Single-stranded Telomeric DNA Research Articles

High-Resolution Insight into G-Overhang Architecture

NMR and fluorescence spectroscopy were used to address the effect of intracellular molecular crowding and related hydration on a model telomeric G-quadruplex (G4) DNA structure (d(AG(3)(TTAGGG)(3))). d(AG(3)(TTAGGG)(3)) prevalently adopted the hybrid-1 conformation in vivo, ex vivo, and in dilute potassium-based solution, while it formed the parallel propeller fold in water-depleted potassium-based solution, a commonly used model system for studying intracellular molecular crowding. The dilute potassium-based solution appeared to imitate the properties of the cellular environment required for d(AG(3)(TTAGGG)(3)) folding under in vivo and ex vivo conditions. High-resolution NMR investigations of site-specifically (15)N-labeled G4 units in native-like single-stranded telomeric DNA revealed that the 3'-terminal and internal G4 unit predominantly coexist in 2-tetrad antiparallel basket and hybrid-2 structures that are arranged in "beads-on-a-string"-like fashion. Our data provide the first high-resolution insight into the telomeric G-overhang architecture under essentially physiological conditions and identify the 2-tetrad antiparallel basket and hybrid-2 topologies as the structural targets for the development of telomere-specific G4 ligands.

Caenorhabditis elegans POT-1 and POT-2 Repress Telomere Maintenance Pathways

Telomeres are composed of simple tandem DNA repeats that protect the ends of linear chromosomes from replicative erosion or inappropriate DNA damage response mechanisms. The mammalian Protection Of Telomeres (POT1) protein interacts with single-stranded telomeric DNA and can exert positive and negative effects on telomere length. Of four distinct POT1 homologs in the roundworm Caenorhabditis elegans, deficiency for POT-1 or POT-2 resulted in progressive telomere elongation that occurred because both proteins negatively regulate telomerase. We created a POT-1::mCherry fusion protein that forms discrete foci at C. elegans telomeres, independent of POT-2, allowing for live analysis of telomere dynamics. Transgenic pot-1::mCherry repressed telomerase in pot-1 mutants. Animals deficient for pot-1, but not pot-2, displayed mildly enhanced telomere erosion rates in the absence of the telomerase reverse transcriptase, trt-1. However, trt-1; pot-1 double mutants exhibited delayed senescence in comparison to trt-1 animals, and senescence was further delayed in trt-1; pot-2; pot-1 triple mutants, some of which survived robustly in the absence of telomerase. Our results indicate that POT-1 and POT-2 play independent roles in suppressing a telomerase-independent telomere maintenance pathway but may function together to repress telomerase.

Characterization of the intramolecular G-quadruplex promoting activity of Est1

In the budding yeast Saccharomyces cerevisiae, telomeric DNA includes TG1–3/C1–3A double-stranded DNA and a protruding G-rich overhang. Our previous studies revealed that the telomerase regulatory subunit Est1 promotes telomeric single-stranded DNA to form intermolecular G-quadruplex in vitro, and this activity is required for telomere replication and protection in vivo. In this study, we further characterized the G-quadruplex promoting activity of Est1. Here we report that Est1 is able to promote the single-stranded oligonucleotide of (TGTGTGGG)4, which mimics the natural telomeric DNA, to form intramolecular G-quadruplex. Therefore, it remains possible that the intramolecular G-quadruplex promoting activity of Est1 is biologically relevant in telomere replication in vivo.

The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity

Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase1–3. Shelterin includes the heterodimeric POT1-TPP1 protein, which binds the telomeric single-stranded DNA tail4–9. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event)9–14. Determining the mechanisms of these activities has been difficult, especially because genetic perturbations also tend to affect the essential chromosome end-protection function of TPP115–17. Here we identify separation-of-function mutants of TPP1 that retain full telomere-capping function in vitro and in vivo, yet are defective in binding telomerase. The seven separation-of-function mutations map to a patch of amino acids on the surface of TPP1, the TEL patch, that both recruits telomerase to telomeres and promotes high-processivity DNA synthesis, indicating that these two activities are manifestations of the same molecular interaction. Given that the interaction between telomerase and TPP1 is required for telomerase function in vivo, the TEL patch of TPP1 provides a new target for anti-cancer drug development.

Chromosome end protection by blunt-ended telomeres

Single-stranded telomeric DNA protrusions are considered to be evolutionarily conserved structural elements essential for chromosome end protection. Their formation at telomeres replicated by the leading strand mechanism is thought to involve poorly understood post-replicative processing of blunt ends. Unexpectedly, we found that angiosperm plants contain blunt-ended and short (1- to 3-nucleotide) G-overhang-containing telomeres that are stably retained in post-mitotic tissues, revealing a novel mechanism of chromosome end protection. The integrity of blunt-ended telomeres depends on the Ku70/80 heterodimer but not on another telomere capping protein, STN1. Curiously, Ku-depleted telomeres are fully functional. They are resected by exonuclease 1, promoting intrachromatid recombination, which may facilitate formation of an alternative capping structure. These data challenge the view that telomeres require ssDNA protrusions for forming a functional capping structure and demonstrate flexibility in solutions to the chromosome end protection problem.

Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit

Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA–DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5′-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5′-end of the RNA template. The upstream DNA–TBE interaction may also function as an anchor for the subsequent realignment of the 3′-end of the DNA with the 3′-end of the template to enable initiation of synthesis of a new telomeric repeat.

Specific binding of telomeric G-quadruplexes by hydrosoluble perylene derivatives inhibits repeat addition processivity of human telomerase

Telomerase is responsible for the immortal phenotype of cancer cells and telomerase inhibition may specifically target cancer cell proliferation. Ligands able to selectively bind to G-quadruplex telomeric DNA have been considered as telomerase inhibitors but their mechanisms of action have often been deduced from a non-quantitative telomerase activity assay (TRAP assay) that involves a PCR step and that does not provide insight on the mechanism of inhibition. Furthermore, quadruplex ligands have also been shown to exert their effects by affecting association of telomere binding proteins with telomeres. Here, we use quantitative direct telomerase activity assays to evaluate the strength and mechanism of action of hydrosoluble perylene diimides (HPDIs). HPDIs contain a perylene moiety and different numbers of positively charged side chains. Side chain features vary with regard to number and distances of the charges. IC50 values of HPDIs were in the low micromolar (0.5–5 μM) range depending on the number and features of the side chains. HPDIs having four side chains emerged as the best compounds of this series. Analysis of primer elongation products demonstrated that at low HPDI concentrations, telomerase inhibition involved formation of telomeric G-quadruplex structures, which inhibited further elongation by telomerase. At high HPDI concentrations, telomerase inhibition occurred independently of G-quadruplex formation of the substrate. The mechanism of action of HPDIs and their specific binding to G-quadruplex DNA was supported by PAGE analysis, CD spectroscopy and ESI-MS. Finally, competition Telospot experiments with duplex DNA indicated specific binding of HPDIs to the single-stranded telomeric substrates over double stranded DNA, a result supported by competitive ESI-MS. Altogether, our results indicate that HPDIs act by stabilizing G-quadruplex structures in single-stranded telomeric DNA, which in turn prevents repeat addition processivity of telomerase.

The telomeric Cdc13 protein interacts directly with the telomerase subunit Est1 to bring it to telomeric DNA ends in vitro

In Saccharomyces cerevisiae, a Cdc13-Est1 interaction is proposed to mediate recruitment of telomerase to DNA ends. Here we provide unique in vitro evidence for this model by demonstrating a direct interaction between purified Cdc13 and Est1. The Cdc13-Est1 interaction is specific and requires the in vivo defined Cdc13 recruitment domain. Moreover, in the absence of this interaction, Est1 is excluded from telomeric single-stranded (ss)DNA. The apparent association constand (K(d)) between Est1 and a Cdc13-telomeric ssDNA complex was ∼250 nM. In G2 phase cells, where telomerase is active, Cdc13 and Est1 were sufficiently abundant (∼420 and ∼110 copies per cell, respectively) to support complex formation. Interaction between Cdc13 and Est1 was unchanged by three telomerase-deficient mutations, Cdc13(E252K) (cdc13-2), Est1(K444E) (est1-60), and Cdc13(S249,255D), indicating that their telomerase null phenotypes are not due to loss of the Cdc13-Est1 interaction. These data recapitulate in vitro the first step in telomerase recruitment to telomeric ssDNA and suggest that this step is necessary to recruit telomerase to DNA ends.

DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

Eukaryotic chromosome ends are protected from illicit DNA joining by protein–DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.

Gold nanorod-based FRET assay for selection of G-quadruplex-binding ligands

Single-stranded telomeric DNA tends to fold into G-quadruplexes made of a stack of G-quartets. Ligands that target G-quadruplex DNA have received considerable attention because of their potential anticancer activity. In this paper, we designed a fluorescence resonance energy transfer system to identify drugs that form G-quadruplexes with human telomeric DNA in a homogeneous medium using gold nanorods (GNRs) as fluorescence quencher. Fluorescein-tagged human telomeric DNA (F-GDNA) adsorbed on the surface of GNRs, which can quench the fluorescence of F-GDNA. In the presence of G-quadruplex-binding drugs, the formation of G-quadruplex further quenched the fluorescence of F-GDNA, which can be utilized to select G-quadruplex-binding ligands. Thirteen natural drugs were studied by this proposed method. G-quadruplex formation induced by drugs was investigated by circular dichroism measurements. Results of research indicated that it offers a simple, sensitive and effective method to identify ligands with potential anticancer activity.

Silencing of the hPOT1 gene by RNA inference promotes apoptosis and inhibits proliferation and aggressive phenotype of gastric cancer cells, likely through up-regulating PinX1 expression

BackgroundThe human protection of telomeres 1 (hPOT1) protein, a single-strand telomeric DNA binding protein, plays an important role in telomere protection and telomere length regulation. However, its effect on invasion...

Sequence-specific binding to telomeric DNA is not a conserved property of the Cdc13 DNA binding domain.

In the budding yeast Saccharomyces cerevisiae, chromosome end protection is provided by a heterotrimeric complex composed of Cdc13 in association with the RPA-like proteins Stn1 and Ten1. We report here that the high affinity and specificity of the S. cerevisiae Cdc13 DNA binding domain for single-stranded telomeric DNA are not widely shared by other fungal Cdc13 proteins, suggesting that restriction of this complex to telomeres may be limited to the Saccharomyces clade. We propose that the evolutionarily conserved task of Stn1 and Ten1 (and their associated large subunit) is a genome-wide role in DNA replication rather than a telomere-dedicated activity.

Human telomerase model shows the role of the TEN domain in advancing the double helix for the next polymerization step.

Telomerases constitute a group of specialized ribonucleoprotein enzymes that remediate chromosomal shrinkage resulting from the "end-replication" problem. Defects in telomere length regulation are associated with several diseases as well as with aging and cancer. Despite significant progress in understanding the roles of telomerase, the complete structure of the human telomerase enzyme bound to telomeric DNA remains elusive, with the detailed molecular mechanism of telomere elongation still unknown. By application of computational methods for distant homology detection, comparative modeling, and molecular docking, guided by available experimental data, we have generated a three-dimensional structural model of a partial telomerase elongation complex composed of three essential protein domains bound to a single-stranded telomeric DNA sequence in the form of a heteroduplex with the template region of the human RNA subunit, TER. This model provides a structural mechanism for the processivity of telomerase and offers new insights into elongation. We conclude that the RNADNA heteroduplex is constrained by the telomerase TEN domain through repeated extension cycles and that the TEN domain controls the process by moving the template ahead one base at a time by translation and rotation of the double helix. The RNA region directly following the template can bind complementarily to the newly synthesized telomeric DNA, while the template itself is reused in the telomerase active site during the next reaction cycle. This first structural model of the human telomerase enzyme provides many details of the molecular mechanism of telomerase and immediately provides an important target for rational drug design.

Put on your thinking cap: G-quadruplexes, helicases, and telomeres

Understanding how G-quadruplex (G4) DNA structures that form in G-rich tracts of the genome affect chromosomal stability and processes such as copying the genetic information (DNA replication) or decoding the information (RNA transcription) has posed a significant challenge to researchers in the field. Although historically there has been some controversy over the existence of G4 DNA structures in vivo, emerging evidence suggests that they are indeed likely to form and have cellular consequences. In a recent study, Smith et al. investigated a role of G4 DNA in telomere capping [1], i.e., the adaptation of a nucleoprotein structure that prevents the chromosomal DNA ends from being recognized as DNA breaks and protects them from becoming degraded or fused. Telomere capping is a fairly complex process since a number of proteins have been shown to bind telomeric single-stranded or double-stranded DNA at the chromosome end. Moreover, the ability of telomeric DNA to form a variety of conformations including t-loops and G-quadruplexes adds to the complexity of how competing proteins and DNA structures influence the structural topology and metabolism of chromosome ends [2]. Using genetic and pharmacological approaches, Smith et al. showed that under conditions that stabilize G4 DNA structures, which form from a guanine-rich telomeric ssDNA exposed in a yeast temperature sensitive cdc13-1 mutant, telomere capping is in turn enhanced and phenotypes associated with capping defects are suppressed [1]. Conversely, telomere uncapping occurs under conditions that dissuade the formation of telomeric G4 DNA in the cdc13-1 mutant. The authors proposed a model in which G4 DNA structure enables G4 DNA binding proteins to further stabilize the telomere end by binding to G-quadruplex DNA, thereby preventing 5′ to 3′ exonucleolytic resection when the normal protein that blocks telomeric end processing is defective. Rad53-mediated checkpoint activation is also dampened, permitting suppression of the growth defects characteristic of the cdc13-1 mutant at the nonpermissive temperature.

Rif1 Supports the Function of the CST Complex in Yeast Telomere Capping

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex.

TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA

Maintenance of telomeres requires both DNA replication and telomere ‘capping’ by shelterin. These two processes employ two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres1, 2, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to efficiently prevent RPA binding to telomeric ssDNA. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres3. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the reaccumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA, and POT1 act in concert to displace RPA from telomeric ssDNA following DNA replication, and promote telomere capping to preserve genomic integrity.

Est1 Protects Telomeres and Inhibits Subtelomeric Y′-Element Recombination

In the budding yeast Saccharomyces cerevisiae, the structure and function of telomeres are maintained by binding proteins, such as Cdc13-Stn1-Ten1 (CST), Yku, and the telomerase complex. Like CST and Yku, telomerase also plays a role in telomere protection or capping. Unlike CST and Yku, however, the underlying molecular mechanism of telomerase-mediated telomere protection remains unclear. In this study, we employed both the CDC13-EST1 fusion gene and the separation-of-function allele est1-D514A to elucidate that Est1 provided a telomere protection pathway that was independent of both the CST and Yku pathways. Est1's ability to convert single-stranded telomeric DNA into a G quadruplex was required for telomerase-mediated telomere protection function. Additionally, Est1 maintained the integrity of telomeres by suppressing the recombination of subtelomeric Y' elements. Our results demonstrate that one major functional role that Est1 brings to the telomerase complex is the capping or protection of telomeres.

Single Molecule Studies of Physiologically Relevant Telomeric Tails Reveal POT1 Mechanism for Promoting G-quadruplex Unfolding

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.

Single- and double-stranded DNA binding proteins act in concert to conserve a telomeric DNA core sequence

BackgroundTelomeres are protective cap structures at the ends of the linear eukaryotic chromosomes, which provide stability to the genome by shielding from degradation and chromosome fusions. The cap consists of telomere-specific proteins binding to the respective single- and double-stranded parts of the telomeric sequence. In addition to the nucleation of the chromatin structure the telomere-binding proteins are involved in the regulation of the telomere length. However, the telomeric sequences are highly diverged among yeast species. During the evolution this high rate of divergency presents a challenge for the sequence recognition of the telomere-binding proteins.ResultsWe found that the Saccharomyces castellii protein Rap1, a negative regulator of telomere length, binds a 12-mer minimal binding site (MBS) within the double-stranded telomeric DNA. The sequence specificity is dependent on the interaction with two 5 nucleotide motifs, having a 6 nucleotide centre-to-centre spacing. The isolated DNA-binding domain binds the same MBS and retains the same motif binding characteristics as the full-length Rap1 protein. However, it shows some deviations in the degree of sequence-specific dependence in some nucleotide positions. Intriguingly, the positions of most importance for the sequence-specific binding of the full-length Rap1 protein coincide with 3 of the 4 nucleotides utilized by the 3' overhang binding protein Cdc13. These nucleotides are very well conserved within the otherwise highly divergent telomeric sequences of yeasts.ConclusionsRap1 and Cdc13 are two very distinct types of DNA-binding proteins with highly separate functions. They interact with the double-stranded vs. the single-stranded telomeric DNA via significantly different types of DNA-binding domain structures. However, we show that they are dependent on coinciding nucleotide positions for their sequence-specific binding to telomeric sequences. Thus, we conclude that during the molecular evolution they act together to preserve a core sequence of the telomeric DNA.

Telomere protein complexes and interactions with telomerase in telomere maintenance

Telomeres are the termini of linear chromosomes. They are composed of DNA and DNA-binding proteins critical for maintaining chromosome integrity and cellular function. Telomere binding proteins regulate the structure and function of telomeres through the formation of different complexes with telomeric DNA. Double- and single-stranded telomeric DNA binding protein complexes have shared and unique functions that regulate telomere homeostasis. Recent studies have shown that telomerase interacts with several telomere-binding protein complexes including shelterin, CST, DNA-dependent protein kinase (DNA-PK) and MRN. The present review describes the recognised telomere-binding protein complexes, sub-complex exchanges and inter-complex molecular interactions. It also discusses the evidence suggesting that telomerase reverse transcriptase (TERT) switches between different complexes. Studies of the telomere protein inter-complex interactions and the switching of components between complexes provide insight into their fundamental roles of programming telomere length and configuration, and thus cell proliferative potential.