Single-stranded Telomeric DNA Research Articles

POT1 deficiency alters telomere length and telomere-associated gene expression in human gastric cancer cells

Telomeres are the end structures of linear chromosomes in eukaryotic cells. The integrity of a telomere is essential for the overall stability of the chromosome. The human protection of telomeres 1 (hPOT1) protein, a single-stranded telomeric DNA binding protein, plays an important role in telomere protection and telomere length regulation. Here, we show that the loss of hPOT1 by RNA interference in BGC823 (poorly differentiated human gastric adenocarcinoma) cells leads to an increase in multinucleated giant cells, a decrease in cell proliferation and colony formation, induction of senescence and apoptosis, shortened telomere length, upregulation of the TRF1 gene and downregulation of the TRF2, tankyrase1 and hTERT genes. These results suggest that the loss of hPOT1 results in a decrease in the viability of BGC823 cells; hPOT1 regulates telomere length positively and has an influence on the expression of other telomere-associated genes in the cells.

Tobacco GTBP1, a Homolog of Human Heterogeneous Nuclear Ribonucleoprotein, Protects Telomeres from Aberrant Homologous Recombination

Telomeres are nucleoprotein complexes essential for the integrity of eukaryotic chromosomes. Cellular roles of single-stranded telomeric DNA binding proteins have been extensively described in yeast and animals, but our knowledge about plant single-strand telomeric factors is rudimentary. Here, we investigated Nicotiana tabacum G-strand-specific single-stranded telomere binding proteins (GTBPs), homologs of a human heterogeneous nuclear ribonucleoprotein. GTBPs bound specifically to the plant single-stranded (TTTAGGG)(4) telomeric repeat element in vitro and were associated with telomeric sequences in tobacco BY-2 suspension cells. Transgenic plants (35S:RNAi-GTBP1), in which GTBP1 was suppressed, exhibited severe developmental anomalies. In addition, the chromosomes of 35S:RNAi-GTBP1 cells displayed elongated telomeres, frequent formation of extrachromosomal telomeric circles, and numerous abnormal anaphase bridges, indicating that GTBP1 knockdown tobacco plants experienced genome instability. GTBP1 inhibited strand invasion, an initial step in interchromosomal homologous recombination. We propose that GTBP1 plays a critical role in telomere structure and function by preventing aberrant interchromosomal telomeric homologous recombination in tobacco.

Telomeric DNA-binding activities of heterogeneous nuclear ribonucleoprotein A3 in vitro and in vivo

Telomeres are dynamic DNA–protein complexes that protect the ends of linear chromosome. Telomere-binding proteins play crucial role in the maintenance of telomeres. HnRNP A3 has been shown recently to bind specifically to single-stranded telomeric DNA in vitro, although its in vivo telomere function remains unknown. In this study, the DNA-binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. The minimal sequence for hnRNP A3 binding to DNA was determined as an undecamer with the following consensus sequence 5′-[T/C]AG[G/T]NN[T/C]AG[G/T]N-3′. Confocal microscopy and chromatin-immunoprecipitation (ChIP) analyses showed that hnRNP A3 is associated with telomere in vivo. Knocking-down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation. In contrast, overexpression of hnRNP A3 resulted in the production of steady-state short telomeres in OECM1 cells. These results suggest that hnRNP A3 is associated with telomere in vivo and acts as a negative regulator of telomere length maintenance.

Protection of Telomeres 1 Is Required for Telomere Integrity in the Moss Physcomitrella patens

In vertebrates, the single-stranded telomeric DNA binding protein Protection of Telomeres 1 (POT1) shields chromosome ends and prevents them from eliciting a DNA damage response. By contrast, Arabidopsis thaliana encodes two divergent full-length POT1 paralogs that do not exhibit telomeric DNA binding in vitro and have evolved to mediate telomerase regulation instead of chromosome end protection. To further investigate the role of POT1 in plants, we established the moss Physcomitrella patens as a new model for telomere biology and a counterpoint to Arabidopsis. The sequence and architecture of the telomere tract is similar in P. patens and Arabidopsis, but P. patens harbors only a single-copy POT1 gene. Unlike At POT1 proteins, Pp POT1 efficiently bound single-stranded telomeric DNA in vitro. Deletion of the P. patens POT1 gene resulted in the rapid onset of severe developmental defects and sterility. Although telomerase activity levels were unperturbed, telomeres were substantially shortened, harbored extended G-overhangs, and engaged in end-to-end fusions. We conclude that the telomere capping function of POT1 is conserved in early diverging land plants but is subsequently lost in Arabidopsis.

Purification and characterization of Stn1p, a single-stranded telomeric DNA binding protein

In Saccharomyces cerevisiae, Stn1p and Ten1p are required for telomere maintenance. These two proteins and another telomeric single-stranded DNA binding protein, Cdc13p, have been proposed to form a complex to control telomere integrity. In this work, we purified the recombinant Stn1p in Escherichia coli and found that the purified protein could specifically interact with single-stranded telomeric DNA in vitro. Co-fractionation of co-overexpressed Stn1p and Ten1p in insect cells revealed their stable association. A Stn1p/Ten1p binary complex was reconstituted with purified recombinant proteins in vitro. These results indicated that Stn1p and Ten1p interact with each other directly, which is important in telomere length regulation and end protection.

Telomerase and Chromosome End Protection In Vivo: the TPP1 Connection

In an article published in this issue of Developmental Cell, Maria Blasco's group shows that the telomere end-binding protein TPP1 is involved in both end protection and telomerase regulation in vivo. Importantly, they highlight the relevance of telomerase activity in highly proliferative tissues and in reprogramming of cells to induced pluripotency (iPS).

DNA‐binding properties and telomere function of hnRNP A3

Human telomeric DNA consists of hundreds to thousands of tandem repeat hexanucleotides (TTAGGG) and is bound by telomere‐associated proteins to form specialized structure that maintains the integrity of chromosome. These telomere‐associated proteins serve as structural and/or regulatory components for telomere functions. Dysfunctional telomeres are known to trigger growth arrest, senescence, and apoptosis. Previously, we have identified hnRNP A3, a poorly understood member of the hnRNPs A/B, as a novel telomere‐associated protein that binds specifically to single‐stranded telomeric DNA. In this study, the DNA‐binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. Here, we showed that the minimal binding sequence (MBS) for hnRNP A3 is 5′‐TAGGGTTAGGG‐3′, and the position of MBS in the single‐strand DNA had little or no effect on the binding by hnRNP A3. The consensus binding sequence in the 11‐mer MBS was characterized as 5′‐[T/C]AG[G/T]NN[T/C]AG[G/T]N‐3′. Furthermore, confocal microscopy and chromatin‐immunoprecipitation (ChIP) studies revealed that hnRNP A3 are associated with telomere in vivo. Knocking‐ down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation capacity. However, overexpression of hnRNPA3 resulted in the production of steady‐state short telomeres in several cell lines. These findings indicate that hnRNP A3 can participate in telomere function and acts as a negative regulator of telomere length maintenance. This work was supported by research grant CMRPD 170541.

Telomere Protection by TPP1 Is Mediated by POT1a and POT1b

Mammalian telomeres are protected by the shelterin complex, which contains single-stranded telomeric DNA binding proteins (POT1a and POT1b in rodents, POT1 in other mammals). Mouse POT1a prevents the activation of the ATR kinase and contributes to the repression of the nonhomologous end-joining pathway (NHEJ) at newly replicated telomeres. POT1b represses unscheduled resection of the 5'-ended telomeric DNA strand, resulting in long 3' overhangs in POT1b KO cells. Both POT1 proteins bind TPP1, forming heterodimers that bind to other proteins in shelterin. Short hairpin RNA (shRNA)-mediated depletion had previously demonstrated that TPP1 contributes to the normal function of POT1a and POT1b. However, these experiments did not establish whether TPP1 has additional functions in shelterin. Here we report on the phenotypes of the conditional deletion of TPP1 from mouse embryo fibroblasts. TPP1 deletion resulted in the release of POT1a and POT1b from chromatin and loss of these proteins from telomeres, indicating that TPP1 is required for the telomere association of POT1a and POT1b but not for their stability. The telomere dysfunction phenotypes associated with deletion of TPP1 were identical to those of POT1a/POT1b DKO cells. No additional telomere dysfunction phenotypes were observed, establishing that the main role of TPP1 is to allow POT1a and POT1b to protect chromosome ends.

Yeast telomerase subunit Est1p has guanine quadruplex–promoting activity that is required for telomere elongation

Telomeres are eukaryotic protein-DNA complexes found at the ends of linear chromosomes that are essential for maintaining genome integrity and are implicated in cellular aging and cancer. The guanine (G)-rich strand of telomeric DNA, usually elongated by the telomerase reverse transcriptase, can form a higher-order structure known as a G-quadruplex in vitro and in vivo. Several factors that promote or resolve G-quadruplexes have been identified, but the functional importance of these structures for telomere maintenance is not well understood. Here we show that the yeast telomerase subunit Est1p, known to be involved in telomerase recruitment to telomeres, can convert single-stranded telomeric G-rich DNA into a G-quadruplex structure in vitro in a Mg(2+)-dependent manner. Cells carrying Est1p mutants deficient in G-quadruplex formation in vitro showed gradual telomere shortening and cellular senescence, indicating a positive regulatory role for G-quadruplex in the maintenance of telomere length.

1P133 1D1340 テロメア1本鎖DNA結合蛋白質とテロメア1本鎖DNAの相互作用の熱力学的解析(核酸-相互作用・複合体,口頭発表,第48回日本生物物理学会年会)

1P133 1D1340 テロメア1本鎖DNA結合蛋白質とテロメア1本鎖DNAの相互作用の熱力学的解析(核酸-相互作用・複合体,口頭発表,第48回日本生物物理学会年会)

In Vivo Stoichiometry of Shelterin Components

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1.POT1 heterodimer was present 50-100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1.POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were approximately 10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1 x POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.

The N-terminus of hTERT contains a DNA-binding domain and is required for telomerase activity and cellular immortalization

Telomerase defers the onset of telomere damage-induced signaling and cellular senescence by adding DNA onto chromosome ends. The ability of telomerase to elongate single-stranded telomeric DNA depends on the reverse transcriptase domain of TERT, and also relies on protein:DNA contacts outside the active site. We purified the N-terminus of human TERT (hTEN) from Escherichia coli, and found that it binds DNA with a preference for telomeric sequence of a certain length and register. hTEN interacted with the C-terminus of hTERT in trans to reconstitute enzymatic activity in vitro. Mutational analysis of hTEN revealed that amino acids Y18 and Q169 were required for telomerase activity in vitro, but not for the interaction with telomere DNA or the C-terminus. These mutants did not reconstitute telomerase activity in cells, maintain telomere length, or extend cellular lifespan. In addition, we found that T116/T117/S118, while dispensable in vitro, were required for cellular immortalization. Thus, the interactions of hTEN with telomere DNA and the C-terminus of hTERT are functionally separable from the role of hTEN in telomere elongation activity in vitro and in vivo, suggesting other roles for the protein and nucleic acid interactions of hTEN within, and possibly outside, the telomerase catalytic core.

TEN1Is Essential forCDC13-Mediated Telomere Capping

Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLalpha complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

Telomeric D-loops Containing 8-Oxo-2′-deoxyguanosine Are Preferred Substrates for Werner and Bloom Syndrome Helicases and Are Bound by POT1

8-Oxo-2'-deoxyguanosine (8-oxodG) is one of the most important oxidative DNA lesions, and G-rich telomeric DNA is especially susceptible to oxidative DNA damage. RecQ helicases WRN and BLM and telomere-binding protein POT1 are thought to play roles in telomere maintenance. This study examines the ability of WRN, BLM, and RecQ5 to unwind and POT1 to bind telomeric D-loops containing 8-oxodG. The results demonstrate that WRN and BLM preferentially unwind telomeric D-loops containing 8-oxodG and that POT1 binds with higher affinity to telomeric D-loops with 8-oxodG but shows no preference for telomeric single-stranded DNA with 8-oxodG. We speculate that telomeric D-loops with 8-oxodG may have a greater tendency to form G-quadruplex DNA structures than telomeric DNA lacking 8-oxodG.

POT1 Association with TRF2 Regulates Telomere Length

Deleting the OB folds encoding the telomeric single-stranded DNA (ssDNA)-binding activity of the human telomeric protein POT1 induces significant telomere elongation, suggesting that at least one critical aspect of the regulation of telomere length is disrupted by this POT1(DeltaOB) mutant protein. POT1 is known to associate with two proteins through the protein interaction domain retained in POT1(DeltaOB)-the telomeric double-stranded DNA-binding protein TRF2 and the telomere-associated protein TPP1. We report that introducing a mutation that reduces association of POT1 with TRF2, but not a mutation that reduces the association with TPP1, abrogates the ability of POT1(DeltaOB) to promote telomere elongation. Mechanistically, expression of POT1(DeltaOB) reduced the association of TRF2 with POT1, RAP1, and TIN2; however, of these proteins, only ectopic expression of POT1 suppressed the telomere elongation induced by POT1(DeltaOB). Lastly, replacing endogenous POT1 with a full-length POT1 mutant defective in the association with TRF2 induced telomere elongation. Thus, we conclude that the association of POT1 with both ssDNA and TRF2 is critical for telomere length homeostasis.

OB Fold-containing Protein 1 (OBFC1), a Human Homolog of Yeast Stn1, Associates with TPP1 and Is Implicated in Telomere Length Regulation

The telosome/shelterin, a six-protein complex formed by TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, functions as the core of the telomere interactome, acting as the molecular platform for the assembly of higher order complexes and coordinating cross-talks between various protein subcomplexes. Within the telosome, there are two oligonucleotide- or oligosaccharide-binding (OB) fold-containing proteins, TPP1 and POT1. They can form heterodimers that bind to the telomeric single-stranded DNA, an activity that is central for telomere end capping and telomerase recruitment. Through proteomic analyses, we found that in addition to POT1, TPP1 can associate with another OB fold-containing protein, OBFC1/AAF44. The yeast homolog of OBFC1 is Stn1, which plays a critical role in telomere regulation. We show here that OBFC1/AAF44 can localize to telomeres in human cells and bind to telomeric single-stranded DNA in vitro. Furthermore, overexpression of an OBFC1 mutant resulted in elongated telomeres in human cells, implicating OBFC1/AAF4 in telomere length regulation. Taken together, our studies suggest that OBFC1/AAF44 represents a new player in the telomere interactome for telomere maintenance.

POT1‐independent single‐strand telomeric DNA binding activities in Brassicaceae

Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single-strand protrusion, termed the G-overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plant Arabidopsis thaliana encodes two divergent POT1-like proteins. Here we show that the single-strand telomeric DNA binding activity present in A. thaliana nuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, recombinant POT1a and POT1b proteins from A. thaliana, and from two additional Brassicaceae species, Arabidopsis lyrata and Brassica oleracea (cauliflower), fail to bind single-strand telomeric DNA in vitro under the conditions tested. Finally, although we detected four single-strand telomeric DNA binding activities in nuclear extracts from B. oleracea, partial purification and DNA cross-linking analysis of these complexes identified proteins that are smaller than the predicted sizes of BoPOT1a or BoPOT1b. Taken together, these data suggest that POT1 proteins are not the major single-strand telomeric DNA binding activities in A. thaliana and its close relatives, underscoring the remarkable functional divergence of POT1 proteins from plants and other eukaryotes.

Sua5p a single-stranded telomeric DNA-binding protein facilitates telomere replication

In budding yeast Saccharomyces cerevisiae, telomere length maintenance involves a complicated network as more than 280 telomere maintenance genes have been identified in the nonessential gene deletion mutant set. As a supplement, we identified additional 29 telomere maintenance genes, which were previously taken as essential genes. In this study, we report a novel function of Sua5p in telomere replication. Epistasis analysis and telomere sequencing show that sua5Delta cells display progressively shortened telomeres at early passages, and Sua5 functions downstream telomerase recruitment. Further, biochemical, structural and genetic studies show that Sua5p specifically binds single-stranded telomeric (ssTG) DNA in vitro through a distinct DNA-binding region on its surface, and the DNA-binding ability is essential for its telomere function. Thus, Sua5p represents a novel ssTG DNA-binding protein and positively regulates the telomere length in vivo.

Developmental studies ofXenopusshelterin complexes: the message to reset telomere length is already present in the egg

The 6-protein complex shelterin protects the telomeres of human chromosomes. The recent discovery that telomeres are important for epigenetic gene regulation and vertebrate embryonic development calls for the establishment of model organisms to study shelterin and telomere function under normal developmental conditions. Here, we report the sequences of the shelterin-encoding genes in Xenopus laevis and its close relation Xenopus tropicalis. In vitro expression and biochemical characterization of the Xenopus shelterin proteins TRF1, TRF2, POT1, TIN2, RAP1, TPP1, and the shelterin accessory factor PINX1 indicate that all main functions of their human orthologs are conserved in Xenopus. The XlTRF1 and XtTRF1 proteins bind double-stranded telomeric DNA sequence specifically and interact with XlTIN2 and XtTIN2, respectively. Similarly, the XlTRF2 and XtTRF2 proteins bind double-stranded telomeric DNA and interact with XlRAP1 and XtRAP1, respectively, whereas the XlPOT1 and XtPOT1 proteins bind single-stranded telomeric DNA. Real-time PCR further reveals the gene expression profiles for telomerase and the shelterin genes during embryogenesis. Notably, the composition of shelterin and the formation of its subcomplexes appear to be temporally regulated during embryonic development. Moreover, unexpectedly high telomerase and shelterin gene expression during early embryogenesis may reflect a telomere length-resetting mechanism, similar to that reported for induced pluripotent stem cells and for animals cloned through somatic nuclear transfer.

Insights into the Dynamics of Specific Telomeric Single-Stranded DNA Recognition by Pot1pN

The N-terminal oligonucleotide/oligosaccharide-binding fold domain of the Schizosaccharomyces pombe protection of telomeres 1 (Pot1) protein, Pot1pN (residues 1-187 of full-length Pot1), specifically recognizes telomeric single-stranded DNA (ssDNA) via a complex series of molecular interactions that are punctuated by unusual internucleotide hydrogen bonds. While the structure of ssDNA-bound Pot1pN provides an initial model for understanding how the Pot1pN-ssDNA complex is assembled and how specific nucleotide recognition occurs, further refinement requires knowledge of the ssDNA-free state of Pot1pN and the dynamic changes that accompany the binding of ssDNA. Using NMR strategies, we found that ssDNA-free Pot1pN adopts a similar overall protein backbone topology as ssDNA-bound Pot1pN does. Although the backbone structure remained relatively unchanged, we observed unexpected differential dynamic changes within the ssDNA-binding pockets of Pot1pN upon binding of cognate ssDNA. These studies support a model in which conformational selection and induced fit play important roles in the recognition of ssDNA by Pot1pN. Furthermore, the studies presented here provide a more comprehensive understanding of how specific nucleotide recognition is achieved by the telomere-end protection family of essential proteins.