TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase

Telomere dysfunction may result in chromosomal abnormalities, DNA damage responses, and even cancer. Early studies in lower organisms have helped to establish the crucial role of telomerase and telomeric proteins in maintaining telomere length and protecting telomere ends. In Oxytricha nova, telomere G-overhangs are protected by the TEBP-alpha/beta heterodimer. Human telomeres contain duplex telomeric repeats with 3' single-stranded G-overhangs, and may fold into a t-loop structure that helps to shield them from being recognized as DNA breaks. Additionally, the TEBP-alpha homologue, POT1, which binds telomeric single-stranded DNA (ssDNA), associates with multiple telomeric proteins (for example, TPP1, TIN2, TRF1, TRF2 and RAP1) to form the six-protein telosome/shelterin and other subcomplexes. These telomeric protein complexes in turn interact with diverse pathways to form the telomere interactome for telomere maintenance. However, the mechanisms by which the POT1-containing telosome communicates with telomerase to regulate telomeres remain to be elucidated. Here we demonstrate that TPP1 is a putative mammalian homologue of TEBP-beta and contains a predicted amino-terminal oligonucleotide/oligosaccharide binding (OB) fold. TPP1-POT1 association enhanced POT1 affinity for telomeric ssDNA. In addition, the TPP1 OB fold, as well as POT1-TPP1 binding, seemed critical for POT1-mediated telomere-length control and telomere-end protection in human cells. Disruption of POT1-TPP1 interaction by dominant negative TPP1 expression or RNA interference (RNAi) resulted in telomere-length alteration and DNA damage responses. Furthermore, we offer evidence that TPP1 associates with the telomerase in a TPP1-OB-fold-dependent manner, providing a physical link between telomerase and the telosome/shelterin complex. Our findings highlight the critical role of TPP1 in telomere maintenance, and support a yin-yang model in which TPP1 and POT1 function as a unit to protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA.     

The human CST complex is a terminator of telomerase activity

The lengths of human telomeres, which protect chromosome ends from degradation and end fusions, are crucial determinants of cell lifespan. During embryogenesis and in cancer, the telomerase enzyme counteracts telomeric DNA shortening. As shown in cancer cells, human telomerase binds the shelterin component TPP1 at telomeres during the S phase of the cell cycle, and adds ~60 nucleotides in a single round of extension, after which telomerase is turned off by unknown mechanisms. Here we show that the human CST (CTC1, STN1 and TEN1) complex, previously implicated in telomere protection and DNA metabolism, inhibits telomerase activity through primer sequestration and physical interaction with the protection of telomeres 1 (POT1)–TPP1 telomerase processivity factor. CST competes with POT1–TPP1 for telomeric DNA, and CST–telomeric-DNA binding increases during late S/G2 phase only on telomerase action, coinciding with telomerase shut-off. Depletion of CST allows excessive telomerase activity, promoting telomere elongation. We propose that through binding of the telomerase-extended telomere, CST limits telomerase action at individual telomeres to approximately one binding and extension event per cell cycle. Our findings define the sequence of events that occur to first enable and then terminate telomerase-mediated telomere elongation.     

POT1 as a terminal transducer of TRF1 telomere length control

Human telomere maintenance is essential for the protection of chromosome ends, and changes in telomere length have been implicated in ageing and cancer. Human telomere length is regulated by the TTAGGG-repeat-binding protein TRF1 and its interacting partners tankyrase 1, TIN2 and PINX1 (refs 5-9). As the TRF1 complex binds to the duplex DNA of the telomere, it is unclear how it can affect telomerase, which acts on the single-stranded 3' telomeric overhang. Here we show that the TRF1 complex interacts with a single-stranded telomeric DNA-binding protein--protection of telomeres 1 (POT1)--and that human POT1 controls telomerase-mediated telomere elongation. The presence of POT1 on telomeres was diminished when the amount of single-stranded DNA was reduced. Furthermore, POT1 binding was regulated by the TRF1 complex in response to telomere length. A mutant form of POT1 lacking the DNA-binding domain abrogated TRF1-mediated control of telomere length, and induced rapid and extensive telomere elongation. We propose that the interaction between the TRF1 complex and POT1 affects the loading of POT1 on the single-stranded telomeric DNA, thus transmitting information about telomere length to the telomere terminus, where telomerase is regulated.     

The POT1-TPP1 telomere complex is a telomerase processivity factor

Telomeres were originally defined as chromosome caps that prevent the natural ends of linear chromosomes from undergoing deleterious degradation and fusion events. POT1 (protection of telomeres) protein binds the single-stranded G-rich DNA overhangs at human chromosome ends and suppresses unwanted DNA repair activities. TPP1 is a previously identified binding partner of POT1 that has been proposed to form part of a six-protein shelterin complex at telomeres. Here, the crystal structure of a domain of human TPP1 reveals an oligonucleotide/oligosaccharide-binding fold that is structurally similar to the beta-subunit of the telomere end-binding protein of a ciliated protozoan, suggesting that TPP1 is the missing beta-subunit of human POT1 protein. Telomeric DNA end-binding proteins have generally been found to inhibit rather than stimulate the action of the chromosome end-replicating enzyme, telomerase. In contrast, we find that TPP1 and POT1 form a complex with telomeric DNA that increases the activity and processivity of the human telomerase core enzyme. We propose that POT1-TPP1 switches from inhibiting telomerase access to the telomere, as a component of shelterin, to serving as a processivity factor for telomerase during telomere extension.     

DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA

Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions. The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals. Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation. Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown. Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S. pombe Pot1p complexed with ssDNA. The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding. The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA. Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups. The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus.     

Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1

When telomeres are rendered dysfunctional through replicative attrition of the telomeric DNA or by inhibition of shelterin, cells show the hallmarks of ataxia telangiectasia mutated (ATM) kinase signalling. In addition, dysfunctional telomeres might induce an ATM-independent pathway, such as ataxia telangiectasia and Rad3-related (ATR) kinase signalling, as indicated by the phosphorylation of the ATR target CHK1 in senescent cells and the response of ATM-deficient cells to telomere dysfunction. However, because telomere attrition is accompanied by secondary DNA damage, it has remained unclear whether there is an ATM-independent pathway for the detection of damaged telomeres. Here we show that damaged mammalian telomeres can activate both ATM and ATR and address the mechanism by which the shelterin complex represses these two important DNA damage signalling pathways. We analysed the telomere damage response on depletion of either or both of the shelterin proteins telomeric repeat binding factor 2 (TRF2) and protection of telomeres 1 (POT1) from cells lacking ATM and/or ATR kinase signalling. The data indicate that TRF2 and POT1 act independently to repress these two DNA damage response pathways. TRF2 represses ATM, whereas POT1 prevents activation of ATR. Unexpectedly, we found that either ATM or ATR signalling is required for efficient non-homologous end-joining of dysfunctional telomeres. The results reveal how mammalian telomeres use multiple mechanisms to avoid DNA damage surveillance and provide an explanation for the induction of replicative senescence and genome instability by shortened telomeres.     

Telomeres shorten during ageing of human fibroblasts

The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.     

Telomere-telomere recombination provides an express pathway for telomere acquisition

DNA termini from Tetrahymena and Oxytricha, which bear C4A2 and C4A4 repeats respectively, can support telomere formation in Saccharomyces cerevisiae by serving as substrates for the addition of yeast telomeric C1-3A repeats. Previously, we showed that linear plasmids with 108 base pairs of C4A4 DNA (YLp108CA) efficiently acquired telomeres, whereas plasmids containing 28-64 base pairs of C4A4 DNA also promoted telomere formation, but with reduced efficiency. Although many of the C4A4 termini on these plasmids underwent recombination with a C4A2 terminus, the mechanism of telomere-telomere recombination was not established. We now report the sequence of the C4A4 ends from the linear plasmids. The results provide strong evidence for a novel recombination process involving a gene conversion event that requires little homology, occurs at or near the boundary of telomeric and nontelomeric DNA, and resembles the recombination process involved in bacteriophage T4 DNA replication.     

Defects in mismatch repair promote telomerase-independent proliferation

Mismatch repair has a central role in maintaining genomic stability by repairing DNA replication errors and inhibiting recombination between non-identical (homeologous) sequences. Defects in mismatch repair have been linked to certain human cancers, including hereditary non-polyposis colorectal cancer (HNPCC) and sporadic tumours. A crucial requirement for tumour cell proliferation is the maintenance of telomere length, and most tumours achieve this by reactivating telomerase. In both yeast and human cells, however, telomerase-independent telomere maintenance can occur as a result of recombination-dependent exchanges between often imperfectly matched telomeric sequences. Here we show that loss of mismatch-repair function promotes cellular proliferation in the absence of telomerase. Defects in mismatch repair, including mutations that correspond to the same amino-acid changes recovered from HNPCC tumours, enhance telomerase-independent survival in both Saccharomyces cerevisiae and a related budding yeast with a degree of telomere sequence homology that is similar to human telomeres. These results indicate that enhanced telomeric recombination in human cells with mismatch-repair defects may contribute to cell immortalization and hence tumorigenesis.     

Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase

Telomeres define the ends of chromosomes; they consist of short tandemly repeated DNA sequences loosely conserved in eukaryotes (G1-8(T/A)1-4). Telomerase is a ribonucleoprotein which, in vitro, recognizes a single-stranded G-rich telomere primer and adds multiple telomeric repeats to its 3' end by using a template in the RNA moiety. In conjunction with other components, telomerase may balance the loss of telomeric repeats due to DNA replication. Another role of telomerase may be the de novo formation of telomeres. In eukaryotes like Tetrahymena, this process is an integral part of the formation of macronuclear chromosomes. In other eukaryotes this process stabilizes broken chromosomes. A case of human alpha-thalassaemia is caused by a truncation of chromosome 16 that has been healed by the addition of telomeric repeats (TTAGGG)n. Using an in vitro assay, I show here that human telomerase correctly recognizes the chromosome 16 breakpoint sequence and adds (TTAGGG)n repeats. The DNA sequence requirements are minimal and seem to define two modes of DNA recognition by telomerase.     

Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange

Homologous recombination is crucial for the repair of DNA breaks and maintenance of genome stability. In Escherichia coli, homologous recombination is dependent on the RecA protein. In the presence of ATP, RecA mediates the homologous DNA pairing and strand exchange reaction that links recombining DNA molecules. DNA joint formation is initiated through the nucleation of RecA onto single-stranded DNA (ssDNA) to form helical nucleoprotein filaments. Two RecA-like recombinases, Rad51 and Dmc1, exist in eukaryotes. Whereas Rad51 is needed for both mitotic and meiotic recombination events, the function of Dmc1 is restricted to meiosis. Here we examine human Dmc1 protein (hDmc1) for the ability to promote DNA strand exchange, and show that hDmc1 mediates strand exchange between paired DNA substrates over at least several thousand base pairs. DNA strand exchange requires ATP and is strongly dependent on the heterotrimeric ssDNA-binding molecule replication factor A (RPA). We present evidence that hDmc1-mediated DNA recombination initiates through the nucleation of hDmc1 onto ssDNA to form a helical nucleoprotein filament. The DNA strand exchange activity of hDmc1 is probably indispensable for repair of DNA double-strand breaks during meiosis and for maintaining the ploidy of meiotic chromosomes.     

Molecular cloning of human telomeres in yeast

Telomeres are the DNA sequences found at the ends of linear chromosomes. They define the boundaries of the genetical and physical maps of such chromosomes and so are particularly important for the complete mapping of large genomes that is now being attempted. Telomeres have been intensively studied in the yeast Saccharomyces cerevisiae and in ciliated protozoa: in these organisms the telomeric DNA consists of arrays of tandemly repeated short sequences in which one strand is guanosine-rich and oriented 5' to 3' towards the chromosome end. The conservation of these structural features is reflected in the observation that telomeric DNA from a variety of protozoa will function as telomeres on artificial linear mini-chromosomes in yeast. Tandem arrays of the sequence TTAGGG have been identified at the telomeres of humans and other mammals and also of trypanosomes. This indicates that the structural features of telomeres are conserved between higher and lower eukaryotes and implies that human telomeric DNA could function in yeast. I have used this idea to develop a strategy to isolate a specific human telomere as a molecular clone in yeast and have devised a simple and effective way of cloning other human telomeres and their associated sequences.     

Cloning of human telomeres by complementation in yeast

Telomeres confer stability on chromosomes by protecting them from degradation and recombination and by allowing complete replication of the end. They are genetically important as they define the ends of the linkage map. Telomeres of lower eukaryotes contain short repeats consisting of a G-rich and a C-rich strand, the G-rich strand running 5'-3' towards the telomere and extending at the end. Telomeres of human chromosomes share characteristics with those of lower eukaryotes including sequence similarity as detected by cross-hybridization. Telomeric repeats from many organisms can provide telomere function in yeast. Here we describe a modified yeast artificial chromosome (YAC) vector with only one telomere which we used to clone human telomeres by complementation in yeast. YACs containing human telomeres were identified by hydridization to an oligonucleotide of the trypanosome telomeric repeat. A subcloned human fragment from one such YAC is immediately subtelomeric on at least one human chromosome.     

Telomeric repeat from T. thermophila cross hybridizes with human telomeres

The ends (telomeres) of eukaryotic chromosomes must have special features to ensure their stability and complete replication. Studies in yeast, protozoa, slime moulds and flagellates show that telomeres are tandem repeats of simple sequences that have a G-rich and a C-rich strand. Mammalian telomeres have yet to be isolated and characterized, although a DNA fragment within 20 kilobases of the telomeres of the short arms of the human sex chromosomes has been isolated. Recently we showed that a chromosome from the fission yeast Schizosaccharomyces pombe could, in some cases, replicate as an autonomous mini-chromosome in mouse cells. By extrapolation from other systems, we reasoned that mouse telomeres could be added to the S. pombe chromosome ends in the mouse cells. On setting out to test this hypothesis we found to our surprise that the telomeric probe used (containing both the S. pombe and Tetrahymena thermophila repeats) hybridized to a series of discrete fragments in normal mouse DNA and DNA from a wide range of eukaryotes. We show here that the sequences hybridizing to this probe are located at the telomeres of most, if not all, human chromosomes and are similar to the Tetrahymena telomeric-repeat component of the probe.     

MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans

The germ line is an immortal cell lineage that is passed indefinitely from one generation to the next. To identify the genes that are required for germline immortality, we isolated Caenorhabditis elegans mutants with mortal germ lines--worms that can reproduce for several healthy generations but eventually become sterile. One of these mortal germline (mrt) mutants, mrt-2, exhibits progressive telomere shortening and accumulates end-to-end chromosome fusions in later generations, indicating that the MRT-2 protein is required for telomere replication. In addition, the germ line of mrt-2 is hypersensitive to X-rays and to transposon activity. Therefore, mrt-2 has defects in responding both to damaged DNA and to normal double-strand breaks present at telomeres. mrt-2 encodes a homologue of a checkpoint gene that is required to sense DNA damage in yeast. These results indicate that telomeres may be identified as a type of DNA damage and then repaired by the telomere-replication enzyme telomerase.