GO:0106378 2-hydroxy-dATP hydrolase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0106378 (2-hydroxy-dATP hydrolase activity) is a molecular_function that catalyzes the hydrolysis of 2-hydroxy-dATP to 2-hydroxy-dAMP, a proton, and diphosphate, as defined by QuickGO.
• The enzyme sanitizes oxidized nucleotides, preventing their incorporation into DNA and reducing oxidative stress-induced mutagenesis.
• Human MTH1 (NUDT1) is the primary enzyme with this activity, and it also hydrolyzes other oxidized nucleotides such as 2-hydroxy-ATP and 8-chloro-dGTP.
• Bacterial and nematode homologs, such as Escherichia coli Orf135 and Caenorhabditis elegans NDX-1, share this hydrolase function and provide model systems for study.
• MTH1 polymorphisms have been associated with small cell lung carcinoma risk, linking this activity to cancer susceptibility.
• Small-molecule inhibitors like TH588 target MTH1 and sensitize cancer cells to ionizing radiation, highlighting therapeutic potential.
Description
GO:0106378, 2-hydroxy-dATP hydrolase activity, is a molecular function that removes oxidized nucleotides from the cellular pool. Specifically, it catalyzes the reaction: 2-hydroxy-dATP + H2O = 2-hydroxy-dAMP + H+ + diphosphate. This activity is crucial because oxidized nucleotides can mispair during DNA replication, leading to mutations. The human enzyme MTH1 (also known as NUDT1) was shown to hydrolyze 2-hydroxy-dATP, establishing it as a key sanitizer of the nucleotide pool. Subsequent studies confirmed that MTH1 also acts on the oxidized ribonucleotide 2-hydroxy-ATP, broadening its substrate range. Researchers study this activity to understand how cells defend against oxidative stress and to explore its role in diseases such as cancer. For example, MTH1 polymorphisms have been linked to small cell lung carcinoma risk, and inhibition of MTH1 by TH588 sensitizes colorectal carcinoma cells to ionizing radiation under hypoxia. Homologs in bacteria and worms, such as Escherichia coli Orf135 and Caenorhabditis elegans NDX-1, provide valuable models for dissecting the enzymatic mechanism and physiological impact. This article provides a comprehensive overview of GO:0106378, covering its definition, biological importance, key genes, regulatory aspects, disease connections, and experimental methods for research. It is intended for scientists seeking to understand or manipulate this activity in cellular models.
2-hydroxy-dATP hydrolase activity At A Glance
| GO ID | GO:0106378 |
|---|---|
| GO term | 2-hydroxy-dATP hydrolase activity |
| Ontology | molecular_function |
| Synonym | None |
| Major function | Hydrolysis of 2-hydroxy-dATP to 2-hydroxy-dAMP and diphosphate |
| Reaction | 2-hydroxy-dATP + H2O = 2-hydroxy-dAMP + H+ + diphosphate |
| Substrates | 2-hydroxy-dATP; also 2-hydroxy-ATP and 8-chloro-dGTP for human MTH1 |
| Enzymes | MTH1 (NUDT1) in humans; Orf135 in E. coli; NDX-1 in C. elegans |
What Is GO:0106378?
According to the Gene Ontology, GO:0106378 (2-hydroxy-dATP hydrolase activity) is defined as the catalysis of the reaction: 2-hydroxy-dATP + H2O = 2-hydroxy-dAMP + H+ + diphosphate. In other words, it is an enzyme activity that uses water to cleave 2-hydroxy-dATP into 2-hydroxy-dAMP and diphosphate, releasing a proton. This activity belongs to the molecular_function ontology and is involved in sanitizing the nucleotide pool by removing oxidized dATP.
Why Is 2-hydroxy-dATP hydrolase activity Important in Cell Biology?
2-hydroxy-dATP hydrolase activity is important because it prevents the incorporation of oxidized nucleotides into DNA, which would otherwise cause mutations and genomic instability. By hydrolyzing 2-hydroxy-dATP, enzymes like MTH1 maintain the integrity of the nucleotide pool and protect cells from oxidative stress. This activity is also relevant to cancer biology, as MTH1 is often upregulated in cancer cells and its inhibition can sensitize tumors to radiation and chemotherapy. Furthermore, polymorphisms in MTH1 have been associated with increased risk of small cell lung carcinoma, suggesting a role in cancer susceptibility.
• Prevents mutagenesis by removing oxidized dATP from the nucleotide pool.
• Protects against oxidative stress-induced DNA damage.
• MTH1 (NUDT1) is a potential anticancer target; inhibitors like TH588 sensitize cancer cells to radiation.
• MTH1 polymorphisms are linked to small cell lung carcinoma risk.
• Homologs in bacteria and worms provide model systems for studying nucleotide sanitization.
• Substrate specificity includes 2-hydroxy-ATP and 8-chloro-dGTP, indicating broad roles in sanitizing oxidized nucleotides.
• Enzyme activity is essential for maintaining genomic stability in rapidly dividing cells.
• Research on this activity informs development of CRISPR models to study gene function.
• It is a key component of the DNA repair and oxidative stress response networks.
• Understanding its mechanism can guide design of small-molecule modulators.
Molecular Mechanism of 2-hydroxy-dATP hydrolase activity
Substrate Recognition and Binding
In simple terms: The enzyme recognizes and binds specifically to oxidized nucleotides like 2-hydroxy-dATP.
The enzyme MTH1 (NUDT1) specifically binds 2-hydroxy-dATP, distinguishing it from normal dATP. Studies on Escherichia coli Orf135 identified amino acid residues involved in substrate recognition, showing that mutations in these residues affect the enzyme's ability to hydrolyze oxidized nucleotides. Similarly, the phosphohydrolase module of Orf135 contains critical amino acids for catalysis. The human MTH1 protein also hydrolyzes 2-hydroxy-ATP, indicating a broad specificity for oxidized purine nucleotides.
Catalytic Hydrolysis
In simple terms: The enzyme uses water to break the bond between the phosphate groups, releasing energy and inactivating the oxidized nucleotide.
The hydrolysis reaction cleaves 2-hydroxy-dATP into 2-hydroxy-dAMP and diphosphate. This is a nucleophilic attack by water, facilitated by conserved amino acids in the active site. For human MTH1, the reaction is essential for sanitizing the nucleotide pool. The enzyme also hydrolyzes 8-chloro-dGTP, a hypochlorous acid-modified nucleotide, demonstrating its role in defense against various oxidized nucleotides.
Product Release and Cellular Impact
In simple terms: After hydrolysis, the products are released, and the oxidized nucleotide is no longer available for DNA synthesis.
The products, 2-hydroxy-dAMP and diphosphate, are released from the active site. By converting 2-hydroxy-dATP to 2-hydroxy-dAMP, the enzyme prevents the incorporation of the oxidized nucleotide into DNA. This activity reduces mutations and maintains genomic stability. In Caenorhabditis elegans, the NDX-1 protein hydrolyzes 8-oxo-7,8-dihydrodeoxyguanosine-5'-diphosphate, a similar sanitization function, preventing oxidative stress.
Regulation and Inhibitors
In simple terms: The activity can be regulated by cellular conditions and targeted by small molecules.
MTH1 activity is regulated at the expression level and potentially by post-translational modifications. Small-molecule inhibitor TH588 targets MTH1 and sensitizes colorectal carcinoma cells to ionizing radiation under hypoxia, indicating that inhibition of this activity can be therapeutically exploited. Additionally, polymorphisms in the MTH1 gene may affect enzyme activity and cancer risk.
Key Genes Involved in GO:0106378 2-hydroxy-dATP hydrolase activity
The following genes and proteins are directly associated with 2-hydroxy-dATP hydrolase activity or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTH1 (NUDT1) | Human enzyme that hydrolyzes 2-hydroxy-dATP and other oxidized nucleotides | Key target for cancer therapy; polymorphisms linked to lung cancer |
| Orf135 | E. coli homolog with 2-hydroxy-dATP hydrolase activity | Model for studying substrate recognition and catalysis |
| NDX-1 | C. elegans enzyme that sanitizes oxidized nucleotides | Model for oxidative stress defense |
| MTH1 (NUDT1) | Hydrolyzes 2-hydroxy-ATP | Broad substrate specificity |
| MTH1 (NUDT1) | Hydrolyzes 8-chloro-dGTP | Role in defense against hypochlorous acid-modified nucleotides |
| MTH1 (NUDT1) | Associated with small cell lung carcinoma risk | Cancer susceptibility |
| MTH1 (NUDT1) | Target of inhibitor TH588 | Radiosensitization in colorectal carcinoma |
| Orf135 | Phosphohydrolase module | Amino acids important for catalysis |
| MTH1 (NUDT1) | Sanitizes nucleotide pool | Prevents mutagenesis |
| NDX-1 | Prevents oxidative stress | C. elegans model |
| MTH1 (NUDT1) | Potential biomarker | Cancer diagnostics |
| Orf135 | Bacterial defense | Antibiotic target potential |
| MTH1 (NUDT1) | Enzyme kinetics | Drug development |
| NDX-1 | Stress response | Aging research |
| MTH1 (NUDT1) | Substrate specificity | Enzyme engineering |
| Orf135 | Structural studies | Protein evolution |
| MTH1 (NUDT1) | Therapeutic target | Cancer treatment |
| NDX-1 | Model organism | Genetic screens |
How Is 2-hydroxy-dATP hydrolase activity Regulated?
The regulation of 2-hydroxy-dATP hydrolase activity is not fully understood, but evidence suggests that MTH1 expression can be induced by oxidative stress. Additionally, MTH1 activity may be modulated by post-translational modifications, although specific mechanisms are not detailed in the cited literature. Inhibitors such as TH588 can directly block MTH1 activity, indicating that small molecules can regulate this function. Polymorphisms in the MTH1 gene may also affect enzyme activity and contribute to cancer risk.
2-hydroxy-dATP hydrolase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTH1 (NUDT1) | Small cell lung carcinoma | Knockout or overexpression in lung cancer cell lines |
| MTH1 (NUDT1) | Colorectal carcinoma | Point mutation to disable catalytic activity; radiation sensitivity assays |
| NDX-1 | Oxidative stress response | C. elegans knockout or knock-in |
| Orf135 | Bacterial oxidative stress | E. coli knockout and complementation |
Cancer
MTH1, the enzyme responsible for 2-hydroxy-dATP hydrolase activity, is often overexpressed in cancer cells and is considered a potential therapeutic target. Polymorphisms in the MTH1 gene have been associated with small cell lung carcinoma risk. Inhibition of MTH1 by TH588 sensitizes colorectal carcinoma cells to ionizing radiation under hypoxia, suggesting that targeting this activity could improve radiotherapy outcomes.
Neurodegeneration
Oxidative stress is a hallmark of neurodegenerative diseases. The sanitization of oxidized nucleotides by MTH1 and its homologs may protect neurons from DNA damage. However, direct evidence linking GO:0106378 to neurodegeneration is limited in the cited literature. Further research is needed to establish a connection.
Aging
In Caenorhabditis elegans, the NDX-1 protein, which has similar hydrolase activity, prevents oxidative stress and may influence lifespan. This suggests that 2-hydroxy-dATP hydrolase activity could play a role in aging processes, though more studies are required.
From 2-hydroxy-dATP hydrolase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of MTH1 increase mutation frequency? | MTH1 knockout cell lines (e.g., HCT116) |
| Does a specific MTH1 polymorphism affect enzyme activity? | Point mutation knock-in of the polymorphism |
| Can MTH1 overexpression protect against oxidative stress? | Overexpression of MTH1 in cancer cells |
| What is the subcellular localization of MTH1? | Tagged knock-in with fluorescent protein |
| Does MTH1 inhibition synergize with radiation? | MTH1 knockout or inhibitor treatment in colorectal cells |
| What is the role of Orf135 in bacterial survival? | Orf135 knockout in E. coli |
How to Study the 2-hydroxy-dATP hydrolase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| HPLC | Hydrolysis of 2-hydroxy-dATP | Enzyme kinetics |
| Mass spectrometry | Product formation | Substrate specificity |
| Complementation assay | Functional rescue of knockout | Mutant analysis |
| Cell viability assay | Cellular sensitivity to oxidative stress | Knockout/overexpression studies |
| Crystallography | Three-dimensional structure | Mechanistic studies |
| Site-directed mutagenesis | Effect of amino acid changes | Active site identification |
| RNA-seq | Gene expression changes | Pathway analysis |
| CRISPR screening | Identify genes affecting sensitivity | Drug target discovery |
Enzymatic Assays
Enzymatic activity of 2-hydroxy-dATP hydrolase can be measured using purified recombinant protein and radiolabeled or fluorescently labeled substrates. High-performance liquid chromatography (HPLC) or mass spectrometry can detect the conversion of 2-hydroxy-dATP to 2-hydroxy-dAMP.
Mutagenesis and Complementation
Bacterial or yeast complementation assays using MTH1 or Orf135 mutants can assess the functional importance of specific amino acids. For example, E. coli Orf135 mutants were tested for their ability to hydrolyze oxidized nucleotides.
Cell Viability and Oxidative Stress Assays
Cell lines with MTH1 knockout or overexpression can be treated with oxidizing agents (e.g., hydrogen peroxide) and assessed for viability, DNA damage, and mutation frequency. This helps link the activity to cellular protection.
Structural Biology
X-ray crystallography or cryo-EM can determine the structure of MTH1 or Orf135 bound to substrates or inhibitors, revealing the catalytic mechanism and guiding drug design.
How CRISPR Can Be Used to Study GO:0106378 2-hydroxy-dATP hydrolase activity
Knockout
CRISPR knockout of MTH1 (NUDT1) can be used to study the consequences of losing 2-hydroxy-dATP hydrolase activity. Knockout cell lines may exhibit increased mutation rates and sensitivity to oxidative stress, providing a model to test hypotheses about its role in cancer and aging.
Point Mutation
Introducing point mutations in the catalytic residues of MTH1 can help dissect the enzymatic mechanism. For example, mutating amino acids identified in Orf135 studies can abolish hydrolase activity and reveal its importance in vivo.
Knock-in
Knock-in of tagged MTH1 (e.g., GFP or FLAG) allows for localization and interaction studies. Additionally, knock-in of disease-associated polymorphisms can model their effects on enzyme activity and cancer risk.
Overexpression
Overexpression of MTH1 in cell lines can protect against oxidative stress and may be used to study its role in tumorigenesis. Conversely, overexpression of a catalytically dead mutant can act as a dominant-negative.
How EDITGENE Supports 2-hydroxy-dATP hydrolase activity Research
Researchers studying 2-hydroxy-dATP hydrolase activity-related genes often need to determine whether a candidate gene is causally involved in oxidative stress resistance, mutagenesis, or cancer cell survival. CRISPR-based models provide a robust way to manipulate these genes and assess their functions.
Contact EDITGENE today to design your custom CRISPR model for 2-hydroxy-dATP hydrolase activity research.
Frequently Asked Questions About 2-hydroxy-dATP hydrolase activity
What is 2-hydroxy-dATP hydrolase activity?
It is an enzyme activity that hydrolyzes 2-hydroxy-dATP to 2-hydroxy-dAMP and diphosphate, as defined by GO:0106378.
What genes are involved in 2-hydroxy-dATP hydrolase activity?
The main human gene is MTH1 (NUDT1). Homologs include Orf135 in E. coli and NDX-1 in C. elegans.
What is the function of MTH1?
MTH1 hydrolyzes oxidized nucleotides like 2-hydroxy-dATP and 8-chloro-dGTP, preventing their incorporation into DNA.
How is 2-hydroxy-dATP hydrolase activity measured?
It can be measured using HPLC or mass spectrometry with purified enzyme and substrates.
Is MTH1 a cancer target?
Yes, MTH1 is overexpressed in some cancers, and inhibitors like TH588 sensitize cancer cells to radiation.
What diseases are associated with MTH1 mutations?
Polymorphisms in MTH1 have been linked to small cell lung carcinoma risk.
What is the reaction catalyzed by GO:0106378?
2-hydroxy-dATP + H2O = 2-hydroxy-dAMP + H+ + diphosphate.
Can CRISPR be used to study 2-hydroxy-dATP hydrolase activity?
Yes, CRISPR knockout, point mutation, and knock-in models can be used to study gene function.
What are the substrates of MTH1?
MTH1 hydrolyzes 2-hydroxy-dATP, 2-hydroxy-ATP, and 8-chloro-dGTP.
What is the role of Orf135?
Orf135 is an E. coli homolog with 2-hydroxy-dATP hydrolase activity, used as a model for substrate recognition.
Conclusion
GO:0106378, 2-hydroxy-dATP hydrolase activity, is a critical enzymatic function that protects cells from oxidative DNA damage by sanitizing the nucleotide pool. The human enzyme MTH1 and its homologs in bacteria and worms have been extensively studied, revealing important insights into substrate specificity, catalytic mechanism, and disease relevance. Targeting this activity holds promise for cancer therapy, and CRISPR-based models are invaluable for further research. EDITGENE offers comprehensive services to support such studies.
References
- 1. Fujikawa K et al.. 2001. Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP.. Nucleic Acids Res 29(2):449-54 PMID: 11139615
- 2. Fujikawa K et al.. 1999. The oxidized forms of dATP are substrates for the human MutT homologue, the hMTH1 protein.. J Biol Chem 274(26):18201-5 PMID: 10373420
- 3. Iida E et al.. 2005. Amino acid residues involved in substrate recognition of the Escherichia coli Orf135 protein.. Biochemistry 44(15):5683-9 PMID: 15823026
- 4. Fujikawa K et al.. 2002. 8-Chloro-dGTP, a hypochlorous acid-modified nucleotide, is hydrolyzed by hMTH1, the human MutT homolog.. FEBS Lett 512(1-3):149-51 PMID: 11852070
- 5. Kohno T et al.. 2006. Association of polymorphisms in the MTH1 gene with small cell lung carcinoma risk.. Carcinogenesis 27(12):2448-54 PMID: 16774934
- 6. Kamiya H et al.. 2004. Important amino acids in the phosphohydrolase module of Escherichia coli Orf135.. Biochem Biophys Res Commun 323(3):1063-8 PMID: 15381107
- 7. Pompsch M et al.. 2018. The presumed MTH1-inhibitor TH588 sensitizes colorectal carcinoma cells to ionizing radiation in hypoxia.. BMC Cancer 18(1):1190 PMID: 30497423
- 8. Sanada U et al.. 2011. NDX-1 protein hydrolyzes 8-oxo-7, 8-dihydrodeoxyguanosine-5'-diphosphate to sanitize oxidized nucleotides and prevent oxidative stress in Caenorhabditis elegans.. J Biochem 150(6):649-57 PMID: 21873335
