by different repair mechanisms. These alternative routes involve either a cleavage of the bond between the methylated cytosine base and the deoxyribose (2) or nucleotide excision (3) (Fig. 1). We have recently shown that mamMalian cancer-cell lines bear a bona fide demethylase activity and we defined the reactants and products of the demethylation reaction. The demethylation reaction involves the hydrolytic cleavage of the bond between the methyl-carbon and the carbon at the 5 position of the cytosine ring (Fig. 1 and Fig. 2) producing unmethylated cytosine while the methyl group is released as methanol (4).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kafri, T., Gao, X., and Razin, A. (1993) Mechanistic aspects of genome-wide demethylation in the preimplantation mouse embryo. Proc. Natl. Acad. Sci. USA 90,10,558–10,562.
Razin, A., Szyf, M., Kafri, T., Roll, M., Giloh, H., Scrapa, S., et al. (1986) Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation. Proc. Natl. Acad. Sci. USA 83, 2827–2831.
Weiss, A., Keshet, I., Razin, A., and Cedar, H. (1996) DNA demethylation in vitro: involvement of RNA. Cell 87, 709–718.
Ramchandani, S., Bhattacharya, S. K., Cervoni, N., and Szyf, M. (1999) DNA methylation is a reversible biological signal. Proc. Natl. Acad. Sci. USA 96,6107–6112.
Waalwijk, C. and Flavell, R. A. (1978) DNA methylation at a CCGG sequence in the large intron of the rabbit b-globin gene: tissue specific variations. Nucleic Acids. Res. 5, 4631–4641.
Clark, S. J., Harrison, C. L., Paul, C. L., and Fromer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22,2990–2997.
Cervoni, N., Bhattacharya, S. K., and Szyf, M. (1999) DNA demethylase is a processive enzyme. J. Biol. Chem. 274, 8363–8366.
Szyf, M., Theberge, J., and Bozovic, V. (1995) Ras induces a general DNA demethylation activity in mouse embryonal P19 cells. J. Biol. Chem. 270, 12,690–12,696.
Terwilliger, T. C., Bogoonez, E., Wang, E. A., and Koshland Jr., D. E. (1983) Sites of methyl esterification on the aspartate receptor involved in bacterial chemotaxis. J. Biol. Chem. 258,9608–9611.
Bhattacharya, S. K., Ramchandani, S., Cervoni, N., and Szyf, M. (1999) A mamalian protein with specific demethylase activity for mCpG DNA. Nature 397,579–583.
Hendrich B. and Bird A. Identification and characterization of a family of mamalian methyl-CpG binding proteins. (1998) Mol. Cell. Biol. 18, 6538–6547.
Ng, H. H., Zhang, Y., Hendrich, B., Johnson, C. A., Turner, B. M., Erdjument-Bromage, H., et al. (1999) MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nature Genet. 23,58–61.
Ausaubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Stairwell, K. (eds.) (1988) Current Protocols in Molecular Biology. John Wiley & Sons, New York.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Szyf, M., Bhattacharya, S.K. (2002). Extracting DNA Demethylase Activity from Mammalian Cells. In: Mills, K.I., Ramsahoye, B.H. (eds) DNA Methylation Protocols. Methods in Molecular Biology™, vol 200. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-182-5:163
Download citation
DOI: https://doi.org/10.1385/1-59259-182-5:163
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-618-5
Online ISBN: 978-1-59259-182-4
eBook Packages: Springer Protocols