GO:0032143 single thymine insertion binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0032143 (single thymine insertion binding) is a molecular function defined as binding to a double-stranded DNA region containing a single thymine insertion or a deletion that produces an unpaired thymine.
• This binding activity is experimentally associated with mismatch-repair proteins such as the human MSH2-MSH6 heterodimer, which recognizes thymine- or uracil-containing UV photoproducts opposite mismatched bases.
• Single thymine insertion binding is mechanistically linked to DNA damage recognition, because unpaired thymines arise from UV photoproducts and other lesions that distort the double helix.
• APOBEC3A and APOBEC3B provide structural and mechanistic insight into how enzymes engage thymine-containing single-stranded and mismatch contexts during cytosine deamination and mutagenesis.
• The function is relevant to cancer biology and mutagenesis research, because defective or altered DNA lesion recognition can contribute to mutation accumulation and cancer predisposition.
• CRISPR-based knockout, point-mutation, knock-in, and overexpression models enable causal testing of genes implicated in single thymine insertion binding and related DNA repair pathways.
Description
GO:0032143, single thymine insertion binding, is a molecular function term describing the selective binding of a protein or protein complex to a double-stranded DNA region that contains a single thymine insertion or a deletion that leaves an unpaired thymine. This type of DNA lesion is structurally distinct from a simple mismatch because the extra thymine is not paired with a complementary base, creating a local distortion that can be recognized by dedicated DNA-binding and repair factors. The term is therefore important for researchers who study how cells detect and process abnormal DNA structures generated by damage, replication errors, or recombination intermediates. The best-characterized experimental example of single thymine insertion binding comes from the human mismatch-repair heterodimer MSH2-MSH6, which was shown to bind specifically to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases. This finding connects GO:0032143 to nucleotide-level damage recognition and to the broader question of how cells distinguish unpaired or mispaired thymines from normal Watson-Crick base pairs. Related structural work on APOBEC3A and APOBEC3B has revealed how enzymes can engage thymine-containing DNA contexts during cytosine deamination and mutagenesis, providing a mechanistic framework for understanding lesion-specific DNA binding. For biomedical researchers, GO:0032143 matters because unpaired thymine structures can arise from UV photoproducts, alkylation damage, and other helix-distorting lesions that are relevant to cancer, mutagenesis, and genome stability. Studying this function helps clarify how DNA repair and damage-response proteins achieve specificity for abnormal thymine configurations, and it provides a functional annotation target for CRISPR-based perturbation experiments.
single thymine insertion binding At A Glance
| GO ID | GO:0032143 |
|---|---|
| GO term | single thymine insertion binding |
| Ontology | molecular_function |
| Synonym | none listed in QuickGO |
| Definition | Binding to a double-stranded DNA region containing a single thymine insertion or a deletion that results in an unpaired thymine |
| Major function | Recognition and binding of DNA containing an unpaired or inserted thymine, often in the context of DNA damage or mismatch repair |
| Example protein | Human MSH2-MSH6 mismatch-repair heterodimer |
| Related DNA lesion | Thymine- or uracil-containing UV light photoproducts opposite mismatched bases |
| Research relevance | DNA damage recognition, mutagenesis, cancer predisposition, and genome stability |
What Is GO:0032143?
In plain terms, GO:0032143 describes the ability of a protein to stick to a double-stranded DNA site where one thymine base is extra, either because it was inserted or because a deletion left it unpaired. The official QuickGO definition states that this is binding to a double-stranded DNA region containing a single thymine insertion or a deletion that results in an unpaired thymine. Unlike ordinary sequence-specific DNA binding, this function depends on the presence of an abnormal, unpaired thymine that distorts the local DNA structure. Experimentally, this activity has been demonstrated for the human MSH2-MSH6 mismatch-repair heterodimer, which binds DNA containing thymine- or uracil-containing UV photoproducts opposite mismatched bases.
Why Is single thymine insertion binding Important in Cell Biology?
GO:0032143 is important because it defines a precise molecular recognition event for an abnormal DNA structure that can arise from UV damage, alkylation, or replication errors. Proteins that bind single thymine insertions or unpaired thymines help cells detect helix-distorting lesions and initiate repair or signaling responses, and failure of such recognition can contribute to mutation accumulation and cancer. Understanding this function also supports the interpretation of mutagenesis studies, because the ability to discriminate an unpaired thymine from a normal base pair is central to damage-specific DNA binding.
• Provides a defined molecular function for annotating proteins that recognize unpaired or inserted thymines in double-stranded DNA.
• Links DNA damage recognition to mismatch repair through the MSH2-MSH6 heterodimer, a well-characterized binder of thymine-containing UV photoproducts.
• Supports mechanistic studies of UV-induced mutagenesis and photoproduct processing.
• Connects to APOBEC3A and APOBEC3B biology, where thymine-containing DNA contexts influence cytosine deamination and mutagenesis.
• Relevant to cancer research because altered DNA repair and damage recognition can contribute to cancer predisposition.
• Helps interpret alkylation damage repair studies, including alkyltransferase-like factors that enhance nucleotide excision repair.
• Useful for functional genomics and CRISPR screens that test DNA repair gene causality.
• Guides structural and biochemical assays for lesion-specific DNA binding.
• Provides a framework for comparing thymine versus uracil recognition in damaged DNA.
• Supports development of experimental models for genome stability and mutagenesis research.
Molecular Mechanism of single thymine insertion binding
Recognition of an unpaired thymine in double-stranded DNA
In simple terms: The protein looks for a thymine that has no partner base, which makes the DNA look abnormal.
Single thymine insertion binding begins with the detection of a double-stranded DNA region in which a thymine is inserted or left unpaired by a deletion. This unpaired thymine creates a local structural distortion that distinguishes the site from normal Watson-Crick paired DNA. The human MSH2-MSH6 heterodimer has been shown to bind DNA incorporating thymine-containing UV light photoproducts opposite mismatched bases, demonstrating that mismatch-repair proteins can engage thymine-containing lesions with high specificity.
Substrate specificity for thymine- and uracil-containing lesions
In simple terms: The binder can tell the difference between a normal base and a damaged or mismatched thymine.
Specific binding of human MSH2-MSH6 heterodimers to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases shows that the function can discriminate among related lesion structures. This specificity is important because thymine and uracil are chemically similar but biologically distinct, and their recognition influences downstream repair or processing decisions. Structural studies of APOBEC3A and APOBEC3B further illustrate how enzymes achieve targeted engagement with thymine-containing DNA contexts during cytosine deamination and mutagenesis.
Coupling to DNA damage response and repair pathways
In simple terms: Once the abnormal thymine is bound, the cell can trigger repair or signaling.
Binding of single thymine insertions is functionally connected to DNA damage response and repair, because the MSH2-MSH6 heterodimer is a mismatch-repair factor that recognizes UV photoproducts containing thymine or uracil opposite mismatched bases. Related repair pathways, such as nucleotide excision repair, can be enhanced by accessory factors that process alkylation damage, as shown for the alkyltransferase-like ybaZ gene product in E. coli. These connections place GO:0032143 within a broader network of lesion recognition and repair coordination.
Structural and biochemical determinants of binding
In simple terms: The shape and chemistry of the DNA site determine whether the protein can hold on.
The structural basis for targeted DNA engagement in related systems has been illuminated by studies of APOBEC3A and APOBEC3B, which reveal how protein-DNA contacts accommodate thymine-containing contexts during deamination and mutagenesis. For MSH2-MSH6, binding to thymine- or uracil-containing UV photoproducts opposite mismatched bases indicates that the heterodimer can accommodate lesion-induced distortions while maintaining specificity. These observations support a model in which single thymine insertion binding depends on both the abnormal thymine and the surrounding double-stranded DNA architecture.
Regulation and pathway context
In simple terms: Other proteins and damage signals can influence how strongly or when this binding happens.
Single thymine insertion binding does not occur in isolation; it is embedded in DNA damage response and repair pathways that can be modulated by accessory factors and lesion context. For example, the alkyltransferase-like ybaZ gene product enhances nucleotide excision repair of O6-alkylguanine adducts in E. coli, illustrating how repair efficiency can be tuned by auxiliary proteins. In human cells, mismatch-repair heterodimers such as MSH2-MSH6 provide a regulated recognition step for thymine-containing lesions. Structural insights from APOBEC3A and APOBEC3B further suggest that enzyme conformation and substrate context influence lesion engagement.
Key Genes Involved in GO:0032143 single thymine insertion binding
The following genes and proteins are experimentally or mechanistically linked to single thymine insertion binding, DNA lesion recognition, or related repair and mutagenesis pathways.
| Gene | Major Role | Research Relevance |
|---|---|---|
| MSH2 | Component of the MSH2-MSH6 mismatch-repair heterodimer that binds thymine- or uracil-containing UV photoproducts opposite mismatched bases | Core experimental model for single thymine insertion binding and mismatch repair |
| MSH6 | Partner of MSH2 in the mismatch-repair heterodimer that recognizes thymine-containing lesions | Directly implicated in lesion-specific DNA binding assays |
| APOBEC3A | Cytosine deaminase with structurally characterized DNA engagement relevant to thymine-containing contexts | Provides structural insight into targeted DNA deamination and mutagenesis |
| APOBEC3B | Cytosine deaminase implicated in mutagenesis and DNA lesion processing | Model for understanding how enzymes engage thymine-containing DNA |
| ATM | DNA damage response kinase; mutations identified in cancer families | Links DNA damage recognition pathways to cancer predisposition |
| ybaZ | Alkyltransferase-like gene product that enhances nucleotide excision repair of O6-alkylguanine adducts in E. coli | Bacterial model for accessory repair factor function |
| DNA polymerase genes of Sulfolobus solfataricus | DNA replication and lesion bypass in a crenarchaeal model | Comparative model for DNA replication and damage processing |
| phi29 DNA packaging nanomotor components | Real-time sensing and discrimination of single chemicals through a channel | Nanotechnology and sensing model for single-molecule discrimination |
| Actinomycin D binding targets | Binding to single-stranded DNA sequence motifs d(TGTCT(n)G) and d(TGT(n)GTCT) | Chemical probe for thymine-rich DNA interactions |
| ParB-associated T-dioxygenases | Bacteriophage T-dioxygenase subclasses with ParB-dependent and -independent behavior | Model for thymine-related DNA modification and phage biology |
| MSH2-MSH6 heterodimer | Functional mismatch-repair complex that binds thymine-containing UV photoproducts | Primary biochemical system for GO:0032143 |
| APOBEC3 family | Enzymes that deaminate cytosine in DNA and contribute to mutagenesis | Structural and mechanistic models for lesion-specific DNA engagement |
| Nucleotide excision repair factors | Repair pathway enhanced by alkyltransferase-like factors for O6-alkylguanine adducts | Pathway context for lesion recognition and repair |
| Mismatch repair pathway components | Recognize and process mispaired or unpaired bases | Functional network surrounding single thymine insertion binding |
| DNA damage response kinases | Signal DNA lesions and coordinate repair | Cancer-relevant pathway linked to ATM mutations |
| Crenarchaeal DNA replication machinery | Replication and accessory protein functions in Sulfolobus solfataricus | Comparative model for DNA processing |
| Single-stranded DNA binding probes | Actinomycin D binding to thymine-rich motifs | Chemical biology tool for thymine-rich DNA |
| Bacteriophage T-dioxygenases | Thymine-modifying enzymes with ParB-dependent and -independent subclasses | Phage model for thymine DNA modification |
How Is single thymine insertion binding Regulated?
Single thymine insertion binding is regulated at the level of lesion recognition and pathway coordination rather than by a single dedicated regulator. The MSH2-MSH6 heterodimer provides a mismatch-repair recognition step for thymine- or uracil-containing UV photoproducts opposite mismatched bases, and its activity is embedded in the broader DNA damage response. Accessory factors such as the alkyltransferase-like ybaZ gene product can enhance nucleotide excision repair of O6-alkylguanine adducts in E. coli, showing that repair efficiency is modulated by auxiliary proteins. In addition, DNA damage response kinases such as ATM, which is mutated in cancer families, coordinate cellular responses to DNA lesions and can influence repair pathway choice. Structural and biochemical studies of APOBEC3A and APOBEC3B further indicate that enzyme conformation and substrate context regulate lesion engagement and downstream mutagenesis.
single thymine insertion binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MSH2 | Mismatch repair and UV photoproduct recognition | Knockout and point-mutation cell models for lesion binding assays |
| MSH6 | Mismatch repair and thymine-containing lesion recognition | Knock-in reporter models for binding specificity |
| ATM | Cancer predisposition and DNA damage response | Knockout models for DNA damage sensitivity |
| APOBEC3A | Mutagenesis and DNA deamination | Overexpression models for structural and mutagenesis studies |
| APOBEC3B | Mutagenesis and DNA lesion processing | Knockout and overexpression models for deamination assays |
Cancer predisposition and DNA repair defects
Defects in DNA damage recognition and repair pathways can contribute to cancer predisposition, as illustrated by ATM mutations identified in cancer families. Because single thymine insertion binding is linked to mismatch-repair recognition of thymine-containing UV photoproducts, altered function of proteins such as MSH2-MSH6 could influence mutation accumulation and cancer risk. Studying GO:0032143 therefore helps connect lesion-specific DNA binding to cancer biology.
UV-induced mutagenesis and skin cancer biology
UV light generates photoproducts that can contain thymine or uracil opposite mismatched bases, and the human MSH2-MSH6 heterodimer binds such structures. This places single thymine insertion binding in the mechanistic landscape of UV-induced mutagenesis, which is relevant to skin cancer and genome instability. Structural work on APOBEC3A and APOBEC3B further links thymine-containing DNA contexts to mutagenic processes.
Alkylation damage and repair pathway crosstalk
Alkylation damage produces adducts such as O6-alkylguanine, and the alkyltransferase-like ybaZ gene product enhances nucleotide excision repair of these adducts in E. coli. Although this is a bacterial model, it illustrates how accessory factors modulate repair of helix-distorting lesions that can involve thymine-containing contexts. This pathway crosstalk is relevant to understanding how single thymine insertion binding integrates with broader repair networks.
From single thymine insertion binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of MSH2 or MSH6 abolish single thymine insertion binding? | Knockout cell models |
| Which residues are required for thymine-containing lesion recognition? | Point-mutation knock-in models |
| Can a tagged repair protein be tracked at lesion sites? | Tagged knock-in models |
| Does overexpression of APOBEC3A or APOBEC3B increase mutagenesis at thymine-containing sites? | Overexpression models |
| Does ATM loss alter sensitivity to DNA-damaging agents? | Knockout models |
| Can accessory repair factors enhance lesion processing? | Bacterial or mammalian overexpression models |
How to Study the single thymine insertion binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Electrophoretic mobility shift assay | Direct binding of proteins to thymine-containing DNA | Testing MSH2-MSH6 binding specificity |
| Structural biology (crystallography/cryo-EM) | Three-dimensional protein-DNA contacts | Defining lesion recognition interfaces |
| Mutagenesis reporter assays | Mutation frequency after DNA damage | Linking binding function to genome stability |
| DNA damage sensitivity assays | Cell survival after UV or alkylating agents | Testing repair pathway defects |
| CRISPR knockout screening | Gene requirement for lesion processing | Identifying factors needed for single thymine insertion binding |
| Overexpression studies | Effect of increased protein levels on mutagenesis | Testing APOBEC3A/APOBEC3B activity |
| Comparative model-organism genetics | Conserved repair factor function | Dissecting pathway conservation |
| Single-molecule sensing | Discrimination of individual chemicals or DNA features | Nanotechnology-based detection |
Biochemical DNA binding assays
Electrophoretic mobility shift assays and related biochemical approaches can test whether purified proteins such as MSH2-MSH6 bind double-stranded DNA containing a single thymine insertion or an unpaired thymine. These assays are essential for directly demonstrating GO:0032143 activity and for comparing wild-type and mutant proteins.
Structural biology of lesion recognition
Structural studies of APOBEC3A and APOBEC3B have revealed how enzymes engage thymine-containing DNA contexts during cytosine deamination and mutagenesis. Similar structural approaches can be applied to mismatch-repair heterodimers to define the contacts that mediate single thymine insertion binding.
Mutagenesis and damage sensitivity assays
Cell-based mutagenesis assays and damage sensitivity tests can measure the functional consequences of altering genes involved in single thymine insertion binding. For example, ATM mutations are linked to cancer families and DNA damage response defects, providing a framework for interpreting sensitivity phenotypes.
Comparative and model-organism studies
Bacterial and archaeal systems offer tractable models for studying DNA repair and replication factors, such as the alkyltransferase-like ybaZ gene product in E. coli and DNA polymerases of Sulfolobus solfataricus. These models can help dissect conserved principles of lesion recognition that relate to single thymine insertion binding.
How CRISPR Can Be Used to Study GO:0032143 single thymine insertion binding
Knockout
CRISPR knockout of MSH2, MSH6, or related repair genes can test whether these factors are required for single thymine insertion binding and downstream lesion processing. Loss-of-function models help establish causality between a candidate gene and the DNA binding phenotype.
Point Mutation
Point-mutation knock-in models can introduce specific amino acid substitutions in DNA-binding domains to determine which residues are essential for recognizing thymine-containing lesions. Such models are valuable for separating binding activity from other protein functions.
Knock-in
Tagged knock-in of repair proteins allows localization and interaction studies at sites of DNA damage containing unpaired thymines. These models support imaging and proteomic analyses of lesion recognition complexes.
Overexpression
Overexpression of APOBEC3A, APOBEC3B, or repair factors can reveal gain-of-function effects on mutagenesis and lesion processing. Overexpression models are useful for testing whether increased protein levels alter single thymine insertion binding dynamics.
How EDITGENE Supports single thymine insertion binding Research
Researchers studying single thymine insertion binding-related genes often need to determine whether a candidate gene is causally involved in lesion recognition, repair, or mutagenesis. EDITGENE provides CRISPR-based cell model services that enable precise perturbation of these genes in relevant cellular contexts.
Contact EDITGENE today to design your custom CRISPR model for single thymine insertion binding research.
Frequently Asked Questions About single thymine insertion binding
What is single thymine insertion binding?
Single thymine insertion binding (GO:0032143) is a molecular function defined as binding to a double-stranded DNA region containing a single thymine insertion or a deletion that results in an unpaired thymine.
What genes are involved in single thymine insertion binding?
The human MSH2 and MSH6 genes, which form the MSH2-MSH6 mismatch-repair heterodimer, are experimentally linked to binding of thymine-containing UV photoproducts opposite mismatched bases. APOBEC3A and APOBEC3B are also relevant to thymine-containing DNA contexts.
Which protein complex binds thymine-containing UV photoproducts?
The human MSH2-MSH6 mismatch-repair heterodimer binds DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases.
Why is GO:0032143 important for cancer research?
DNA damage recognition and repair defects can contribute to cancer predisposition, as shown by ATM mutations in cancer families, and single thymine insertion binding is part of lesion recognition biology.
How can I study single thymine insertion binding in the lab?
Biochemical DNA binding assays, structural biology, mutagenesis reporters, and CRISPR-based perturbation models can be used to study this function.
What is the difference between single thymine insertion binding and mismatch repair?
Single thymine insertion binding is a specific molecular function for recognizing an unpaired or inserted thymine, while mismatch repair is a broader pathway that includes recognition and processing of mispaired bases.
Can CRISPR knockout help identify genes required for single thymine insertion binding?
Yes, CRISPR knockout of candidate genes such as MSH2 or MSH6 can test whether they are required for binding thymine-containing lesions and for downstream repair.
What experimental models are available for GO:0032143 research?
Knockout, point-mutation, knock-in, tagged knock-in, and overexpression cell models are suitable for testing genes involved in single thymine insertion binding.
Is single thymine insertion binding related to UV mutagenesis?
Yes, because UV photoproducts can contain thymine or uracil opposite mismatched bases, and the MSH2-MSH6 heterodimer binds such structures, linking this function to UV mutagenesis.
What databases define GO:0032143?
GO:0032143 is defined in the Gene Ontology as a molecular function: binding to a double-stranded DNA region containing a single thymine insertion or a deletion that results in an unpaired thymine.
Conclusion
GO:0032143, single thymine insertion binding, defines a precise molecular recognition event for double-stranded DNA containing an inserted or unpaired thymine. Experimental evidence from the human MSH2-MSH6 mismatch-repair heterodimer connects this function to UV photoproduct recognition and DNA damage response biology. Related structural and mechanistic studies of APOBEC3A and APOBEC3B further illuminate how enzymes engage thymine-containing DNA contexts during mutagenesis. Together, these findings make GO:0032143 a valuable annotation for research on genome stability, cancer predisposition, and DNA repair. CRISPR-based knockout, point-mutation, knock-in, and overexpression models provide powerful tools for causally testing genes implicated in single thymine insertion binding and related pathways. By combining biochemical binding assays with cellular perturbation, researchers can clarify how lesion recognition contributes to mutagenesis and disease.
References
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