GO:0106431 N6-methyl-(d)ATP hydrolase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0106431 describes the enzymatic activity that hydrolyzes N6-methyl-(d)ATP to N6-methyl-(d)AMP and diphosphate, thereby removing a methylated nucleotide from the nucleotide pool.
• The best-characterized enzyme carrying this activity is MTH1 (NUDT1), which sanitizes the dNTP pool by eliminating oxidized and methylated nucleotides.
• N6-methyl-dATP can be incorporated into DNA, causing mutations; its removal by MTH1 protects genome integrity.
• Loss of N6-methyl-(d)ATP hydrolase activity may contribute to cancer and neurodegeneration through accumulation of damaged nucleotides.
• Studying this activity requires sensitive biochemical assays, CRISPR knockout models, and analytical methods such as LC-MS/MS.
• EDITGENE provides CRISPR services to interrogate the genes and pathways linked to N6-methyl-(d)ATP hydrolase activity.
Description
N6-methyl-(d)ATP hydrolase activity (GO:0106431) is a molecular function that catalyzes the hydrolysis of N6-methyl-(d)ATP to N6-methyl-(d)AMP and diphosphate. This activity is part of the cellular machinery that maintains the integrity of the nucleotide pool by removing methylated nucleotides that can otherwise be misincorporated into DNA. The enzyme MTH1 (also known as NUDT1) has been shown to remove N6-methyl-dATP from the dNTP pool, highlighting its role in sanitizing nucleotide precursors. Understanding this activity is important because methylated nucleotides can lead to mutations and genomic instability, which are hallmarks of cancer and other diseases. Researchers study GO:0106431 to elucidate how cells prevent the incorporation of damaged nucleotides and to explore therapeutic strategies that target these pathways.
N6-methyl-(d)ATP hydrolase activity At A Glance
| GO ID | GO:0106431 |
|---|---|
| GO term | N6-methyl-(d)ATP hydrolase activity |
| Ontology | molecular_function |
| Synonym | none |
| Major function | Hydrolysis of N6-methyl-(d)ATP to N6-methyl-(d)AMP and diphosphate |
| Reaction | N6-methyl-(d)ATP + H2O = N6-methyl-(d)AMP + diphosphate + H+ |
| Representative enzyme | MTH1 (NUDT1) |
| Biological context | Sanitization of the dNTP pool to prevent incorporation of methylated nucleotides into DNA |
What Is GO:0106431?
According to the Gene Ontology, GO:0106431 N6-methyl-(d)ATP hydrolase activity is defined as the catalysis of the reaction: N6-methyl-(d)ATP + H2O = N6-methyl-(d)AMP + diphosphate + H+. In other words, it is an enzymatic activity that uses water to cleave N6-methyl-(d)ATP, releasing N6-methyl-(d)AMP and diphosphate.
Why Is N6-methyl-(d)ATP hydrolase activity Important in Cell Biology?
N6-methyl-(d)ATP hydrolase activity is crucial for maintaining genomic stability because it eliminates N6-methyl-dATP, a methylated nucleotide that can be incorporated into DNA and cause mutations. MTH1, the enzyme responsible for this activity, prevents the accumulation of such damaged nucleotides, and its dysfunction has been linked to cancer and neurodegenerative disorders. Therefore, understanding GO:0106431 provides insights into cellular defense mechanisms against DNA damage and offers potential targets for therapeutic intervention.
• Prevents incorporation of N6-methyl-dATP into DNA, reducing mutation load.
• Maintains dNTP pool purity and genome integrity.
• Dysfunction of MTH1 is associated with cancer progression.
• May play a role in neurodegeneration due to oxidative stress.
• Potential target for cancer therapy, as MTH1 inhibitors are being explored.
• Provides a model for studying nucleotide sanitization pathways.
• Relevant to understanding mutagenesis and carcinogenesis.
• Helps explain cellular responses to alkylating agents.
What Happens During N6-methyl-(d)ATP hydrolase activity?
Substrate recognition and binding
In simple terms: The enzyme finds and grabs the methylated nucleotide.
The enzyme MTH1 recognizes N6-methyl-(d)ATP as a substrate and binds it in its active site. This binding is specific to the methylated form, allowing the enzyme to distinguish it from normal nucleotides.
Catalytic hydrolysis
In simple terms: Water is used to break the nucleotide into smaller pieces.
Once bound, the enzyme catalyzes the hydrolysis of N6-methyl-(d)ATP, using a water molecule to cleave the phosphoanhydride bond, releasing N6-methyl-(d)AMP and diphosphate.
Product release and pool sanitization
In simple terms: The broken pieces are released, and the harmful nucleotide is removed.
After the reaction, N6-methyl-(d)AMP and diphosphate are released, effectively removing the methylated nucleotide from the dNTP pool and preventing its incorporation into DNA.
Biological impact
In simple terms: This process protects DNA from mutations.
By eliminating N6-methyl-dATP, the activity reduces the chance of mutations caused by misincorporation of methylated nucleotides during DNA replication.
Key Genes Involved in GO:0106431 N6-methyl-(d)ATP hydrolase activity
The following genes and proteins are directly or indirectly involved in N6-methyl-(d)ATP hydrolase activity and related nucleotide sanitation pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MTH1 (NUDT1) | Hydrolyzes N6-methyl-(d)ATP and other oxidized nucleotides | Primary enzyme for GO:0106431; knockout models show increased mutation load |
| NUDT1 | Alternative symbol for MTH1 | Same as above |
| MTH1 variants | May alter substrate specificity | Studied for cancer susceptibility |
| OGG1 | DNA glycosylase involved in base excision repair | Works downstream of MTH1 to repair oxidized bases |
| MUTYH | DNA glycosylase that removes adenine mispaired with 8-oxoguanine | Related to oxidative DNA damage response |
| TP53 | Tumor suppressor, guardian of the genome | Mutations in TP53 may synergize with MTH1 loss |
| KRAS | Oncogene frequently mutated in cancers | MTH1 inhibitors show efficacy in KRAS-mutant cancers |
| NRF2 | Transcription factor regulating antioxidant response | May regulate MTH1 expression |
| ATM | DNA damage response kinase | Coordinates with MTH1 in response to oxidative stress |
| ATR | DNA damage response kinase | Similar to ATM |
| PARP1 | Poly(ADP-ribose) polymerase involved in DNA repair | Synthetic lethality with MTH1 inhibition |
| BRCA1 | DNA repair protein | Potential synthetic lethal interaction |
| BRCA2 | DNA repair protein | Potential synthetic lethal interaction |
| POLB | DNA polymerase involved in base excision repair | May incorporate N6-methyl-dATP if not removed |
| POLH | Translesion synthesis polymerase | Can bypass lesions caused by methylated nucleotides |
| REV1 | Translesion synthesis polymerase | Similar to POLH |
| PCNA | Proliferating cell nuclear antigen | Coordinates DNA replication and repair |
| RPA | Single-stranded DNA binding protein | Involved in DNA damage response |
How Is N6-methyl-(d)ATP hydrolase activity Regulated?
The expression and activity of MTH1, the primary enzyme for GO:0106431, are regulated at multiple levels. Transcription of MTH1 is induced by oxidative stress through the NRF2 pathway. Additionally, MTH1 activity can be modulated by post-translational modifications, although specific details remain to be fully elucidated. The PI3K/AKT pathway has also been implicated in regulating MTH1 expression in cancer cells.
N6-methyl-(d)ATP hydrolase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MTH1 (NUDT1) | Cancer | MTH1 knockout cancer cell lines, xenograft models |
| MTH1 (NUDT1) | Neurodegeneration | Neuronal cell lines with MTH1 knockout, primary neurons |
| MTH1 (NUDT1) | Aging | MTH1 knockout mice, cellular senescence models |
| MTH1 (NUDT1) | Inflammation | Macrophage cell lines with MTH1 knockdown |
| MTH1 (NUDT1) | Metabolic disorders | MTH1 knockout hepatocytes |
Cancer
MTH1 is overexpressed in many cancers and is considered a promising target for cancer therapy. Cancer cells rely on MTH1 to survive oxidative stress and avoid mutations that could be detrimental, but also to maintain genomic stability for proliferation. Inhibitors of MTH1 have shown efficacy in preclinical models, particularly in cancers with high oxidative stress.
Neurodegeneration
Oxidative stress is a hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's. MTH1 dysfunction may contribute to neuronal death by allowing accumulation of oxidized nucleotides, leading to DNA damage.
Aging
Accumulation of DNA damage is a hallmark of aging. Reduced MTH1 activity could accelerate aging by increasing mutation load and cellular senescence.
From N6-methyl-(d)ATP hydrolase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does MTH1 knockout increase mutation frequency? | MTH1 knockout cell lines (e.g., HAP1, HeLa) |
| Does MTH1 point mutation affect substrate specificity? | Point-mutant MTH1 knock-in cell lines |
| Can MTH1 overexpression protect against oxidative stress? | MTH1 overexpression cell lines |
| What is the subcellular localization of MTH1? | Tagged knock-in of MTH1 with fluorescent protein |
| Does MTH1 inhibition synergize with PARP inhibitors? | MTH1 knockout or inhibitor-treated cancer cells |
| What is the role of MTH1 in neurodegeneration? | MTH1 knockout neurons or mouse models |
How to Study the N6-methyl-(d)ATP hydrolase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| LC-MS/MS | N6-methyl-dATP levels | Quantification of nucleotide pools |
| HPLC | Enzymatic activity | In vitro hydrolase assays |
| CRISPR knockout | Gene function | Loss-of-function studies |
| Comet assay | DNA damage | Assessment of genomic instability |
| Gamma-H2AX staining | DNA double-strand breaks | DNA damage response |
| Mutation reporter assay | Mutation frequency | Genotoxicity studies |
| RNA-seq | Gene expression changes | Transcriptomic profiling |
| Proteomics | Protein interactions | Identification of MTH1 binding partners |
Biochemical assays for hydrolase activity
Enzymatic activity of N6-methyl-(d)ATP hydrolase can be measured using purified MTH1 protein and N6-methyl-(d)ATP as substrate, followed by detection of products via HPLC or LC-MS/MS.
CRISPR-based genetic screens
Genome-wide CRISPR knockout screens can identify genes that modulate sensitivity to MTH1 inhibition or that are synthetic lethal with MTH1 loss.
Analytical detection of N6-methyl-dATP
Levels of N6-methyl-dATP in cells can be quantified using LC-MS/MS after nucleotide extraction.
DNA damage and mutation assays
Comet assay, gamma-H2AX staining, and mutation reporter assays can assess the impact of MTH1 loss on DNA integrity.
How CRISPR Can Be Used to Study GO:0106431 N6-methyl-(d)ATP hydrolase activity
Knockout
CRISPR knockout of MTH1 (NUDT1) can be used to study the consequences of losing N6-methyl-(d)ATP hydrolase activity, including increased mutation load and sensitivity to oxidative stress.
Point Mutation
Introducing point mutations in the catalytic site of MTH1 can help dissect the enzymatic mechanism and identify residues critical for substrate binding and hydrolysis.
Knock-in
Knock-in of tagged MTH1 (e.g., GFP or FLAG) allows for visualization and immunoprecipitation of the enzyme to study its localization and interactions.
Overexpression
Overexpression of MTH1 can protect cells from oxidative stress and may be used to study its role in cancer cell survival.
How EDITGENE Supports N6-methyl-(d)ATP hydrolase activity Research
Researchers studying N6-methyl-(d)ATP hydrolase activity-related genes often need to determine whether a candidate gene is causally involved in nucleotide pool sanitation, DNA damage responses, or disease progression. EDITGENE provides a comprehensive suite of CRISPR services to enable such investigations.
Contact EDITGENE today to design your custom CRISPR model for N6-methyl-(d)ATP hydrolase activity research.
Frequently Asked Questions About N6-methyl-(d)ATP hydrolase activity
What is N6-methyl-(d)ATP hydrolase activity?
It is an enzymatic activity that hydrolyzes N6-methyl-(d)ATP to N6-methyl-(d)AMP and diphosphate, as defined by GO:0106431.
What genes are involved in N6-methyl-(d)ATP hydrolase activity?
The primary gene is MTH1 (NUDT1), which encodes the enzyme responsible for this activity.
What is the role of MTH1 in DNA repair?
MTH1 sanitizes the dNTP pool by removing oxidized and methylated nucleotides, preventing their incorporation into DNA.
How is N6-methyl-(d)ATP hydrolase activity measured?
It can be measured using biochemical assays with purified enzyme and LC-MS/MS detection of products.
What diseases are associated with N6-methyl-(d)ATP hydrolase dysfunction?
Cancer, neurodegeneration, and aging have been linked to impaired MTH1 function.
Can CRISPR be used to study N6-methyl-(d)ATP hydrolase activity?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models are valuable tools.
What are the substrates of N6-methyl-(d)ATP hydrolase?
The substrate is N6-methyl-(d)ATP, and the products are N6-methyl-(d)AMP and diphosphate.
Is N6-methyl-(d)ATP hydrolase activity specific to certain tissues?
MTH1 is ubiquitously expressed, but its levels may vary among tissues.
What are MTH1 inhibitors?
Small molecules that inhibit MTH1 activity are being developed as anticancer agents.
How does N6-methyl-(d)ATP hydrolase activity affect mutation rates?
By removing N6-methyl-dATP, it reduces the chance of misincorporation and subsequent mutations.
Conclusion
N6-methyl-(d)ATP hydrolase activity (GO:0106431) is a critical molecular function that protects cells from the mutagenic effects of methylated nucleotides. The enzyme MTH1 (NUDT1) is the key player in this process, and its dysfunction is associated with cancer, neurodegeneration, and aging. Continued research using CRISPR models and advanced analytical techniques will further illuminate the biological significance of this activity and its potential as a therapeutic target.
References
- 1. Scaletti ER et al.. 2020. MutT homologue 1 (MTH1) removes N6-methyl-dATP from the dNTP pool.. J Biol Chem 295(15):4761-4772 PMID: 32144205
