GO:0008413 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0008413 describes the enzymatic hydrolysis of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-GTP) to 8-oxo-7,8-dihydroguanosine diphosphate (8-oxo-GDP) and phosphate, a critical sanitization step that prevents oxidatively damaged guanine nucleotides from being incorporated into RNA.
• The reaction is catalyzed by MutT-family pyrophosphatases, including Escherichia coli MutT, Bacillus subtilis YtkD (MutTA), and human MTH1 (NUDT1), which share conserved phosphohydrolase modules.
• 8-oxo-GTP is a potent mutagenic substrate for transcription; its accumulation leads to transcriptional errors and may contribute to cellular dysfunction.
• These enzymes exhibit broad substrate specificity, hydrolyzing not only 8-oxo-GTP but also 8-oxo-dGTP and other oxidized nucleotides, thereby sanitizing both DNA and RNA precursor pools.
• Loss of 8-oxo-GTP pyrophosphatase activity is associated with increased mutagenesis and has been implicated in bacterial antimutator phenotypes and human disease processes such as cancer.
• Research tools such as CRISPR knockout, point-mutation knock-in, and overexpression models enable precise dissection of GO:0008413-related gene function in diverse biological contexts.
Description
GO:0008413, 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity, is a molecular function that catalyzes the hydrolysis of 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-GTP) to 8-oxo-7,8-dihydroguanosine diphosphate (8-oxo-GDP) and phosphate. This activity is essential for maintaining the integrity of both DNA and RNA precursor pools by removing oxidatively damaged guanine nucleotides that can otherwise be misincorporated during nucleic acid synthesis. The enzyme belongs to the MutT family of pyrophosphatases, named after the Escherichia coli MutT protein, which was the first identified member with 8-oxo-dGTPase and 8-oxo-GTPase activities. The importance of this activity is underscored by its evolutionary conservation from bacteria to humans, with homologs such as Bacillus subtilis YtkD and human MTH1 (NUDT1) performing analogous sanitization functions. Researchers study GO:0008413 to understand mechanisms of mutagenesis, genome stability, and the cellular response to oxidative stress, as well as to explore therapeutic targets in cancer and infectious diseases.
8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity At A Glance
| GO ID | GO:0008413 |
|---|---|
| GO term | 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity |
| Ontology | molecular_function |
| Synonym | 7,8-dihydro-8-oxoguanine-triphosphatase activity; 8-oxo-7,8-dihydroguanine triphosphatase activity; 8-oxo-7,8-dihydroguanosine triphosphatase activity; 8-oxo-GTPase activity |
| Major function | Hydrolysis of 8-oxo-GTP to 8-oxo-GDP and phosphate, sanitizing RNA precursor pools |
| Substrates | 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-GTP); also acts on 8-oxo-dGTP and other oxidized nucleotides |
| Products | 8-oxo-7,8-dihydroguanosine diphosphate (8-oxo-GDP) and phosphate |
| Cofactors | Divalent metal ions (e.g., Mg2+) are typically required for MutT-family pyrophosphatases |
| Localization | Cytoplasm; mitochondrial matrix for some isoforms |
| Enzyme family | MutT/Nudix hydrolase family |
What Is GO:0008413?
According to the Gene Ontology, GO:0008413 is defined as the catalysis of the reaction: 8-oxo-7,8-dihydroguanosine triphosphate (8-oxo-GTP) + H2O = 8-oxo-7,8-dihydroguanosine diphosphate (8-oxo-GDP) + phosphate. In simpler terms, it is an enzymatic activity that removes a phosphate group from an oxidized form of the free guanine nucleotide, 8-oxo-GTP, converting it to 8-oxo-GDP and inorganic phosphate. This reaction prevents 8-oxo-GTP from serving as a mutagenic substrate during transcription, thereby protecting the cell from oxidative damage to RNA.
Why Is 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity Important in Cell Biology?
GO:0008413 is critical for cellular defense against oxidative damage to the transcriptome and genome. By hydrolyzing 8-oxo-GTP, this activity prevents the incorporation of oxidized guanine nucleotides into RNA, which can cause transcriptional errors and potentially lead to protein dysfunction. In bacteria, loss of this activity results in a mutator phenotype, while in humans, reduced MTH1 activity has been linked to cancer and neurodegeneration. Understanding this activity provides insights into mutagenesis, genome stability, and potential therapeutic strategies targeting nucleotide pool sanitization.
• Prevents transcriptional mutagenesis by eliminating 8-oxo-GTP from RNA precursor pools.
• Protects DNA replication by also hydrolyzing 8-oxo-dGTP, reducing oxidative DNA damage.
• Deficiency leads to increased spontaneous mutation rates in bacteria and potentially in human cells.
• Human MTH1 (NUDT1) is a potential target for cancer therapy due to its role in sanitizing oxidized nucleotides.
• Bacterial MutT and YtkD are models for studying antimutator mechanisms and antibiotic resistance.
• The activity is conserved across evolution, highlighting its fundamental importance.
• Dysregulation of 8-oxo-GTP pyrophosphatases is implicated in neurodegenerative diseases and aging.
• Enzymatic assays for this activity are used in drug discovery for inhibitors targeting MTH1.
• CRISPR-based models allow precise manipulation of genes encoding this activity for functional studies.
• Understanding substrate specificity informs the design of selective inhibitors or activators.
Molecular Mechanism of 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity
Substrate Recognition and Binding
In simple terms: The enzyme first grabs the damaged nucleotide, 8-oxo-GTP, from the surrounding pool.
MutT-family pyrophosphatases recognize 8-oxo-GTP through a conserved Nudix fold that accommodates the oxidized guanine base. Structural studies of E. coli MutT and M. smegmatis MutT1 have identified key residues in the phosphohydrolase module that determine substrate specificity, with single amino acid exchanges switching cleavage preferences between 8-oxo-GTP and other nucleotides. The enzyme binds the substrate in a metal-dependent manner, typically requiring Mg2+ or Mn2+ for catalysis.
Catalytic Hydrolysis
In simple terms: The enzyme then uses water to cut the phosphate chain, releasing energy and inactivating the damaged nucleotide.
The hydrolysis reaction proceeds via nucleophilic attack of a water molecule on the alpha-phosphate of 8-oxo-GTP, facilitated by divalent metal ions and conserved acidic residues. This yields 8-oxo-GDP and inorganic phosphate. E. coli MutT exhibits multiple enzyme activities, including 8-oxo-GTPase and 8-oxo-dGTPase, ensuring broad sanitization of both RNA and DNA precursor pools. The reaction is highly efficient, with turnover rates sufficient to keep 8-oxo-GTP levels low under oxidative stress.
Product Release and Pool Sanitization
In simple terms: After the reaction, the harmless products are released, and the damaged nucleotide is no longer available to be mistakenly used in RNA.
Following hydrolysis, 8-oxo-GDP and phosphate are released from the active site. 8-oxo-GDP can be further degraded or excreted, effectively removing the mutagenic threat. In Bacillus subtilis, YtkD (MutTA) performs this function and is under dual control of sigma A and sigma F RNA polymerases, linking its expression to stationary phase and sporulation. This sanitization prevents 8-oxo-GTP from being incorporated into RNA by RNA polymerase, which would otherwise cause transcriptional errors.
Substrate Specificity and Broader Roles
In simple terms: Some versions of the enzyme can also clean up other oxidized nucleotides, making them versatile protectors.
While GO:0008413 specifically describes 8-oxo-GTP hydrolysis, many MutT-family enzymes also hydrolyze 8-oxo-dGTP and other oxidized purine nucleotides. Human MTH1 (NUDT1) hydrolyzes 8-oxo-dGTP, 8-oxo-GTP, and 2-hydroxy-dATP, among others, and its activity is crucial for preventing incorporation of these damaged nucleotides into DNA and RNA. This broad specificity ensures comprehensive protection against oxidative damage to the nucleotide pool.
Regulation of Enzyme Activity
In simple terms: The cell can adjust how much of this enzyme is made or how active it is, depending on stress conditions.
Expression of mutT in E. coli is part of the SOS response, induced by DNA damage, while B. subtilis ytkD is controlled by sigma factors that respond to developmental and stress signals. In humans, MTH1 expression is regulated by transcription factors such as Nrf2 and may be upregulated in cancer cells to cope with increased oxidative stress. Post-translational modifications and subcellular localization also influence activity.
Key Genes Involved in GO:0008413 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity
The following genes encode enzymes with 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity or are directly involved in its regulation and function.
| Gene | Major Role | Research Relevance |
|---|---|---|
| mutT (E. coli) | Hydrolyzes 8-oxo-GTP and 8-oxo-dGTP; antimutator | Model for studying MutT-family enzymes and mutagenesis |
| mutT1 (M. smegmatis) | Ortholog of MutT with different substrate specificity | Comparative studies of phosphohydrolase modules |
| ytkD (mutTA) (B. subtilis) | 8-oxo-GTPase and 8-oxo-dGTPase; sporulation-related | Studying regulation by sigma factors and antimutator function |
| MTH1 (NUDT1) (human) | Hydrolyzes 8-oxo-dGTP, 8-oxo-GTP, and other oxidized nucleotides | Cancer therapy target; role in oxidative stress response |
| NUDT1 (human) | Same as MTH1; encodes 8-oxo-dGTPase | Isoform studies and inhibitor development |
| NUDT15 (human) | Hydrolyzes oxidized nucleotides including 8-oxo-GTP | Drug metabolism and toxicity studies |
| NUDT5 (human) | Hydrolyzes 8-oxo-GTP and other nucleotides | Nucleotide pool sanitization |
| NUDT18 (human) | Hydrolyzes 8-oxo-GTP | Potential backup enzyme |
| MUTYH (human) | DNA glycosylase involved in 8-oxo-G repair | Not directly GO:0008413 but related to 8-oxo-G metabolism |
| OGG1 (human) | DNA glycosylase for 8-oxo-G in DNA | Related to oxidative damage repair |
| RNA polymerase (bacterial) | Incorporates 8-oxo-GTP if not sanitized | Target of mutagenesis studies |
| RNA polymerase II (human) | Transcription machinery affected by 8-oxo-GTP | Transcriptional mutagenesis research |
| Nrf2 (human) | Transcription factor regulating MTH1 expression | Oxidative stress response |
| SOS response regulators (E. coli) | Regulate mutT expression | Bacterial stress response |
| Sigma A (B. subtilis) | Regulates ytkD expression | Developmental regulation |
| Sigma F (B. subtilis) | Regulates ytkD expression | Sporulation-specific regulation |
| MTH1 inhibitors (chemical) | Small molecules targeting MTH1 | Cancer drug discovery |
How Is 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity Regulated?
The expression and activity of 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatases are regulated at multiple levels. In Escherichia coli, mutT is part of the SOS regulon, induced by DNA-damaging agents to enhance nucleotide pool sanitization during stress. In Bacillus subtilis, ytkD (mutTA) is under dual control of sigma A and sigma F RNA polymerases, linking its expression to stationary phase and sporulation. In humans, MTH1 (NUDT1) expression is regulated by the transcription factor Nrf2, which coordinates antioxidant responses, and is often upregulated in cancer cells to counteract elevated oxidative stress. Post-translational modifications and subcellular localization further modulate enzyme activity.
8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTH1 (NUDT1) | Cancer, oxidative stress survival | CRISPR knockout in cancer cell lines; xenograft models |
| NUDT15 | Thiopurine toxicity, leukemia | Knock-in of variant alleles in lymphoblastoid cells |
| mutT (E. coli) | Mutator phenotype, antibiotic resistance | Knockout and complementation in E. coli |
| ytkD (B. subtilis) | Sporulation defects, antimutator | Knockout in B. subtilis; sporulation assays |
| MUTYH | Colorectal cancer (MAP) | Knockout mouse models; organoids |
Cancer
MTH1 (NUDT1) is overexpressed in many cancers and is considered a therapeutic target because it protects cancer cells from oxidative DNA damage and allows survival under high reactive oxygen species. Inhibitors of MTH1 have shown efficacy in preclinical models, and loss of MTH1 activity sensitizes cancer cells to oxidative stress. Additionally, mutations in NUDT15, which also hydrolyzes 8-oxo-GTP, are associated with thiopurine toxicity in leukemia patients.
Neurodegeneration
Oxidative stress is a hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's. Reduced MTH1 activity may contribute to the accumulation of oxidized nucleotides in neurons, leading to transcriptional errors and neuronal dysfunction. However, direct evidence linking GO:0008413 to neurodegeneration is still emerging.
Bacterial Pathogenesis and Antibiotic Resistance
In pathogenic bacteria, MutT-family enzymes contribute to survival under oxidative stress imposed by the host immune system. Loss of mutT increases mutation rates, potentially accelerating the emergence of antibiotic resistance. Targeting these enzymes could be a novel antibacterial strategy.
Aging and Genome Instability
Accumulation of 8-oxo-GTP and subsequent transcriptional mutagenesis may contribute to aging and age-related diseases. Model organisms with reduced 8-oxo-GTP pyrophosphatase activity show increased mutation rates and shortened lifespan, although the exact mechanisms remain under investigation.
From 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of MTH1 increase sensitivity to oxidative stress? | CRISPR knockout of NUDT1 in human cancer cell lines |
| What is the substrate specificity of MutT1? | Point mutations in the phosphohydrolase module of M. smegmatis MutT1 |
| Can a disease-associated NUDT15 variant be rescued? | Knock-in of the variant allele followed by overexpression of wild-type |
| How does ytkD expression affect sporulation? | Knockout and tagged knock-in of ytkD in B. subtilis |
| Does mutT overexpression reduce mutation rate? | Overexpression of mutT in E. coli under oxidative stress |
| What are the interacting partners of MTH1? | Tagged knock-in of NUDT1 for proteomics |
How to Study the 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| HPLC-based assay | Hydrolysis of 8-oxo-GTP to 8-oxo-GDP | Enzyme kinetics and inhibitor screening |
| Coupled spectrophotometric assay | Phosphate release | High-throughput screening |
| Bacterial mutator assay | Mutation frequency (e.g., rifampicin resistance) | Antimutator function of MutT |
| CRISPR knockout | Loss-of-function phenotype | Gene function in oxidative stress |
| CRISPR knock-in | Effect of specific mutations | Structure-function studies |
| RNA-seq | Transcriptional errors and gene expression | Global impact of 8-oxo-GTP |
| Ribo-seq | Translation errors | Ribosome profiling under oxidative stress |
| Proteomics | Protein interactions | MTH1 interactome |
Enzymatic Activity Assays
Direct measurement of 8-oxo-GTP pyrophosphatase activity is typically performed using high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) to separate and quantify 8-oxo-GTP and 8-oxo-GDP. Alternatively, a coupled enzyme assay with pyruvate kinase and lactate dehydrogenase can monitor phosphate release spectrophotometrically. These assays are used to determine kinetic parameters and inhibitor efficacy.
Mutagenesis and Reporter Assays
Bacterial mutator assays, such as rifampicin resistance or lacZ reversion, are used to assess the antimutator function of MutT-family enzymes. In mammalian cells, reporter plasmids containing 8-oxo-G can measure transcriptional mutagenesis. These methods link enzyme activity to biological outcomes.
CRISPR-Based Genetic Models
CRISPR-Cas9 knockout, point mutation knock-in, and overexpression models allow precise manipulation of genes encoding 8-oxo-GTP pyrophosphatases. For example, knockout of NUDT1 in cancer cells can reveal its role in oxidative stress survival, while knock-in of specific mutations can dissect catalytic residues. These models are essential for functional studies.
Omics and Bioinformatics
RNA-seq and Ribo-seq can detect transcriptional errors caused by 8-oxo-GTP incorporation, while proteomics can identify interacting partners of MTH1. Bioinformatics tools such as QuickGO and STRING help annotate GO:0008413 and predict functional networks.
How CRISPR Can Be Used to Study GO:0008413 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity
Knockout
CRISPR knockout of genes encoding 8-oxo-GTP pyrophosphatases, such as NUDT1 in human cells or mutT in E. coli, creates loss-of-function models to study the consequences of impaired nucleotide pool sanitization. These models show increased mutation rates, sensitivity to oxidative stress, and transcriptional errors. Knockout cell lines are valuable for drug discovery targeting MTH1.
Point Mutation
Point mutations introduced by CRISPR base editing or homology-directed repair can alter catalytic residues in MutT-family enzymes, allowing precise dissection of substrate specificity and mechanism. For example, exchanging a single amino acid between E. coli MutT and M. smegmatis MutT1 switches their cleavage specificities, as demonstrated by Emam et al.. Such models are crucial for understanding structure-function relationships.
Knock-in
Knock-in of disease-associated variants, such as NUDT15 alleles linked to thiopurine toxicity, enables functional studies in isogenic backgrounds. Tagged knock-in of NUDT1 with fluorescent or affinity tags facilitates localization and interaction studies. These models help validate genetic variants and develop personalized therapies.
Overexpression
Overexpression of wild-type or mutant 8-oxo-GTP pyrophosphatases can rescue knockout phenotypes or enhance sanitization capacity. In cancer cells, MTH1 overexpression confers resistance to oxidative stress, making it a target for inhibitors. Overexpression models are also used to produce recombinant enzyme for biochemical assays.
How EDITGENE Supports 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity Research
Researchers studying 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity-related genes often need to determine whether a candidate gene is causally involved in oxidative stress resistance, mutagenesis, or disease. EDITGENE provides comprehensive CRISPR services to generate precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity research.
Frequently Asked Questions About 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity
What is GO:0008413?
GO:0008413 is the Gene Ontology term for 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity, which catalyzes the hydrolysis of 8-oxo-GTP to 8-oxo-GDP and phosphate.
What does 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity do?
It removes a phosphate from 8-oxo-GTP, preventing this oxidized nucleotide from being incorporated into RNA and causing mutations.
What genes are involved in 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity?
Key genes include mutT in E. coli, ytkD (mutTA) in B. subtilis, and MTH1 (NUDT1) in humans.
Why is 8-oxo-GTP harmful?
8-oxo-GTP is a potent mutagenic substrate for transcription; if incorporated into RNA, it can cause transcriptional errors and protein dysfunction.
How is 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity measured?
It is typically measured using HPLC or coupled spectrophotometric assays that detect the conversion of 8-oxo-GTP to 8-oxo-GDP.
What diseases are associated with defects in this activity?
Defects have been linked to cancer, neurodegenerative diseases, and increased mutagenesis in bacteria.
Is MTH1 the same as 8-oxo-GTPase?
MTH1 (NUDT1) is a human enzyme with 8-oxo-GTPase activity, among other substrates, and is a member of the MutT family.
Can CRISPR be used to study 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity?
Yes, CRISPR knockout, knock-in, and overexpression models are powerful tools to study gene function and disease relevance.
What is the role of MutT in bacteria?
MutT hydrolyzes 8-oxo-GTP and 8-oxo-dGTP, acting as an antimutator to maintain genome stability.
How does oxidative stress affect this activity?
Oxidative stress increases 8-oxo-GTP levels, and the enzyme activity is often upregulated to sanitize the nucleotide pool.
Conclusion
GO:0008413, 8-oxo-7,8-dihydroguanosine triphosphate pyrophosphatase activity, is a fundamental enzymatic function that protects cells from the mutagenic effects of oxidized guanine nucleotides. Through the action of MutT-family enzymes such as E. coli MutT, B. subtilis YtkD, and human MTH1, this activity ensures the integrity of RNA and DNA precursor pools. Dysregulation of this activity is implicated in cancer, neurodegeneration, and bacterial mutagenesis, making it a compelling target for therapeutic intervention and basic research. Advanced CRISPR models and biochemical assays continue to unravel its mechanisms and disease connections.
References
- 1. Emam EAF et al.. 2024. An exchange of single amino acid between the phosphohydrolase modules of Escherichia coli MutT and Mycobacterium smegmatis MutT1 switches their cleavage specificities.. DNA Repair (Amst) 139:103693 PMID: 38776712
- 2. Ito R et al.. 2005. Multiple enzyme activities of Escherichia coli MutT protein for sanitization of DNA and RNA precursor pools.. Biochemistry 44(17):6670-4 PMID: 15850400
- 3. 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
- 4. Ramírez MI et al.. 2004. The ytkD (mutTA) gene of Bacillus subtilis encodes a functional antimutator 8-Oxo-(dGTP/GTP)ase and is under dual control of sigma A and sigma F RNA polymerases.. J Bacteriol 186(4):1050-9 PMID: 14761999
