Extracting DNA Demethylase Activity from Mammalian Cells
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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 mamM alian 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 ).
Fig. 1. Possible demethylation mechanisms. The figures shows a methylated CpG dinucleotide. DNA could be demethylated either directly or indirectly. Indirect demethylation might involve either removing the methyl-C base by a glycosylase or removing the methyl-CpG dinucleotide by a nucleotide excision mechanism. The possible cleavage points are indicated. The damaged DNA is repaired by the repair machinery, introducing unmethylated cytosines into the damaged region. Alternatively, the methyl group per se could be removed by cleavage of the carbon-carbon bond between the carbon at the 5′ position of cytosine and the methyl group. The methyl group leaves as a volatile residue.
Fig. 2. The direct demethylation reaction. As shown in ref. 4 the methyl residue in methylated cytosine can react with water in a reaction catalyzed by the demethylase enzyme to form methanol and nonmethylated cytosines.
