Loss of cap structure causes mitotic defect in Tetrahymena thermophila telomerase mutants

Petcherskaia, Marina; McGuire, Jennifer M.; Pherson, James M.; Kirk, Karen E.

doi:10.1007/s00412-003-0233-9

Cited by 7 publications

(20 citation statements)

References 44 publications

(75 reference statements)

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“…TBE substitutions reduce the fidelity of template 5Ј boundary definition in Tetrahymena telomerase RNPs reconstituted in vitro (2,24) and in vivo (above). The synthesis of mutant telomeric repeats in vivo can impact Tetrahymena telomere length maintenance, chromosome segregation, and cell growth (22,34,44). Tetrahymena strains expressing the TBE variant hpTERs grew normally, however, suggesting that altered telomeric repeats were not abundant or were tolerated.…”

Section: Discussionmentioning

confidence: 99%

Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

Cunningham

Collins

2005

Molecular and Cellular Biology

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Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.Telomeres protect authentic chromosome termini from aberrant resection or rearrangement. In most eukaryotes, a stable telomere is composed of a tandem array of simple-sequence repeats assembled into specialized chromatin (37). The overall length of the telomeric simple-sequence repeat tract is maintained in a dynamic balance of sequence loss and addition. To compensate for the inevitable loss from incomplete replication by DNA-dependent DNA polymerases, telomeres recruit the telomerase ribonucleoprotein (RNP) reverse transcriptase in coordination with other DNA replication factors (18). Telomerase copies a defined sequence within its integral RNA subunit (TER) to extend chromosome 3Ј ends. Sequence substitutions within the TER template can rapidly impose defects in cell division and viability due to the synthesis of altered repeats (6).During a catalytic cycle of repeat synthesis, the telomerase active site must proceed through a remarkable number of distinct states of template positioning and pairing (Fig. 1A). Substrates unable to base-pair with the template are extended in alignment with the template 3Ј end at its "default" position, defined as the position of template in the absence of a template-primer hybrid (41). For Tetrahymena thermophila TER, default template positioning places C49 in the active site (Fig. 1A, top). As deoxynucleoside triphosphates (dNTPs) are added, a template-product hybrid forms, and successively more 5Ј positions of unpaired template must thread into the active site (Fig. 1A, middle). Product synthesis halts at the template 5Ј boundary, corresponding to position C43 of T. thermophila TER (Fig. 1A, bottom). Upon eventual dissociation of the template-product hybrid, the liberated template repositions to recover its default placement. Product either dissociates from the enzyme or r...

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Section: Discussionmentioning

confidence: 99%

Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

Cunningham

Collins

2005

Molecular and Cellular Biology

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“…Although it was shown previously that the sequences of the macronuclear telomeres in this mutant are altered (40), the micronuclear telomeres had not been examined. We show here that the micronuclear telomeres are dramatically shortened.…”

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confidence: 93%

“…A number of telomere mutants have been constructed in T. thermophila, for example, by mutating the telomerase RNA and consequently the telomeric DNA itself (40) or by depleting a protein that binds the telomeres (24). The telomeric DNA is mutated in ter1-43AA cells by altering the telomerase RNA template such that it synthesizes a combination of G4T3 repeats, which are harmless, and G4T4 repeats, which are toxic (40).…”

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confidence: 99%

“…The telomeric DNA is mutated in ter1-43AA cells by altering the telomerase RNA template such that it synthesizes a combination of G4T3 repeats, which are harmless, and G4T4 repeats, which are toxic (40). Even with this sequence alteration, the macronuclear telomere length is only subtly altered.…”

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confidence: 99%

“…Even with this sequence alteration, the macronuclear telomere length is only subtly altered. Nonetheless, a dramatic arrest appears in micronuclear mitosis, where the chromatin appears elongated during anaphase (27,40), which takes about 30 times longer than wild-type (WT) anaphase. In a different type of telomere mutant, where the G-strand telomeric binding protein POT1a is depleted, the cells undergo a growth arrest where the macronucleus is enlarged and the macronuclear telomeres become extremely lengthened (24).…”

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Abnormal Micronuclear Telomeres Lead to an Unusual Cell Cycle Checkpoint and Defects in Tetrahymena Oral Morphogenesis

et al. 2008

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Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a "dynamic pause" in the program of cortical development, which may reveal an unusual cell cycle checkpoint.Telomeres have been implicated in a number of diverse cellular processes. Normal telomeres serve as the means by which chromosomes can be replicated completely, and they function as a cap, thereby protecting the natural ends from inappropriate fusion. Telomeric mutants can be generated by making predictable changes in the RNA template of the enzyme telomerase, which synthesizes telomeres, such that the corresponding complementary mutation is made at the telomeres. Although these types of telomeric DNA mutants in complex eukaryotes are poorly studied, particularly with respect to the downstream developmental consequences, those in simplified yeasts and the ciliated protozoa are better understood.Ciliated protozoa, such as Tetrahymena thermophila, provide excellent model systems for studying telomerase, telomeric DNA mutants, and the downstream developmental consequences to changing the telomeric repeat sequence. This is because the components of telomeres and telomerase have been exceptionally well documented (5) and the cells are large enough (roughly 60 to 70 m) to allow a glimpse of their unique developmental processes.Interestingly, T. thermophila, like other ciliates, bears two nuclei distinct in function and mode of division (reviewed in reference 25). The micronucleus represents the germ line nucleus and is transcriptionally silent, whereas the macronucleus serves as the vegetative nucleus of the cell. The micronucleus contains five pairs of chromosomes, which are passed on through mitosis and meiosis. In contrast, the macronucleus contains many subchromosomal fragments, each of which is amplified to ϳ45 copies, excluding the ribosomal DNA, which has thousands of copies. The macronuclear chromosomes are not faithfully inherited, as the macronucleus divides amitotically.The telomeres of the two T. thermophila nuclei are dramatically different. The macronuclear telomeres, present at a copy number of roughly 40,000 per cell, contain about 0.3 to 0.5 kb of G4T2 repeats (3). Mic...

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Asparagales Telomerases which Synthesize the Human Type of Telomeres

2006

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The order of monocotyledonous plants Asparagales is attractive for studies of telomere evolution as it includes three phylogenetically distinct groups with telomeres composed of TTTAGGG (Arabidopsis-type), TTAGGG (human-type) and unknown alternative sequences, respectively. To analyze the molecular causes of these switches in telomere sequence (synthesis), genes coding for the catalytic telomerase subunit (TERT) of representative species in the first two groups have been cloned. Multiple alignments of the sequences, together with other TERT sequences in databases, suggested candidate amino acid substitutions grouped in the Asparagales TERT synthesizing the human-type repeat that could have contributed to the changed telomere sequence. Among these, mutations in the C motif are of special interest due to its functional importance in TERT. Furthermore, two different modes of initial elongation of the substrate primer were observed in Asparagales telomerases producing human-like repeats, which could be attributed to interactions between the telomerase RNA subunit (TR) and the substrate.

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Loss of cap structure causes mitotic defect in Tetrahymena thermophila telomerase mutants

Cited by 7 publications

References 44 publications

Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

Abnormal Micronuclear Telomeres Lead to an Unusual Cell Cycle Checkpoint and Defects in Tetrahymena Oral Morphogenesis

Asparagales Telomerases which Synthesize the Human Type of Telomeres

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