Extracting DNA Demethylase Activity from Mammalian Cells
Bibliographic record
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 (), 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 () or nucleotide excision () (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 (). Open image in new window 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. Open image in new window Fig. 2. The direct demethylation reaction. As shown in ref. the methyl residue in methylated cytosine can react with water in a reaction catalyzed by the demethylase enzyme to form methanol and nonmethylated cytosines.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".