Extracting DNA Demethylase Activity from Mammalian Cells
Part of the Methods in Molecular Biology™ book series (MIMB, volume 200)
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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).
KeywordsNuclear Extract Glass Wool Scintillation Vial Microfuge Tube Unmethylated Cytosine
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