Surprises from the chromosome front: lessons from Arabidopsis on telomeres and telomerase.

Telomeres serve two vital functions: They act as a buffer against the end-replication problem, and they prevent chromosome ends from being recognized as double-strand DNA (dsDNA) breaks. These functions are orchestrated by the telomerase reverse transcriptase and a variety of telomere protein complexes. Here, we discuss our recent studies with Arabidopsis thaliana that uncovered a new and highly conserved telomere complex called CST (Cdc13/CTC1, STN1, TEN1). Formerly believed to be yeast specific, CST has now been identified as a key component of both plant and vertebrate telomeres, which is essential for genome integrity and stem cell viability. We also describe the unexpected discovery of alternative telomerase ribonucleoprotein complexes in Arabidopsis. Fueled by duplication and diversification of the telomerase RNA subunit and telomerase accessory proteins, these telomerase complexes act in concert to maintain genome stability. In addition to the canonical telomerase enzyme, one of two alternative telomerase ribonucleoprotein (RNP) complexes functions as a novel negative regulator of enzyme activity in response to genotoxic stress. These contributions highlight the immense potential of Arabidopsis in probing the depths of the chromosome end.

Analysis of Carica papaya Telomeres and Telomere-Associated Proteins: Insights into the Evolution of Telomere Maintenance in Brassicales.

Telomeres are terminal regions of linear eukaryotic chromosomes that are critical for genome stability and continued cell proliferation. The draft assembly of the papaya genome provides an opportunity to analyze and compare the evolution of telomeric DNA sequence composition and telomere maintenance machinery in this and other organisms of the Brassicales Order, which includes Arabidopsis. Here we investigate telomere size and sequence variation at papaya chromosome ends. As with most other plant species, papaya telomeres consist of TTTAGGG repeats. However, in contrast to members of the closely related Brassicaceae family, telomeres in papaya are ~10-fold longer. Sequence analysis reveals that many centromereproximal telomere repeats in papaya harbor nucleotide substitutions and insertions of Gs and Ts. In contrast, we found very few N-to-C substitutions, and even fewer instances of nucleotide deletion, suggesting that a six-nucleotide telomere repeat is not well tolerated. The papaya genome encodes single-copy sequence homologues of several genes involved in telomere maintenance and chromosome end protection, including the Telomerase Reverse Transcriptase (TERT) and Protection Of Telomeres (POT1). Notably, unlike Arabidopsis, which encodes six Telomere Repeat binding Factor-like (TRFL) proteins that bind double-stranded telomere DNA, papaya appears to encode only two such proteins. Thus, the more streamlined genome of papaya will provide an excellent resource for comparative and functional analysis of telomeres in plants.

Different modes of de novo telomere formation by plant telomerases.

The telomerase reverse transcriptase can recognize broken chromosome ends and add new telomeres de novo in a reaction termed "chromosome healing". Here we investigate new telomere formation in vitro by telomerases from a variety of flowering plant species. Comparing the electrophoretic mobilities and nucleotide sequences of the products, we uncovered three different modes of new telomere formation. The soybean telomerase, designated a Class I enzyme, only elongated DNA primers ending in telomeric nucleotides. Arabidopsis and maize telomerases, designated Class II enzymes, efficiently extended completely non-telomeric sequences by positioning the 3' terminus at a preferred site on the RNA template. Silene latifolia and sorghum telomerases constituted class III enzymes that elongated non-telomeric DNA primers by annealing them at alternative sites on the RNA template. For all enzymes, errors were prevalent during synthesis of the first two repeats, likely reflecting lateral instability of the primer 3' terminus on the template during the initial rounds of elongation. Class III telomerases, however, were five- to 13-fold more error prone than class II, generating more mistakes in distal repeats added to the primers. This remarkable variability in enzyme-DNA interactions among plant telomerases does not reflect phylogenetic relationships, and therefore implies that the telomerase active site can evolve rapidly.

Living with genome instability: plant responses to telomere dysfunction.

Loss of telomere function in metazoans results in catastrophic damage to the genome, cell cycle arrest, and apoptosis. Here we show that the mustard weed Arabidopsis thaliana can survive up to 10 generations without telomerase. The last five generations of telomerase-deficient plants endured increasing levels of cytogenetic damage, which was correlated with developmental anomalies in both vegetative and reproductive organs. Mutants ultimately arrested at a terminal vegetative state harboring shoot meristems that were grossly enlarged, disorganized, and in some cases, dedifferentiated into a callusoid mass. Unexpectedly, late-generation mutants had an extended life-span and remained metabolically active. The differences in plant and animal responses to dysfunctional telomeres may reflect the more plastic nature of plant development and genome organization.

Analysis of the G-overhang structures on plant telomeres: evidence for two distinct telomere architectures.

Telomeres are highly conserved structures essential for maintaining the integrity of eukaryotic genomes. In yeast, ciliates and mammals, the G-rich strand of the telomere forms a 3' overhang on the chromosome terminus. Here we investigate the architecture of telomeres in the dicot plants Silene latifolia and Arabidopsis thaliana using the PENT (primer extension/nick translation) assay. We show that both Arabidopsis and Silene telomeres carry G-overhangs longer than 20-30 nucleotides. However, in contrast to yeast and ciliate telomeres, only half of the telomeres in Silene seedlings possess detectable G-overhangs. PENT reactions using a variety of primers and reaction conditions revealed that the remaining fraction of Silene telomeres carries either no overhangs or overhangs less than 12 nucleotides in length. G-overhangs were observed in Silene seeds and leaves, tissues that lack telomerase activity. These findings suggest that incomplete DNA replication of the lagging strand, rather than synthesis by telomerase, is the primary mechanism for G-overhang synthesis in plants. Unexpectedly, we found that the fraction of telomeres with detectable G-overhangs decreased from 50% in seedlings to 35% in leaves. The difference may reflect increased susceptibility of the G-overhangs to nuclease attack in adult leaves, an event that could act as a precursor for the catabolic processes accompanying leaf senescence

Telomerase enzyme activity as a diagnostic tool to distinguish effusions of malignant and benign origin.

Telomerase enzyme activity is high in populations of cells that are dividing, and is low or undetectable in quiescent cell populations. Activation of telomerase in tissues that normally lack the capacity for self-renewal is strongly correlated with neoplasia. Telomerase activity can be detected in samples containing very small numbers of cells and studies of human patients suggest that measurement of telomerase activity may be useful for the evaluation of samples that can be obtained in a minimally invasive manner. This study compares the presence or absence of telomerase activity with cytologic evaluation of body cavity effusions, to determine if neoplasia is the underlying cause for the effusion in dogs and cats. Detection of telomerase in effusions was no more sensitive than cytologic evaluation for the identification of underlying neoplasia, and was less specific (telomerase assay: sensitivity = 50%, specificity = 83%; cytology: sensitivity = 50%, specificity = 100%). We conclude that although the telomerase assay may constitute a useful adjunctive test for the diagnosis of neoplasia in some dogs and cats with body cavity effusions, the results of this assay are not sufficiently reliable to be used as a sole diagnostic test.

Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA.

Telomerase is an essential enzyme that maintains telomeres on eukaryotic chromosomes. In mammals, telomerase is required for the lifelong proliferative capacity of normal regenerative and reproductive tissues and for sustained growth in a dedifferentiated state. Although the importance of telomeres was first elucidated in plants 60 years ago, little is known about the role of telomeres and telomerase in plant growth and development. Here we report the cloning and characterization of the Arabidopsis telomerase reverse transcriptase (TERT) gene, AtTERT. AtTERT is predicted to encode a highly basic protein of 131 kDa that harbors the reverse transcriptase and telomerase-specific motifs common to all known TERT proteins. AtTERT mRNA is 10-20 times more abundant in callus, which has high levels of telomerase activity, versus leaves, which contain no detectable telomerase. Plants homozygous for a transfer DNA insertion into the AtTERT gene lack telomerase activity, confirming the identity and function of this gene. Because telomeres in wild-type Arabidopsis are short, the discovery that telomerase-null plants are viable for at least two generations was unexpected. In the absence of telomerase, telomeres decline by approximately 500 bp per generation, a rate 10 times slower than seen in telomerase-deficient mice. This gradual loss of telomeric DNA may reflect a reduced rate of nucleotide depletion per round of DNA replication, or the requirement for fewer cell divisions per organismal generation. Nevertheless, progressive telomere shortening in the mutants, however slow, ultimately should be lethal.

Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila.

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes and maintains telomeric DNA. Studies of telomeres and telomerase are facilitated by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase III-directed gene encoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing the Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahymena regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element (PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated expression of the chimeric gene. In addition, mutation of an A/T-rich element at -25 decreased expression markedly. A gel mobility shift assay and protein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular mass of approximately 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.

Developmentally programmed assembly of higher order telomerase complexes with distinct biochemical and structural properties.

In Euplotes crassus, telomerase is responsible for telomere maintenance during vegetative growth and de novo telomere synthesis during macronuclear development. Here we show that telomerase in the vegetative stage of the life cycle exists as a 280-kD complex that can add telomeric repeats only onto telomeric DNA primers. Following the initiation of macronuclear development, telomerase assembles into larger complexes of 550 kD, 1600 kD, and 5 MD. In the 1600-kDa and 5-MDa complexes, telomerase is more processive than in the two smaller complexes and can add telomeres de novo onto nontelomeric 3' ends. Assembly of higher order telomerase complexes is accompanied by an extended region of RNase V1 and RNase T1 protection in the telomerase RNA subunit that is not observed with telomerase from vegetatively growing cells. The protected residues encompass a highly conserved region previously proposed to serve as a platform for formation of higher order structures. These findings provide the first direct demonstration of developmentally regulated higher order telomerase complexes with unique biochemical and structural properties.

Reiterative dG addition by Euplotes crassus telomerase during extension of non-telomeric DNA.

Telomerase from the ciliate Euplotes crassus incorporates G4T4telomeric repeats onto both telomeric and non-telomeric single-stranded DNA 3'-ends via reverse transcription of a templating domain in its RNA subunit. Here we describe an unusual mode of template copying that is characteristic of DNA synthesis onto non-telomeric 3'-ends in vitro . When dTTP was eliminated from telomerase reactions, telomeric primers or DNA products generated from the telomerase endonuclease were extended by precise copying of the RNA template. In contrast, telomerase catalyzed the addition of up to 13 dG residues onto primers with non-telomeric 3'-ends under the same reaction conditions. Introducing mismatches in the 3'-terminus of telomeric primers that reduced primer complementarity to the RNA template induced reiterative dG incorporation, indicating that the reaction is influenced by Watson-Crick base pair formation between the primer and the RNA template. Unexpectedly, the reiterative dG addition mode was confined to telomerase derived from developing cells that undergo new telomere formation. This reaction was not observed in vegetatively growing cells. We postulate that indiscriminate dG addition by telomerase occurs by reiterative copying of C residues in the telomerase RNA templating domain and reflects lateral instability of the primer-template interaction during de novo telomere formation.

Flexible positioning of the telomerase-associated nuclease leads to preferential elimination of nontelomeric DNA.

In addition to a reverse transcriptase activity, telomerase is associated with a DNA endonuclease that removes nucleotides from a primer 3' terminus prior to telomere repeat addition. Here we examine the DNA specificity of the primer cleavage-elongation reaction carried out by the Euplotes crassus telomerase. We show that the primer cleavage activity copurified with the E. crassus telomerase polymerase, indicating that it either is an intrinsic property of telomerase or is catalyzed by a tightly associated factor. Using chimeric primers containing stretches of telomeric DNA that could be precisely positioned on the RNA template, we found that the cleavage site is more flexible than originally proposed. Primers harboring mismatches in dT tracts that aligned opposite nucleotides 37 to 40 in the RNA template were cleaved to eliminate the mismatched residues along with the adjacent 3' sequence. The cleaved product was then elongated to generate perfect telomeric repeats. Mismatches in dG tracts were not removed, implying that the nuclease does not track coordinately with the polymerase active site. Our data indicate that the telomerase-associated nuclease could provide a rudimentary proofreading function in telomere synthesis by eliminating mismatches between the DNA primer and the 5' region of the telomerase RNA template.

Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation.

Telomerase serves a dual role at telomeres, maintaining tracts of telomere repeats and forming telomeres de novo on broken chromosomes in a process called chromosome healing. In ciliates, both mechanisms are readily observed. Vegetatively growing cells maintain pre-existing telomeres, while cells undergoing macronuclear development fragment their chromosomes and form telomeres de novo. Here we provide the first evidence for developmentally regulated initiation of DNA synthesis by telomerase. In vitro assays were conducted with telomerase from vegetative and developing Euplotes macronuclei using chimeric primers that contained non-telomeric 3' ends and an upstream stretch of telomeric DNA. In developing macronuclei, chimeric primers had two fates: nucleotides were either polymerized directly onto the 3' terminus or residues were removed from the 3' end by endonucleolytic cleavage before polymerization began. In contrast, telomerase from vegetative macronuclei used only the cleavage pathway. Telomere repeat addition onto non-telomeric 3' ends was lost when developing macronuclei were lysed and the contents purified on glycerol gradients. However, when fractions from the glycerol gradient were added back to partially purified telomerase, telomere synthesis was restored. The data indicate that a dissociable chromosome healing factor (CHF) collaborates with telomerase to initiate developmentally programmed de novo telomere formation.

Plant telomeres and telomerases. A review.

Barbara McClintock began investigating plant telomeres during the 1930s, but little additional work was done in this area until a telomeric DNA sequence was isolated and characterized from Arabidopsis thaliana in 1988. This sequence, a simple repeat of the heptanucleotide 5'-TTTAGGG-3', has been found in telomeres of almost all plants analyzed. Telomere length in plants, which can be a long as 75 kb or as short as 2 kb, is controlled by both genetic and developmental factors. The major mechanism for synthesis of telomeres is telomerase, a ribonucleoprotein with reverse transcriptase activity. Telomerase expression is highly regulated in both plants and animals. For example, there is little or no detectable expression of telomerase in most vegetative tissues of plants nor in most somatic tissues of animals. In contrast to animals, plants do not specify a germ line until late in development, but telomerase is reactivated during flowering, possibly to ensure that gametes and embryos arising from them inherit fully functional chromosomes. Telomerase is also highly expressed in plant tissue culture cells, as might be expected for cells with an unlimited capacity for proliferation. Despite recent progress in investigating plant telomeres and telomerase at the molecular level, there is still much more to learn, especially concerning the developmental control of telomerase activity.

Characterization and developmental patterns of telomerase expression in plants.

Telomerase activity is developmentally regulated in mammals. Here we examine telomerase activity in plants, whose development differs in fundamental ways from that of animals. Using a modified version of the telomere repeat amplification protocol (TRAP) assay, we detected an activity in extracts from carrots, cauliflower, soybean, Arabidopsis, and rice with all the characteristics expected for a telomerase synthesizing the plant telomere repeat sequence TTTAGGG. The activity was dependent on RNA and protein components, required dGTP, dATP, and dTTP, but not dCTP, and generated products with a seven nucleotide periodicity. Telomerase activity was abundant in cauliflower meristematic tissue and undifferentiated cells from Arabidopsis, soybean, and carrot suspension cultures, but was low or not detectable in a sampling of differentiated tissues from mature plants. Telomerase from cauliflower meristematic tissues exhibited relaxed DNA sequence requirements, which might reflect the capacity to form telomeres on broken chromosomes in vivo. The dramatic differences in telomerase expression and their correlation with cellular proliferation capacity mirror changes in human telomerase levels during differentiation and immortalization. Hence, telomerase activation appears to be a conserved mechanism involved in conferring long-term proliferation capacity.

Processing of nontelomeric 3' ends by telomerase: default template alignment and endonucleolytic cleavage.

Telomerase is a specialized reverse transcriptase that maintains telomeres at chromosome ends by extending preexisting tracts of telomeric DNA and forming telomeres de novo on broken chromosomes. Whereas the interaction of telomerase with telomeric DNA has been studied in some detail, relatively little is known about how this enzyme processes nontelomeric DNA. In this study we recruited the Euplotes telomerase to nontelomeric 3' termini in vitro using chimeric DNA primers that carried one repeat of a telomeric sequence at various positions upstream of a nontelomeric 3' end. Such primers were processed in two distinct pathways. First, nontelomeric 3' ends could be elongated directly by positioning a primer terminus at a specific site on the RNA template. Delivery to this default site was precise, always resulting in the addition of 4 dG residues to the non-telomeric 3' ends. These same residues initiate new telomeres formed in vivo. Alternatively, 3' nontelomeric nucleotides were removed from primers prior to initiating the first elongation cycle. As with default positioning of nontelomeric 3' ends, the cleavage event was extremely precise and was followed by the addition of dG residues to the primer 3' ends. The specificity of the cleavage reaction was mediated by primer interaction with the RNA template and, remarkably, proceeded by an endonucleolytic mechanism. These observations suggest a mechanism for the precision of developmentally regulated de novo telomere formation and expand our understanding of the enzymatic properties of telomerase.

Chromosome healing: spontaneous and programmed de novo telomere formation by telomerase.

Telomeres are protective caps for chromosome ends that are essential for genome stability. Broken chromosomes missing a telomere will not be maintained unless the chromosome is 'healed' with the formation of a new telomere. Chromosome healing can be a programmed event following developmentally regulated chromosome fragmentation, or it may occur spontaneously when a chromosome is accidentally broken. In this article we discuss the consequences of telomere loss and the possible mechanisms that the enzyme telomerase employs to form telomeres de novo on broken chromosome ends.

Sequence of the macronuclear DNA encoding large subunit ribosomal protein 29 (L29) in Euplotes crassus and cycloheximide sensitivity.

As a first step towards developing a DNA transformation method for the ciliated protozoan Euplotes crassus we determined the minimum inhibitory concentration (MIC) for cell division in the presence of cycloheximide (Chx) for several cell lines and the range of Chx sensitivity for 106 different progeny cell lines derived by mating two lines. All of the cell lines are highly sensitive to Chx. Progeny cell lines show a wider range of sensitivities than the parental lines. Because site-directed mutagenesis of the RPL29 gene encoding the large subunit ribosomal protein 29 (L29) has been used to generate a Chx-resistance marker (ChxR) for another ciliate, Tetrahymena thermophila [Yao and Yao, Proc. Natl. Acad. Sci. USA 88 (1991) 9493-9497], we isolated and sequenced the entire E. crassus macronuclear DNA carrying RPL29. The encoded peptide is 52-73% identical in sequence to L29 sequences from organisms ranging from T. thermophila and Saccharomyces cerevisiae to mouse. In E. crassus, the codon that has been mutated to confer Chx resistance in both S. cerevisiae and T. thermophila already encodes the amino-acid residue of one of the mutant forms identified in these other organisms. Thus, E. crassus RPL29 is not a convenient source of a selectable marker. Notable features of the macronuclear DNA carrying RPL29 are its extremely short non-coding regions and a TAG stop codon.

Oligonucleotides complementary to the Oxytricha nova telomerase RNA delineate the template domain and uncover a novel mode of primer utilization.

The telomerase reverse transcriptase uses an essential RNA subunit as a template to direct telomeric DNA synthesis. The 190-nucleotide Oxytricha nova telomerase RNA was identified by using an oligonucleotide probe complementary to the predicted CCCCAAAA template. This RNA displays extensive sequence similarity to the Euplotes crassus telomerase RNA and carries the same 5' CAAAACCCCAAAACC 3' telomeric domain. Antisense oligonucleotides were used to map the boundaries of the functional template and to investigate the mechanism of primer recognition and elongation. On the basis of their ability to inhibit or to prime telomerase, oligonucleotides were classified into three categories. Category 1 oligonucleotides, which extended 5' of residue 42 in the RNA, abolished elongation of (T4G4)3 and (G4T4)3 primers in vitro. In contrast, oligonucleotides terminating between residues 42 and 50 (categories 2 and 3), served as efficient telomerase primers. We conclude that the O. nova template comprises residues 42 to 50 in the 190-nucleotide RNA, a different set of nucleotides than are used by the E. crassus enzyme. Category 2 primer reactions amassed short products, and their abundance could be decreased by altering the 5' sequence of the primer, consistent with the two-primer-binding-site model for telomerase. Category 3 primers generated a bimodal distribution of short and long products, each having a unique elongation profile. The long-product profile is inconsistent with sequence-specific primer alignment. Rather, each primer was extended by the same register of TTTTGGGG repeats, suggesting shuttling to a default position within the template. The parallels between telomerase and RNA polymerase elongation mechanisms are discussed.

DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases.

Macronuclear telomeres in Oxytricha exist as DNA-protein complexes in which the termini of the G-rich strands are bound by a 97-kDa telomere protein. During telomeric DNA replication, the replication machinery must have access to the G-rich strand. However, given the stability of telomere protein binding, it has been unclear how this is accomplished. In this study we investigated the ability of several different DNA polymerases to access telomeric DNA in Oxytricha telomere protein-DNA complexes. Although DNA bound by the telomere protein is not degraded by micrococcal nuclease or labeled by terminal deoxynucleotidyltransferase, this DNA serves as an efficient primer for the addition of telomeric repeats by telomerase, a specialized RNA-dependent DNA polymerase (ribonucleoprotein reverse transcriptase), EC 2.7.7.49. Moreover, in the presence of a suitable complementary C-rich DNA template, AMV reverse transcriptase and the E. coli Klenow fragment will also elongate DNA bound by the telomere protein. These findings indicate that the 3' terminus and the Watson-Crick base pairing positions are exposed in the protein complex. We propose that the telomere protein can serve a dual role at the telomere by protecting the DNA phosphate backbone from degradation while simultaneously exposing the DNA bases for replication.

Telomeres and telomerases.

The ends of eukaryotic chromosomes are defined by specialized nucleoprotein complexes called telomeres. Telomeres impart stability to the genome and are of general interest due to their unique structure and unconventional mode of synthesis. Recent work has identified new components of the telomere complex and expanded our understanding of the role of terminal structures in maintaining cell viability.