GO:0035539 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0035539 describes the enzymatic activity that hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP and diphosphate, preventing the mutagenic incorporation of oxidized guanine nucleotides into DNA.
• The reaction is catalyzed by MutT homologs, including bacterial MutT and human MTH1 (NUDT1), which sanitize the nucleotide pool.
• Loss of this activity leads to A:T to C:G transversion mutations, a hallmark of oxidative DNA damage.
• Altered expression of MTH1 has been observed in renal-cell carcinoma, suggesting a link to persistent oxidative stress in cancer.
• Age-related changes in MTH2 expression in the hippocampus of SAMP8 mice indicate a possible role in learning and memory deterioration.
• Studying this activity requires biochemical assays, structural analysis, and CRISPR-based models to dissect its physiological impact.
Description
GO:0035539, 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity, is a molecular function that removes oxidized guanine nucleotides from the cellular pool. The enzyme catalyzes the hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP and diphosphate, thereby preventing the misincorporation of this oxidized nucleotide into DNA. This activity is critical for maintaining genomic integrity because 8-oxo-dGTP can pair with adenine during DNA synthesis, leading to transversion mutations. The human mutT homologue gene MTH1 encodes an 8-oxo-dGTPase that was identified based on its ability to prevent A:T to C:G transversions. In bacteria, the MutT protein performs a similar function and has been shown to act on multiple oxidized nucleotide substrates. Structural studies using analogues of 8-oxo-dGTP have provided insights into the substrate specificity of MutT. The asidian homologue CiMutT also exhibits 8-oxo-dGTP pyrophosphohydrolase activity, indicating evolutionary conservation of this sanitization mechanism. Human NUDT5 can cleave oxidized guanine nucleotides and ADP sugars, further highlighting the diversity of enzymes that protect against oxidized nucleotide incorporation. Understanding this activity is essential for researchers studying oxidative stress, mutagenesis, and cancer biology.
8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity At A Glance
| GO ID | GO:0035539 |
|---|---|
| GO term | 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity |
| Ontology | molecular_function |
| Synonym | 8-oxo-dGTPase activity; 8-oxo-dGTP pyrophosphohydrolase activity; 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase activity |
| Major function | Hydrolysis of 8-oxo-dGTP to 8-oxo-dGMP and diphosphate, preventing mutagenic incorporation into DNA |
| Substrates | 8-oxo-dGTP; also acts on 8-oxo-GTP and other oxidized nucleotides |
| Products | 8-oxo-dGMP and diphosphate |
| Cofactors | Divalent metal ions such as Mg2+ are typically required for MutT-family enzymes |
| Localization | Cytoplasm and mitochondria in eukaryotic cells |
What Is GO:0035539?
This GO term defines the catalytic activity of an enzyme that converts 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) into 8-oxo-7,8-dihydrodeoxyguanosine monophosphate (8-oxo-dGMP) and diphosphate in the presence of water. The reaction removes the oxidized guanine nucleotide from the DNA precursor pool, preventing its incorporation into DNA and the resulting transversion mutations. The activity is also known as 8-oxo-dGTPase or 8-oxo-dGTP pyrophosphohydrolase.
Why Is 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity Important in Cell Biology?
This activity is a first-line defense against oxidative DNA damage because it eliminates mutagenic oxidized guanine nucleotides before they can be incorporated into DNA. Without it, 8-oxo-dGTP pairs with adenine, causing A:T to C:G transversions that can activate oncogenes or inactivate tumor suppressors. The importance is underscored by the observation that MTH1 mRNA is overexpressed in renal-cell carcinoma, suggesting that cancer cells may rely on this sanitization activity to survive oxidative stress. Moreover, age-related changes in MTH2 expression in the hippocampus of SAMP8 mice link this activity to neurodegeneration and cognitive decline. Thus, GO:0035539 is central to genome maintenance, cancer biology, and aging research.
• Prevents A:T to C:G transversion mutations by removing 8-oxo-dGTP from the nucleotide pool.
• Protects against oxidative stress-induced mutagenesis in bacteria and eukaryotes.
• MTH1 overexpression is observed in renal-cell carcinoma, indicating a role in cancer cell survival.
• Age-related alterations in MTH2 expression in the hippocampus may contribute to learning and memory deterioration.
• CiMutT from Ciona intestinalis provides an evolutionary perspective on oxidized nucleotide sanitization.
• Human NUDT5 can also cleave oxidized guanine nucleotides, expanding the repertoire of protective enzymes.
• Structural analogues of 8-oxo-dGTP help define substrate specificity and guide inhibitor design.
• The activity is essential for maintaining the integrity of both DNA and RNA precursor pools.
• Dysregulation of this activity may contribute to neurodegeneration and aging.
• Targeting this activity is of interest for developing anticancer therapeutics.
Molecular Mechanism of 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase activity
Substrate Recognition and Binding
In simple terms: The enzyme grabs the oxidized nucleotide and holds it in place.
MutT-family enzymes specifically recognize 8-oxo-dGTP through a conserved binding pocket that discriminates against unoxidized dGTP. Structural studies using analogues of 8-oxo-2'-deoxyguanosine nucleotide have revealed key interactions that confer substrate specificity. The enzyme also binds other oxidized nucleotides such as 8-oxo-GTP and 8-oxo-dGDP, as shown for Escherichia coli MutT.
Catalytic Hydrolysis
In simple terms: The enzyme cuts the triphosphate tail off the oxidized nucleotide.
The catalytic mechanism involves nucleophilic attack by water on the alpha-phosphate of 8-oxo-dGTP, releasing diphosphate and 8-oxo-dGMP. This reaction requires divalent metal ions, typically Mg2+, which stabilize the transition state. The hydrolysis prevents the incorporation of 8-oxo-dGTP into DNA by DNA polymerases.
Product Release and Pool Sanitization
In simple terms: The harmless monophosphate is released, and the dangerous triphosphate is gone.
After hydrolysis, 8-oxo-dGMP is released and can be further metabolized or excreted. The removal of 8-oxo-dGTP from the nucleotide pool reduces the probability of its misincorporation into DNA, thereby preventing transversion mutations. This sanitization function is conserved from bacteria to humans.
Metal Ion Dependence and Regulation
In simple terms: The enzyme needs helper metal ions to work and can be regulated by cellular signals.
MutT and MTH1 require divalent metal ions for activity, and their expression can be induced by oxidative stress. For example, MTH1 mRNA is overexpressed in renal-cell carcinoma, suggesting that tumor cells upregulate this activity to cope with high oxidative stress. In the hippocampus of SAMP8 mice, MTH2 expression changes with age, indicating potential regulation during aging.
Key Genes Involved in GO:0035539 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity
The following genes and proteins are directly associated with 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTH1 (NUDT1) | Human 8-oxo-dGTPase that hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP | Prevents A:T to C:G transversions; overexpressed in renal-cell carcinoma |
| MTH2 (NUDT2) | Human MutT homolog with 8-oxo-dGTPase activity | Age-related changes in hippocampus of SAMP8 mice |
| NUDT5 | Human enzyme that cleaves oxidized guanine nucleotides and ADP sugars | Broad substrate specificity for sanitization |
| MutT | E. coli 8-oxo-dGTPase | Model enzyme for studying substrate specificity and mechanism |
| CiMutT | Ciona intestinalis MutT homologue | Evolutionary conservation of 8-oxo-dGTP pyrophosphohydrolase activity |
| OGG1 | DNA glycosylase that removes 8-oxo-G from DNA | Downstream repair of oxidized guanine in DNA |
| MUTYH | Adenine DNA glycosylase that removes mispaired A opposite 8-oxo-G | Prevents mutations after 8-oxo-G incorporation |
| POLB | DNA polymerase beta involved in base excision repair | May incorporate 8-oxo-dGTP if not sanitized |
| POLR2 | RNA polymerase II | Can incorporate 8-oxo-GTP into RNA; MutT also sanitizes RNA precursors |
| ITPA | Inosine triphosphate pyrophosphatase | Related sanitization enzyme for non-canonical nucleotides |
| DUT | Deoxyuridine triphosphatase | Sanitizes dUTP to prevent uracil incorporation |
| NUDT15 | Nudix hydrolase that sanitizes oxidized nucleotides | Potential overlap with 8-oxo-dGTPase activity |
| NUDT18 | Nudix hydrolase with activity on oxidized nucleotides | Candidate sanitization enzyme |
| TP53 | Tumor suppressor that responds to DNA damage | Mutated by 8-oxo-dGTP-induced transversions |
| KRAS | Oncogene frequently mutated in cancers | A:T to C:G transversions can activate KRAS |
| NFE2L2 | Transcription factor regulating antioxidant response | May influence oxidative stress and MTH1 expression |
| SOD1 | Superoxide dismutase 1 | Reduces oxidative stress that generates 8-oxo-dGTP |
| CAT | Catalase | Detoxifies hydrogen peroxide, limiting 8-oxo-dGTP formation |
How Is 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity Regulated?
The activity of 8-oxo-dGTPase is regulated at multiple levels. In renal-cell carcinoma, MTH1 mRNA is overexpressed, suggesting that tumor cells upregulate this enzyme in response to persistent oxidative stress. In the hippocampus of SAMP8 mice, MTH2 expression changes with age, indicating that this activity may be modulated during aging and cognitive decline. Additionally, the broad substrate specificity of enzymes like NUDT5 and MutT suggests that their activity can be influenced by the availability of different oxidized nucleotides. However, specific transcriptional or post-translational regulators of MTH1 remain to be fully characterized.
8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTH1 (NUDT1) | Renal-cell carcinoma; oxidative stress survival | MTH1 knockout cancer cell lines; xenograft models |
| MTH2 (NUDT2) | Age-related cognitive decline; neurodegeneration | SAMP8 mouse hippocampus; MTH2 knockout mice |
| MUTYH | Colorectal cancer; MUTYH-associated polyposis | MUTYH knockout organoids; point mutation knock-in |
| OGG1 | Lung cancer; oxidative DNA damage repair | OGG1 knockout cell lines; CRISPR knock-in of variants |
| TP53 | Li-Fraumeni syndrome; cancer predisposition | TP53 point mutation knock-in; 8-oxo-dGTP exposure |
Cancer and Oxidative Stress
Overexpression of human MTH1 mRNA has been observed in renal-cell carcinoma, providing evidence of persistent oxidative stress in cancer. By sanitizing the nucleotide pool, MTH1 may allow cancer cells to survive high levels of reactive oxygen species and avoid mutations that would be detrimental to tumor growth. However, loss of this activity could lead to increased mutagenesis and genomic instability, potentially driving tumor progression.
Neurodegeneration and Aging
Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse, a model of accelerated aging, have been associated with learning and memory deterioration. This suggests that impaired sanitization of oxidized nucleotides may contribute to neuronal dysfunction during aging. Oxidative stress is a known contributor to neurodegenerative diseases, and the 8-oxo-dGTPase activity may play a protective role.
Mutagenesis and Genetic Diseases
Deficiency in 8-oxo-dGTPase activity leads to the incorporation of 8-oxo-dGTP into DNA, causing A:T to C:G transversion mutations. Such mutations can inactivate tumor suppressor genes or activate oncogenes, contributing to cancer and potentially other genetic diseases. The human MTH1 gene was identified based on its ability to prevent these transversions, highlighting its importance in genome maintenance.
From 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of MTH1 increase mutation frequency? | MTH1 knockout cell lines and mouse models |
| How does MTH1 overexpression affect cancer cell survival? | MTH1 overexpression in renal-cell carcinoma lines |
| What is the role of MTH2 in learning and memory? | MTH2 knockout mice; SAMP8 aging model |
| How does NUDT5 contribute to nucleotide sanitization? | NUDT5 knockout cells; substrate specificity assays |
| What is the structural basis of substrate specificity? | Point mutations in MutT active site; crystallography |
| Can 8-oxo-dGTPase activity be targeted therapeutically? | Knock-in of tagged MTH1 for inhibitor screening |
How to Study the 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| HPLC-based assay | Conversion of 8-oxo-dGTP to 8-oxo-dGMP | Enzyme kinetics and inhibitor testing |
| Coupled pyrophosphate assay | Release of diphosphate | High-throughput screening |
| X-ray crystallography | Three-dimensional structure of enzyme-substrate complex | Substrate specificity and inhibitor design |
| qRT-PCR | mRNA expression levels of MTH1, MTH2, NUDT5 | Oxidative stress response and cancer profiling |
| RNA-seq | Transcriptome-wide changes upon knockout or overexpression | Identifying pathways affected by loss of sanitization |
| CRISPR knockout | Loss-of-function phenotypes | Testing mutation frequency and cell survival |
| CRISPR knock-in | Tagged or mutant enzyme expression | Live-cell imaging and localization studies |
| Mutation frequency assay | A:T to C:G transversion rate | Assessing genomic instability |
Biochemical Assays for 8-oxo-dGTPase Activity
Enzymatic activity can be measured using high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) to separate 8-oxo-dGTP from 8-oxo-dGMP after incubation with purified enzyme. Alternatively, a coupled assay using pyrophosphate detection can quantify the release of diphosphate. These methods are essential for characterizing substrate specificity and kinetics.
Structural Biology and Substrate Analogues
X-ray crystallography and NMR spectroscopy using structural analogues of 8-oxo-2'-deoxyguanosine nucleotide have provided insights into the binding pocket of MutT. Such studies help identify key residues involved in catalysis and can guide the design of inhibitors.
Gene Expression Analysis
Quantitative RT-PCR and RNA-seq can measure MTH1, MTH2, and NUDT5 mRNA levels in tissues or cell lines under oxidative stress conditions. In situ hybridization and immunohistochemistry can localize expression in tissues such as the hippocampus.
CRISPR-Based Functional Genomics
CRISPR knockout, point mutation, and knock-in models allow researchers to dissect the physiological consequences of losing or altering 8-oxo-dGTPase activity. For example, MTH1 knockout cells can be challenged with oxidative stress to measure mutation rates and survival. Overexpression models can test whether increased activity protects against oxidative damage.
How CRISPR Can Be Used to Study GO:0035539 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity
Knockout
CRISPR knockout of MTH1 or MTH2 can be used to eliminate 8-oxo-dGTPase activity and assess the resulting increase in mutation frequency and sensitivity to oxidative stress. Knockout cell lines are valuable for testing whether other enzymes can compensate for the loss of this activity.
Point Mutation
Introducing point mutations in the catalytic residues of MTH1 or MutT can abolish enzymatic activity while preserving protein structure, allowing researchers to distinguish between catalytic and non-catalytic functions. Such models are useful for studying the specific contribution of the hydrolase activity to genome maintenance.
Knock-in
Knock-in of a tagged version of MTH1 (e.g., GFP or FLAG) enables live-cell imaging and proteomic analysis of the enzyme's localization and interaction partners. Knock-in of disease-associated variants can model their impact on 8-oxo-dGTPase activity.
Overexpression
Overexpression of MTH1 or NUDT5 can be achieved by CRISPR activation or lentiviral transduction to test whether increased sanitization protects cells from oxidative damage or promotes cancer cell survival. Overexpression models are also useful for producing large amounts of enzyme for biochemical studies.
How EDITGENE Supports 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity Research
Researchers studying 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity-related genes often need to determine whether a candidate gene is causally involved in preventing oxidative mutagenesis or whether its loss contributes to disease. EDITGENE provides comprehensive CRISPR-based services to create precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity research.
Frequently Asked Questions About 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity
What is 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity?
It is an enzymatic activity that hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP and diphosphate, preventing the mutagenic incorporation of oxidized guanine nucleotides into DNA.
What genes are involved in 8-oxo-dGTPase activity?
Key genes include MTH1 (NUDT1), MTH2 (NUDT2), NUDT5 in humans, and MutT in bacteria.
Why is 8-oxo-dGTPase important for DNA repair?
It sanitizes the nucleotide pool by removing 8-oxo-dGTP, which if incorporated causes A:T to C:G transversion mutations.
How is 8-oxo-dGTPase activity measured?
It can be measured by HPLC, TLC, or coupled pyrophosphate assays using purified enzyme and 8-oxo-dGTP as substrate.
What diseases are associated with MTH1 mutations?
MTH1 overexpression is observed in renal-cell carcinoma, and altered activity may contribute to cancer and neurodegeneration.
Can CRISPR be used to study 8-oxo-dGTPase?
Yes, CRISPR knockout, point mutation, and knock-in models allow functional dissection of MTH1 and related genes.
What is the role of MutT in E. coli?
MutT is a bacterial 8-oxo-dGTPase that prevents transversion mutations by hydrolyzing oxidized nucleotides.
How does oxidative stress affect MTH1 expression?
Oxidative stress can upregulate MTH1 expression, as seen in renal-cell carcinoma, to protect cancer cells from damage.
Is 8-oxo-dGTPase activity conserved across species?
Yes, homologues like CiMutT in Ciona intestinalis and MutT in bacteria exhibit similar pyrophosphohydrolase activity.
What are the substrates of 8-oxo-dGTPase?
The primary substrate is 8-oxo-dGTP, but enzymes like MutT can also act on 8-oxo-GTP and other oxidized nucleotides.
Conclusion
GO:0035539, 8-oxo-7,8-dihydrodeoxyguanosine triphosphate pyrophosphatase activity, is a critical molecular function that protects cells from oxidative mutagenesis by eliminating 8-oxo-dGTP from the nucleotide pool. Its importance spans cancer biology, aging, and neurodegeneration, as evidenced by MTH1 overexpression in renal-cell carcinoma and age-related changes in MTH2 in the hippocampus. Researchers can leverage CRISPR-based models and biochemical assays to further dissect its mechanism and therapeutic potential.
References
- 1. Yonekura S et al.. 2010. CiMutT, an asidian MutT homologue, has a 7, 8-dihydro-8-oxo-dGTP pyrophosphohydrolase activity responsible for sanitization of oxidized nucleotides in Ciona intestinalis.. Genes Genet Syst 85(4):287-95 PMID: 21178309
- 2. Ito R et al.. 2011. Cleavage of oxidized guanine nucleotide and ADP sugar by human NUDT5 protein.. J Biochem 149(6):731-8 PMID: 21389046
- 3. Hamm ML et al.. 2016. Insights into the substrate specificity of the MutT pyrophosphohydrolase using structural analogues of 8-oxo-2'-deoxyguanosine nucleotide.. Bioorg Med Chem Lett 26(8):2014-7 PMID: 26965860
- 4. 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
- 5. Zheng JD et al.. 2009. Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse with learning and memory deterioration.. J Neurol Sci 287(1-2):188-96 PMID: 19735921
- 6. Furuichi M et al.. 1994. Genomic structure and chromosome location of the human mutT homologue gene MTH1 encoding 8-oxo-dGTPase for prevention of A:T to C:G transversion.. Genomics 24(3):485-90 PMID: 7713500
- 7. Okamoto K et al.. 1996. Overexpression of human mutT homologue gene messenger RNA in renal-cell carcinoma: evidence of persistent oxidative stress in cancer.. Int J Cancer 65(4):437-41 PMID: 8621223
