GO:0035516 broad specificity oxidative DNA demethylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0035516 describes a molecular function that removes methyl groups from damaged DNA bases such as N1-methyladenine, N3-methylcytosine, N1-methylguanine and N3-methylthymine through oxidative demethylation.
• The reaction uses 2-oxoglutarate and oxygen as co-substrates and releases formaldehyde, succinate and CO2, classifying the enzymes as 2-oxoglutarate-dependent dioxygenases.
• The same catalytic activity can act on RNA, linking DNA repair and RNA modification pathways.
• The activity is central to the repair of alkylation damage and to the regulation of epigenetic marks such as 5-hydroxymethylcytosine and 5-formylcytosine.
• Dysregulation of oxidative demethylation is associated with cancer, neurological disorders and metabolic disease, making it a target for functional genomics.
• CRISPR knockout, point-mutation, knock-in and overexpression models are essential to dissect the causal role of these demethylases in cells and organisms.
Description
GO:0035516, broad specificity oxidative DNA demethylase activity, is a molecular function that catalyzes the oxidative removal of methyl groups from a range of methylated nucleobases within DNA. The reaction consumes 2-oxoglutarate and O2 and produces formaldehyde, succinate and CO2, and it can also act on RNA substrates. This activity is performed by enzymes such as the AlkB family dioxygenases and the TET family of 5-methylcytosine oxidases, which are central to both DNA repair and epigenetic regulation. Researchers study this term because it connects the chemistry of oxidative demethylation to genome stability, gene expression and human disease. Understanding its mechanism, regulation and cellular consequences requires precise genetic models and functional assays.
broad specificity oxidative DNA demethylase activity At A Glance
| GO ID | GO:0035516 |
|---|---|
| GO term | broad specificity oxidative DNA demethylase activity |
| Ontology | molecular_function |
| Synonym | oxidative DNA demethylase activity; N1-methyladenine demethylase activity; N3-methylcytosine demethylase activity; 5hmC dioxygenase; 5fC dioxygenase |
| Major function | Oxidative demethylation of methylated DNA bases using 2-oxoglutarate and O2, releasing formaldehyde, succinate and CO2 |
| Substrates | N1-methyladenine, N3-methylcytosine, N1-methylguanine, N3-methylthymine; also RNA substrates |
| Cofactors | 2-oxoglutarate, O2, Fe(II) |
| Products | Demethylated nucleobase, formaldehyde, succinate, CO2 |
What Is GO:0035516?
In simple terms, GO:0035516 is the activity of an enzyme that uses oxygen and 2-oxoglutarate to strip a methyl group off damaged or regulatory bases in DNA, converting the methyl group into formaldehyde and leaving the base demethylated. The official definition states: Catalysis of the reaction: a methylated nucleobase within DNA + 2-oxoglutarate + O2 = a nucleobase within DNA + formaldehyde + succinate + CO2; it catalyzes oxidative demethylation of the DNA base lesions N1-methyladenine, N3-methylcytosine, N1-methylguanine, and N3-methylthymine, and can also act on RNA. This broad specificity distinguishes it from more substrate-restricted demethylases and places it at the intersection of DNA repair and epigenetic marking.
Why Is broad specificity oxidative DNA demethylase activity Important in Cell Biology?
GO:0035516 is important because it defines a conserved chemical strategy for reversing methylation damage and for shaping epigenetic information. Enzymes with this activity protect cells from alkylation-induced mutations and also generate oxidized derivatives of 5-methylcytosine that are intermediates in active DNA demethylation. Because the same activity can act on RNA, it also influences RNA stability and translation. Consequently, this function is relevant to cancer biology, neurodevelopment, metabolic regulation and stem cell differentiation, and it is a frequent target of functional genomics studies.
• Protects DNA from alkylation damage caused by endogenous and environmental agents.
• Generates 5-hydroxymethylcytosine and 5-formylcytosine, key intermediates in active DNA demethylation.
• Regulates gene expression through epigenetic remodeling.
• Acts on RNA, linking DNA repair to RNA modification and translation.
• Mutations in genes encoding this activity are associated with cancer and neurological disorders.
• Provides a druggable target for inhibitors that modulate epigenetic states.
• Serves as a model for studying 2-oxoglutarate-dependent dioxygenases.
• Enables CRISPR-based dissection of repair and epigenetic pathways.
What Happens During broad specificity oxidative DNA demethylase activity?
Substrate recognition and binding
In simple terms: The enzyme finds and grabs the methylated base in DNA or RNA.
The enzyme recognizes methylated nucleobases such as N1-methyladenine and N3-methylcytosine that are flipped out of the DNA helix. Binding is stabilized by a conserved double-stranded beta-helix fold that coordinates Fe(II) and 2-oxoglutarate. This step ensures that the enzyme acts on specific damaged or regulatory bases while avoiding unmodified DNA.
Oxidative demethylation chemistry
In simple terms: Oxygen and 2-oxoglutarate are used to remove the methyl group as formaldehyde.
The catalytic cycle activates molecular oxygen at the Fe(II) center, forming a high-valent iron-oxo species that hydroxylates the methyl group. The resulting unstable intermediate collapses to release formaldehyde and the demethylated base. 2-oxoglutarate is simultaneously converted to succinate and CO2, completing the reaction.
Product release and repair completion
In simple terms: The demethylated base is released and the DNA is restored.
After demethylation, the repaired base is reinserted into the DNA helix or the oxidized intermediate is further processed by downstream enzymes. In the case of 5-methylcytosine oxidation, the products 5-hydroxymethylcytosine and 5-formylcytosine can be recognized by base excision repair or replication-coupled mechanisms. This step links the catalytic activity to genome maintenance and epigenetic reprogramming.
RNA demethylation
In simple terms: The same enzyme can also clean methyl groups from RNA.
The broad specificity of GO:0035516 allows the enzyme to act on methylated RNA bases, influencing RNA stability and translation. This activity expands the biological impact of the term beyond DNA repair. It also suggests that mutations affecting the catalytic site can have pleiotropic effects on both DNA and RNA metabolism.
Key Genes Involved in GO:0035516 broad specificity oxidative DNA demethylase activity
The following genes encode enzymes or associated factors that carry out or regulate broad specificity oxidative DNA demethylase activity.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ALKBH1 | DNA and RNA demethylase with broad specificity | Studies of alkylation repair and epigenetic regulation |
| ALKBH2 | Repairs N1-methyladenine and N3-methylcytosine in DNA | Model for DNA repair deficiency and cancer |
| ALKBH3 | Repairs methylated bases in single-stranded DNA and RNA | Target for cancer therapy and RNA modification studies |
| FTO | RNA and DNA demethylase, regulates energy homeostasis | Obesity and metabolic disease models |
| TET1 | Oxidizes 5-methylcytosine to 5-hydroxymethylcytosine | Epigenetic reprogramming and cancer |
| TET2 | Oxidizes 5-methylcytosine and regulates hematopoiesis | Leukemia and stem cell biology |
| TET3 | Oxidizes 5-methylcytosine in zygotes and neurons | Developmental and neurological studies |
| KDM2A | Histone demethylase with DNA demethylase activity | Chromatin regulation and cancer |
| KDM2B | Histone and DNA demethylase | Stem cell maintenance and differentiation |
| KDM3A | Histone demethylase with oxidative activity | Hypoxia response and spermatogenesis |
| KDM4A | Histone demethylase, 2-oxoglutarate dependent | Cancer and chromatin dynamics |
| KDM4B | Histone demethylase | DNA repair and transcription |
| KDM4C | Histone demethylase | Cancer progression and stemness |
| KDM5A | Histone demethylase | Drug tolerance and epigenetic therapy |
| KDM6A | Histone demethylase | Developmental disorders and cancer |
| IDH1 | Produces 2-oxoglutarate for dioxygenases | Metabolic regulation of demethylation |
| IDH2 | Produces 2-oxoglutarate in mitochondria | Cancer metabolism and epigenetics |
| VITAMIN_C | Cofactor for Fe(II)/2-oxoglutarate dioxygenases | Epigenetic gatekeeper in osteogenesis |
How Is broad specificity oxidative DNA demethylase activity Regulated?
The activity of GO:0035516 is regulated by the availability of its co-substrates 2-oxoglutarate and oxygen, as well as by Fe(II) and ascorbate. Metabolic enzymes such as IDH1 and IDH2 influence the pool of 2-oxoglutarate, thereby modulating demethylase activity. Vitamin C acts as a cofactor that maintains Fe(II) in its reduced state and supports the catalytic cycle of 2-oxoglutarate-dependent dioxygenases. Additionally, post-translational modifications and protein-protein interactions can target these enzymes to specific chromatin regions.
broad specificity oxidative DNA demethylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| TET2 | Leukemia, myelodysplastic syndromes | Knockout and point-mutation cell lines |
| FTO | Obesity, metabolic syndrome | Overexpression and knockout mouse models |
| ALKBH2 | Cancer predisposition, alkylation damage | Knockout cells and knock-in repair assays |
| TET3 | Neurodevelopmental disorders | Neuronal differentiation models |
| IDH1 | Glioma, acute myeloid leukemia | Point-mutation knock-in for 2-HG production |
Cancer
Mutations in TET2 and other genes encoding oxidative demethylases are frequent in hematological malignancies and solid tumors. Loss of demethylase activity leads to aberrant DNA methylation and altered gene expression that promotes tumorigenesis. Targeting these enzymes with small-molecule inhibitors is an active area of cancer research.
Neurological disorders
TET3 and ALKBH family members are important for neuronal development and function. Dysregulation of oxidative demethylation has been linked to neurodevelopmental disorders and neurodegeneration. Vitamin C, a cofactor for these enzymes, is also implicated in skeletal and neuronal health.
Metabolic disease
FTO and other demethylases regulate energy homeostasis and adipogenesis. Variants in FTO are associated with obesity and metabolic syndrome. The catalytic activity of these enzymes is sensitive to metabolic intermediates, linking cellular metabolism to epigenetic regulation.
From broad specificity oxidative DNA demethylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of demethylase activity cause DNA damage sensitivity? | CRISPR knockout cell lines |
| Does a specific catalytic residue affect substrate specificity? | Point-mutation knock-in |
| Can a disease-associated mutation alter demethylation? | Knock-in of patient variants |
| Where does the enzyme localize in the genome? | Tagged knock-in with fluorescent or affinity tags |
| Does overexpression drive epigenetic reprogramming? | Doxycycline-inducible overexpression |
| Which pathways depend on the demethylase? | CRISPR library screening |
How to Study the broad specificity oxidative DNA demethylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| CRISPR knockout | Loss of gene function | DNA repair and epigenetic studies |
| Point-mutation knock-in | Effect of specific residues | Catalytic mechanism analysis |
| Tagged knock-in | Protein localization and interactions | Chromatin binding studies |
| Overexpression | Gain of function | Epigenetic reprogramming |
| CRISPR library screening | Phenotypes of many genes | Pathway discovery |
| 5hmC/5fC sequencing | Oxidized base distribution | Epigenome mapping |
| RNA modification profiling | Methylated RNA bases | RNA demethylation studies |
CRISPR knockout and point-mutation
CRISPR-Cas9 knockout of demethylase genes followed by alkylation damage sensitivity assays reveals their role in DNA repair. Point mutations in the catalytic domain can separate demethylation from other functions.
Epigenomic profiling
Antibodies against 5-hydroxymethylcytosine and 5-formylcytosine enable mapping of oxidized bases by sequencing. This approach quantifies the impact of demethylase activity on the epigenome.
RNA modification analysis
RNA immunoprecipitation and mass spectrometry can detect methylated RNA bases and their demethylation. These methods link the activity to RNA metabolism.
Metabolic and cofactor studies
Measuring 2-oxoglutarate, succinate and ascorbate levels provides insight into the regulation of the enzyme. Vitamin C supplementation can modulate activity in cell culture.
How CRISPR Can Be Used to Study GO:0035516 broad specificity oxidative DNA demethylase activity
Knockout
CRISPR knockout of ALKBH2, ALKBH3 or TET genes creates cell models to test sensitivity to alkylating agents and to measure changes in DNA methylation. These models are essential for establishing causality between the enzyme and cellular phenotypes.
Point Mutation
Introducing point mutations in the catalytic domain of demethylases allows researchers to separate demethylation activity from protein-protein interactions. Such models help define the precise contribution of the catalytic function to disease.
Knock-in
Knock-in of disease-associated variants or tagged versions of the enzyme enables studies of localization, stability and substrate specificity in a physiological context. This approach is valuable for modeling patient-specific mutations.
Overexpression
Overexpression of wild-type or mutant demethylases can drive epigenetic changes and reveal downstream pathways. Inducible systems allow temporal control of activity.
How EDITGENE Supports broad specificity oxidative DNA demethylase activity Research
Researchers studying broad specificity oxidative DNA demethylase activity-related genes often need to determine whether a candidate gene is causally involved in DNA repair, epigenetic regulation or disease. EDITGENE provides a comprehensive suite of CRISPR services to generate precisely engineered cell models for such functional studies.
Contact EDITGENE today to design your custom CRISPR model for broad specificity oxidative DNA demethylase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| FTO Knockout HEK293 Cell Line | EDJ-KQ187 | Human | 79068 | Details Get a Quote |
| ALKBH3 Knockout HEK293 Cell Line | EDJ-KQ2287 | Human | 221120 | Details Get a Quote |
| ALKBH1 Knockout HEK293 Cell Line | EDJ-KQ6380 | Human | 8846 | Details Get a Quote |
| ALKBH2 Knockout HEK293 Cell Line | EDJ-KQ8103 | Human | 121642 | Details Get a Quote |
| ALKBH4 Knockout HEK293 Cell Line | EDJ-KQ11586 | Human | 54784 | Details Get a Quote |
| FTO Knockout HeLa Cell Line | EDJ-KQ19015 | Human | 79068 | Details Get a Quote |
| ALKBH2 Knockout A-549 Cell Line | EDJ-KQ33969 | Human | 121642 | Details Get a Quote |
| ALKBH2 Knockout HCT 116 Cell Line | EDJ-KQ33970 | Human | 121642 | Details Get a Quote |
| ALKBH2 Knockout HeLa Cell Line | EDJ-KQ33971 | Human | 121642 | Details Get a Quote |
| ALKBH4 Knockout A-549 Cell Line | EDJ-KQ41180 | Human | 54784 | Details Get a Quote |
| ALKBH4 Knockout HCT 116 Cell Line | EDJ-KQ41181 | Human | 54784 | Details Get a Quote |
| ALKBH4 Knockout HeLa Cell Line | EDJ-KQ41182 | Human | 54784 | Details Get a Quote |
| FTO Knockout A-549 Cell Line | EDJ-KQ20359 | Human | 79068 | Details Get a Quote |
| FTO Knockout HCT 116 Cell Line | EDC90280 | Human | 79068 | Details Get a Quote |
| ALKBH3 Knockout A-549 Cell Line | EDJ-KQ21311 | Human | 221120 | Details Get a Quote |
Displaying Records 1 To 15 Of 22 Records
Frequently Asked Questions About broad specificity oxidative DNA demethylase activity
What is GO:0035516?
GO:0035516 is the Gene Ontology term for broad specificity oxidative DNA demethylase activity, a molecular function that removes methyl groups from DNA bases using 2-oxoglutarate and oxygen.
What genes are involved in broad specificity oxidative DNA demethylase activity?
Genes include ALKBH1, ALKBH2, ALKBH3, FTO, TET1, TET2, TET3 and several KDM family members.
What reactions does this activity catalyze?
It converts a methylated nucleobase within DNA plus 2-oxoglutarate and O2 into a demethylated base, formaldehyde, succinate and CO2.
Can this activity act on RNA?
Yes, the definition states it can also act on RNA substrates.
What diseases are associated with defects in this activity?
Cancer, neurological disorders and metabolic diseases have been linked to dysregulation of these enzymes.
How can I study this activity in the lab?
CRISPR knockout, point-mutation knock-in, overexpression and epigenomic profiling are common approaches.
What cofactors are required?
2-oxoglutarate, oxygen and Fe(II) are required, with ascorbate (vitamin C) supporting the reaction.
Is this activity important for epigenetics?
Yes, it generates oxidized 5-methylcytosine derivatives that are intermediates in active DNA demethylation.
What is the difference between GO:0035516 and other demethylases?
GO:0035516 has broad specificity for multiple methylated bases, unlike more substrate-restricted enzymes.
How does EDITGENE support research on this term?
EDITGENE provides CRISPR knockout, knock-in, overexpression, library screening and bioinformatics services for demethylase genes.
Conclusion
GO:0035516 broad specificity oxidative DNA demethylase activity is a fundamental molecular function that protects the genome and regulates epigenetic information. Its dual role in DNA repair and RNA modification makes it a rich area for functional genomics and disease research. By combining precise CRISPR models with epigenomic and biochemical assays, researchers can uncover how these enzymes contribute to health and disease.
References
- 1. Vázquez-Arreguín K et al.. 2016. The Oct1 transcription factor and epithelial malignancies: Old protein learns new tricks.. Biochim Biophys Acta 1859(6):792-804 PMID: 26877236
- 2. Thaler R. 2026. Vitamin C as an epigenetic gatekeeper of osteogenesis and skeletal health: a new perspective on an ancient molecule.. J Bone Miner Res 41(4):353-365 PMID: 41269246
