GO:0061730 C-rich strand telomeric DNA binding: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0061730 (C-rich strand telomeric DNA binding) is a molecular_function term describing binding to C-rich, single-stranded, telomere-associated DNA.
• The best structurally characterized C-rich strand telomeric DNA-binding module is the KH domain of poly(C)-binding protein 2 (PCBP2), solved in complex with human telomeric C-rich DNA at 1.7 A and 1.6 A resolution.
• C-rich strand recognition is mechanistically linked to telomere end replication, because the C-rich strand is the template for telomerase and for CST-Pol-alpha/primase fill-in.
• POT1 recruits and regulates CST-Pol-alpha/primase at human telomeres, coupling telomeric single-strand handling to C-strand synthesis.
• CST-polymerase alpha-primase solves a second telomere end-replication problem, making C-strand binding proteins central to genome stability.
• C-rich strand telomeric DNA binding is studied with structural biology, electrophoretic mobility shift assays, CRISPR knockout/knock-in models and telomere length assays.
Description
GO:0061730, C-rich strand telomeric DNA binding, is a Gene Ontology molecular_function term defined as binding to C-rich, single-stranded, telomere-associated DNA. Telomeres terminate in a single-stranded G-rich overhang and an internally paired C-rich strand, and proteins that recognize the C-rich strand participate in the replication, protection and regulation of chromosome ends. The term therefore describes a nucleic-acid-binding activity rather than a catalytic activity, and it is assigned to proteins that physically associate with the cytosine-rich telomeric sequence. The best-characterized example is human poly(C)-binding protein 2 (PCBP2), whose first and third KH domains bind the C-rich strand of human telomeric DNA with high specificity. Structural studies have resolved the first KH domain of PCBP2 bound to a C-rich telomeric DNA repeat at 1.7 A and the third KH domain bound to the same type of ligand at 1.6 A, providing direct atomic evidence for this GO activity. In addition, a telomeric DNA-binding protein recognizing the C-rich strand has been identified in the apicomplexan parasite Eimeria tenella, indicating that this activity is not restricted to humans. The biological importance of C-rich strand recognition is underscored by the fact that the C-rich strand is the template for telomerase-mediated G-strand extension and must subsequently be filled in by CST-Pol-alpha/primase, a reaction regulated by POT1 at human telomeres. Consequently, GO:0061730 is relevant to telomere maintenance, end-replication biology and the interpretation of telomere-associated disease variants.
C-rich strand telomeric DNA binding At A Glance
| GO ID | GO:0061730 |
|---|---|
| GO term | C-rich strand telomeric DNA binding |
| Ontology | molecular_function |
| Synonym | None listed in QuickGO |
| Definition | Binding to C-rich, single-stranded, telomere-associated DNA |
| Major function | Sequence-specific recognition of the cytosine-rich single strand of telomeric DNA |
| Representative protein | Poly(C)-binding protein 2 (PCBP2), via KH domains |
| Structural evidence | KH domain-DNA complexes at 1.7 A and 1.6 A resolution |
| Related biology | Telomere end replication and CST-Pol-alpha/primase function |
What Is GO:0061730?
In plain terms, GO:0061730 describes the ability of a protein to stick to the cytosine-rich, single-stranded DNA found at chromosome ends. It is a molecular_function term: it captures a binding event, not an enzymatic reaction. The ligand is telomere-associated DNA that is single-stranded and C-rich, and the annotated proteins are those that selectively recognize this sequence context.
Why Is C-rich strand telomeric DNA binding Important in Cell Biology?
C-rich strand telomeric DNA binding matters because the C-rich strand is the template that must be copied to complete telomere replication after telomerase extends the G-rich strand. Proteins that bind this strand help coordinate the recruitment and regulation of CST-Pol-alpha/primase, and POT1 has been shown to recruit and regulate this complex at human telomeres. A second telomere end-replication problem is solved by CST-polymerase alpha-primase, which depends on proper handling of the C-rich template. Therefore, understanding GO:0061730 provides a mechanistic entry point into telomere maintenance, chromosome-end protection and the cellular response to replication stress at telomeres.
• Defines a specific nucleic-acid-binding activity that distinguishes C-rich telomeric DNA recognition from generic single-stranded DNA binding.
• Provides the structural basis for sequence-specific telomeric C-strand recognition by KH domains.
• Connects telomere end replication to CST-Pol-alpha/primase and POT1 regulation.
• Supports comparative telomere biology across eukaryotes, since telomere structure and protein composition vary between species.
• Extends beyond humans, as a C-rich telomeric DNA-binding protein has been identified in Eimeria tenella.
• Offers a functional annotation target for CRISPR screens and for interpreting variants in telomere-associated proteins.
• Helps explain how the C-rich strand is protected and made available as a template during S phase.
• Links telomeric DNA binding to alternative lengthening of telomeres and TERRA-related regulation.
Molecular Mechanism of C-rich strand telomeric DNA binding
Recognition of the C-rich telomeric repeat
In simple terms: Proteins that carry this activity can read the cytosine-rich sequence at chromosome ends.
The defining event of GO:0061730 is sequence-specific contact between a protein module and C-rich, single-stranded telomeric DNA. Human PCBP2 recognizes the C-rich strand of human telomeric DNA and the RNA template of human telomerase through its first KH domain, establishing that this activity can discriminate the C-rich telomeric sequence from unrelated nucleic acids. The same protein has been structurally characterized in complex with a C-rich strand of human telomeric DNA, confirming direct physical binding.
KH domain architecture and atomic contacts
In simple terms: The binding is performed by a small protein module called a KH domain that grips the DNA.
The first KH domain of human PCBP2 binds a C-rich strand of human telomeric DNA, and the complex was solved at 1.7 A resolution. The third KH domain of the same protein also binds a C-rich strand of human telomeric DNA, with a structure determined at 1.6 A resolution. These structures show that C-rich strand telomeric DNA binding is mediated by a compact, folded nucleic-acid-binding domain rather than by a disordered region.
Coupling to telomere end replication
In simple terms: The C-rich strand is the copy template used to finish telomere replication.
After telomerase extends the G-rich strand, the C-rich strand must be synthesized to complete the telomere. POT1 recruits and regulates CST-Pol-alpha/primase at human telomeres, linking single-strand telomeric DNA handling to C-strand fill-in. CST-polymerase alpha-primase solves a second telomere end-replication problem, so proteins that bind the C-rich template are functionally connected to this reaction.
Regulation by telomere-associated factors
In simple terms: Other telomere proteins control when and where the C-rich strand is engaged.
POT1 acts as a regulator that recruits and controls CST-Pol-alpha/primase at human telomeres, providing a layer of regulation over C-strand synthesis. Telomere elongation pathways, including alternative lengthening of telomeres, are also influenced by TERRA-related mechanisms, indicating that C-rich strand biology is embedded in a broader regulatory network.
Evolutionary and comparative context
In simple terms: Different organisms use different proteins to handle the same kind of telomeric DNA.
Telomere structure varies across eukaryotes, and comparative analyses show that the protein machinery associated with telomeres is not identical between species. A telomeric DNA-binding protein recognizing the C-rich strand has been identified in Eimeria tenella, demonstrating that GO:0061730-type activity occurs outside the human system.
Key Genes Involved in GO:0061730 C-rich strand telomeric DNA binding
The following genes and proteins are directly or functionally associated with C-rich strand telomeric DNA binding and its downstream telomere biology.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PCBP2 | KH-domain protein that binds the C-rich strand of human telomeric DNA and the telomerase RNA template | Primary structural and biochemical model for GO:0061730 |
| POT1 | Recruits and regulates CST-Pol-alpha/primase at human telomeres | Links C-strand handling to telomere end replication |
| CTC1 | Component of the CST complex that acts at telomeres | CST-Pol-alpha/primase function and C-strand fill-in |
| STN1 | Component of the CST complex involved in telomere replication | CST-Pol-alpha/primase function and C-strand fill-in |
| TEN1 | Component of the CST complex involved in telomere replication | CST-Pol-alpha/primase function and C-strand fill-in |
| POLA1 | Catalytic subunit of polymerase alpha, part of CST-Pol-alpha/primase | C-strand synthesis at telomeres |
| PRIM1 | Primase subunit of the Pol-alpha/primase complex | Primer synthesis for C-strand fill-in |
| PRIM2 | Primase subunit of the Pol-alpha/primase complex | Primer synthesis for C-strand fill-in |
| TERC | Telomerase RNA component containing the template for telomeric repeats | Template recognition by C-rich strand-binding proteins |
| TERT | Telomerase reverse transcriptase | Telomere elongation and ALT-related regulation |
| Eimeria tenella telomeric DNA-binding protein | C-rich telomeric DNA-binding protein identified in E. tenella | Comparative evidence for GO:0061730 outside humans |
| Telomere-associated proteins in diverse eukaryotes | Species-specific telomere structure and protein composition | Comparative telomere biology |
How Is C-rich strand telomeric DNA binding Regulated?
C-rich strand telomeric DNA binding is regulated at the level of protein recruitment and telomere end processing. POT1 recruits and regulates CST-Pol-alpha/primase at human telomeres, which means that access of the C-rich template to the replication machinery is controlled by telomere-associated factors rather than occurring constitutively. CST-polymerase alpha-primase activity is required to solve a second telomere end-replication problem, so the timing of C-strand synthesis is coupled to the completion of G-strand extension. In addition, telomere elongation pathways such as alternative lengthening of telomeres and TERRA-related mechanisms influence how telomeric DNA is engaged and processed. Comparative studies further indicate that the protein players and their regulation differ between eukaryotic lineages.
C-rich strand telomeric DNA binding and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| POT1 | Telomere maintenance and genome instability | Knockout and tagged knock-in cell lines with telomere length assays |
| CTC1 | CST-Pol-alpha/primase-dependent C-strand synthesis | Point-mutation models of CST subunits |
| STN1 | Telomere end replication | Knockout and rescue cell models |
| TEN1 | Telomere end replication | Knockout and rescue cell models |
| PCBP2 | C-rich strand telomeric DNA recognition | Domain-specific knock-in and binding assays |
Telomere replication stress and genome instability
Defects in the machinery that completes C-strand synthesis after telomerase action cause a second telomere end-replication problem, which is solved by CST-polymerase alpha-primase. Because POT1 recruits and regulates this complex at human telomeres, perturbation of C-rich strand handling can compromise telomere integrity and chromosome-end stability. These mechanisms are directly relevant to cancer biology, where telomere maintenance is a hallmark of immortalized cells.
Alternative lengthening of telomeres and telomerase-independent maintenance
Telomere elongation can proceed through alternative lengthening of telomeres, and TERRA-related mechanisms influence pathway choice. Proteins that engage the C-rich strand participate in the broader telomere maintenance network that determines whether cells rely on telomerase or ALT. This makes GO:0061730-relevant factors candidate modifiers of telomere maintenance phenotypes in tumors.
Comparative and parasitic disease relevance
A telomeric DNA-binding protein recognizing the C-rich strand has been identified in Eimeria tenella, an apicomplexan parasite. Comparative analyses of telomere structure across eukaryotes show that telomere-associated protein sets differ between organisms, which is relevant when considering species-specific drug or vaccine targets.
From C-rich strand telomeric DNA binding-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is PCBP2 required for C-rich strand telomeric DNA binding in cells? | PCBP2 knockout cell line with binding and telomere assays |
| Which KH domain residues contact the C-rich telomeric repeat? | Point-mutation knock-in of PCBP2 KH domains |
| Does POT1 regulate CST-Pol-alpha/primase recruitment? | POT1 knockout and tagged knock-in models |
| Is CST-Pol-alpha/primase required for C-strand fill-in? | Knockout of CTC1, STN1, TEN1, POLA1, PRIM1 or PRIM2 |
| Does a candidate protein bind the C-rich strand in a species-specific manner? | Overexpression of the candidate protein followed by binding assays |
| How does telomere elongation pathway choice respond to C-strand factors? | Overexpression and knockout models combined with telomere length measurement |
How to Study the C-rich strand telomeric DNA binding Process
| Method | What It Measures | Typical Application |
|---|---|---|
| X-ray crystallography | Atomic contacts between a protein domain and C-rich telomeric DNA | Defining the structural basis of GO:0061730 |
| Electrophoretic mobility shift assay | Direct protein-DNA binding | Testing specificity for the C-rich telomeric strand |
| Recombinant domain binding assay | Binding of isolated KH domains to telomeric DNA or telomerase RNA | Mapping the minimal binding module |
| Telomere length measurement | Changes in telomere maintenance after perturbation | Linking C-strand factors to telomere homeostasis |
| CST complex functional assays | Recruitment and activity of CST-Pol-alpha/primase | Testing C-strand fill-in mechanisms |
| Comparative sequence and protein analysis | Conservation of telomere-associated proteins across eukaryotes | Identifying species-specific C-rich strand binders |
| Candidate protein binding screen | Binding of newly identified proteins to C-rich telomeric DNA | Discovering new GO:0061730 annotations |
Structural biology of C-rich strand recognition
X-ray crystallography has been used to determine the structure of the first KH domain of human PCBP2 in complex with a C-rich strand of human telomeric DNA at 1.7 A resolution, and the third KH domain of the same protein in complex with a C-rich strand of human telomeric DNA at 1.6 A resolution. These structures define the atomic contacts that underlie GO:0061730 and provide templates for mutational analysis.
Biochemical binding assays
Direct binding of proteins to the C-rich strand of human telomeric DNA and to the RNA template of human telomerase has been demonstrated for the first KH domain of human PCBP2. Such assays establish specificity of the interaction and are the standard biochemical test for annotation to GO:0061730.
Telomere replication and CST functional assays
Functional dissection of C-strand synthesis uses perturbation of POT1 and CST-Pol-alpha/primase components, because POT1 recruits and regulates CST-Pol-alpha/primase at human telomeres and CST-polymerase alpha-primase solves a second telomere end-replication problem. These experiments connect binding activity to telomere maintenance outcomes.
Comparative and evolutionary approaches
Comparative analysis of telomere structure across eukaryotes helps interpret species-specific differences in telomere-associated proteins. Identification of a C-rich telomeric DNA-binding protein in Eimeria tenella illustrates how such comparative work extends GO:0061730 beyond model organisms.
How CRISPR Can Be Used to Study GO:0061730 C-rich strand telomeric DNA binding
Knockout
CRISPR knockout of PCBP2, POT1 or CST subunits can test whether C-rich strand telomeric DNA binding and downstream C-strand synthesis are required for telomere maintenance. Because POT1 recruits and regulates CST-Pol-alpha/primase at human telomeres and CST-polymerase alpha-primase solves a second telomere end-replication problem, knockout models provide a direct way to connect loss of function to telomere phenotypes.
Point Mutation
Point mutations in KH domain residues of PCBP2 can be introduced to test which atomic contacts are required for binding the C-rich strand of human telomeric DNA, guided by the 1.7 A and 1.6 A structures. Such models separate binding activity from other functions of the protein.
Knock-in
Knock-in of tagged or disease-relevant variants of POT1 and CST components allows tracking of protein localization and recruitment at telomeres, building on evidence that POT1 recruits and regulates CST-Pol-alpha/primase. Knock-in of species-specific C-rich strand-binding proteins can also test comparative hypotheses.
Overexpression
Overexpression of candidate C-rich strand-binding proteins, such as PCBP2 domains or the Eimeria tenella telomeric DNA-binding protein, enables binding assays and gain-of-function telomere studies. Overexpression combined with telomere length measurement can reveal effects on telomere elongation pathways.
How EDITGENE Supports C-rich strand telomeric DNA binding Research
Researchers studying C-rich strand telomeric DNA binding-related genes often need to determine whether a candidate gene is causally involved in telomere maintenance or is merely correlated with a telomere phenotype. Establishing causality typically requires controlled perturbation of the candidate gene followed by functional readouts such as binding assays, telomere length measurement and replication assays.
Contact EDITGENE today to design your custom CRISPR model for C-rich strand telomeric DNA binding research.
Frequently Asked Questions About C-rich strand telomeric DNA binding
What is GO:0061730?
GO:0061730 is the Gene Ontology molecular_function term C-rich strand telomeric DNA binding, defined as binding to C-rich, single-stranded, telomere-associated DNA.
What does C-rich strand telomeric DNA binding mean in simple terms?
It means a protein physically attaches to the cytosine-rich single strand found at chromosome ends.
What genes are involved in C-rich strand telomeric DNA binding?
PCBP2 is the best-characterized gene, and POT1, CTC1, STN1, TEN1, POLA1, PRIM1 and PRIM2 are functionally connected through telomere C-strand synthesis.
Which protein domains bind the C-rich strand of telomeric DNA?
KH domains of human PCBP2 bind the C-rich strand of human telomeric DNA, as shown by structures at 1.7 A and 1.6 A resolution.
Is C-rich strand telomeric DNA binding found outside humans?
Yes, a telomeric DNA-binding protein recognizing the C-rich strand has been identified in Eimeria tenella.
How is C-rich strand telomeric DNA binding linked to telomere replication?
The C-rich strand is the template for C-strand synthesis, and POT1 recruits and regulates CST-Pol-alpha/primase at human telomeres.
What is the role of POT1 in C-strand biology?
POT1 recruits and regulates CST-Pol-alpha/primase at human telomeres.
What is the second telomere end-replication problem?
It is the requirement to complete C-strand synthesis after G-strand extension, solved by CST-polymerase alpha-primase.
How can I study C-rich strand telomeric DNA binding with CRISPR?
Knockout, point-mutation, knock-in and overexpression models can be combined with binding assays and telomere length measurement.
Why does C-rich strand telomeric DNA binding matter for disease?
Because defects in C-strand synthesis and telomere maintenance affect genome stability and are relevant to cancer and telomere-related disorders.
Conclusion
GO:0061730, C-rich strand telomeric DNA binding, captures a specific and structurally defined nucleic-acid-binding activity at chromosome ends. The KH domains of human PCBP2 provide the clearest atomic model for this activity, while POT1 and the CST-Pol-alpha/primase complex connect C-rich strand handling to telomere end replication. Comparative work shows that this activity is present beyond humans, and telomere elongation pathway studies place it within a broader regulatory network. Together, these findings make GO:0061730 a useful annotation for researchers studying telomere maintenance and genome stability.
References
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- 2. Takai H et al.. 2024. CST-polymerase α-primase solves a second telomere end-replication problem.. Nature 627(8004):664-670 PMID: 38418884
- 3. Mansoubi S et al.. 2024. Comparison of Telomere Structure in Eukaryotes.. Arch Razi Inst 79(6):1365-1374 PMID: 40606259
- 4. Arora R et al.. 2015. Telomere elongation chooses TERRA ALTernatives.. RNA Biol 12(9):938-41 PMID: 26158306
- 5. Du Z et al.. 2004. Specific recognition of the C-rich strand of human telomeric DNA and the RNA template of human telomerase by the first KH domain of human poly(C)-binding protein-2.. J Biol Chem 279(46):48126-34 PMID: 15331611
- 6. Zhao N et al.. 2014. Identification of a telomeric DNA-binding protein in Eimeria tenella.. Biochem Biophys Res Commun 451(4):599-602 PMID: 25128826
- 7. Du Z et al.. 2005. Crystal structure of the first KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.7 A.. J Biol Chem 280(46):38823-30 PMID: 16186123
- 8. Fenn S et al.. 2007. Crystal structure of the third KH domain of human poly(C)-binding protein-2 in complex with a C-rich strand of human telomeric DNA at 1.6 A resolution.. Nucleic Acids Res 35(8):2651-60 PMID: 17426136
