Circular rDNA Replicons Persist in Tetrahymena thermophila Transformants Synthesizing GGGGTC Telomeric Repeats

Site-directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo. The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase RNA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection. Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation.     

A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.     

Humanized telomeres and an attempt to express a functional human telomerase in yeast

The maintenance of telomeric repeat DNA depends on an evolutionarily conserved reverse trans criptase called telomerase. In vitro, only the catalytic subunit and a telomerase-associated RNA are required for the synthesis of species-specific repeat DNA. In an attempt to establish a heterologous system for the study of the human telomerase enzyme, we expressed the two core components and predicted regulatory subunits in the yeast Saccharomyces cerevisiae. We show that adequate substrates for human telomerase can be generated; the expressed enzyme was localized in the nucleus and it had the capacity to synthesize human-specific repeats in vitro. However, there was no evidence for human telomerase activity at yeast telomeres in vivo. Therefore functional replacement of the yeast telomerase by the human enzyme may require additional human-specific components. We also replaced the template region of the yeast telomerase RNA with one that dictates the synthesis of vertebrate repeats and performed a detailed molecular analysis of the composition of the telomeres upon outgrowth of such strains. The results suggest that vertebrate repeats on yeast telomeres are subject to a very high degree of repeat turnover and show that an innermost tract of 50 bp of yeast repeats are resistant to replacement.     

Identification of Kluyveromyces lactis Telomerase: Discontinuous Synthesis along the 30-Nucleotide-Long Templating Domain

Telomeres in the budding yeast Kluyveromyces lactis consist of perfectly repeated 25-bp units, unlike the imprecise repeats at Saccharomyces cerevisiae telomeres and the short (6- to 8-bp) telomeric repeats found in many other eukaryotes. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which uses a portion of its RNA moiety as a template. K. lactis telomerase RNA, encoded by the TER1 gene, is ~1.3 kb long and contains a 30-nucleotide templating domain, the largest ever examined. To examine the mechanism of polymerization by this enzyme, we identified and analyzed telomerase activity from K. lactis whole-cell extracts. In this study, we exploited the length of the template and the precision of copying by K. lactis telomerase to examine primer elongation within one round of repeat synthesis. Under all in vitro conditions tested, K. lactis telomerase catalyzed only one round of repeat synthesis and remained bound to reaction products. We demonstrate that K. lactis telomerase polymerizes along the template in a discontinuous manner and stalls at two specific regions in the template. Increasing the amount of primer DNA-template RNA complementarity results in stalling, suggesting that the RNA-DNA hybrid is not unpaired during elongation in vitro and that lengthy duplexes hinder polymerization through particular regions of the template. We suggest that these observations provide an insight into the mechanism of telomerase and its regulation.     

A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs

The telomerase ribonucleoprotein copies a short template within its integral RNA moiety onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Non-template regions of telomerase RNA (TER) are also crucial for telomerase function, yet they are highly divergent in sequence among species and their roles are largely unclear. Using both phylogenetic and mutational analyses, we predicted secondary structures for TERs from Kluyveromyces budding yeast species. A comparison of these secondary structure models with the published model for the Saccharomyces cerevisiae TER reveals a common arrangement into three long arms, a templating domain in the center and several conserved elements in the same positions within the structure. One of them, a three-way junction element, is highly conserved in budding yeast TERs. This element also shows sequence and structure similarity to the critical CR4-CR5 activating domain of vertebrate TERs. Mutational analysis in Kluyveromyces lactis confirmed that this element, and in particular the residues conserved across yeast and vertebrates, is critical for telomerase action both in vivo and in vitro. These findings demonstrate that despite the extreme divergence of TER sequences from different organisms, they do share conserved elements, which presumably carry out common roles in telomerase function.     

Evidence for an Additional Base-Pairing Element between the Telomeric Repeat and the Telomerase RNA Template in Kluyveromyces lactis and Other Yeasts

In all telomerases, the template region of the RNA subunit contains a region of telomere homology that is longer than the unit telomeric repeat. This allows a newly synthesized telomeric repeat to translocate back to the 3′ end of the template prior to a second round of telomeric repeat synthesis. In the yeast Kluyveromyces lactis, the telomerase RNA (Ter1) template has 30 nucleotides of perfect homology to the 25-bp telomeric repeat. Here we provide strong evidence that three additional nucleotides at positions −2 through −4 present on the 3′ side of the template form base-pairing interactions with telomeric DNA. Mutation of these bases can lead to opposite effects on telomere length depending on the sequence permutation of the template in a manner consistent with whether the mutation increases or decreases the base-pairing potential with the telomere. Additionally, mutations in the −2 and −3 positions that restore base-pairing potential can suppress corresponding sequence changes in the telomeric repeat. Finally, multiple other yeast species were found to also have telomerase RNAs that encode relatively long 7- to 10-nucleotide domains predicted to base pair, often with imperfect pairing, with telomeric DNA. We further demonstrate that K. lactis telomeric fragments produce banded patterns with a 25-bp periodicity. This indicates that K. lactis telomeres have preferred termination points within the 25-bp telomeric repeat.Telomeres are DNA and protein complexes present at the ends of eukaryotic chromosomes that function to protect chromosome ends from terminal sequence losses and fusions (3, 36). Telomeric DNA is typically composed of tandem 5- to 26-bp repeats that are sufficient for telomere function and that serve as binding sites for telomeric proteins (32). The ribonucleoprotein enzyme telomerase adds telomeric repeats to chromosome ends and prevents the gradual shortening that would otherwise occur. Telomerase synthesizes new telomeric repeats onto chromosome ends by using part of its RNA subunit as a template (13, 14, 31). Cells without telomerase encounter growth and viability problems once telomeres begin to become too short to properly function. In most human cells, telomerase activity is greatly reduced or absent and the ensuing telomere shortening functions to inhibit the formation of cancer by limiting the number of divisions that cells can undergo (4, 7, 16, 30).Recognition of a telomeric end by telomerase in vivo is complex and requires a number of different interactions between components of telomerase and components of the telomere (32). Specialized proteins that bind the 3′ single-stranded overhangs of telomeres, including the yeast Cdc13 protein, can interact directly with telomerase (9, 28). A critical aspect of telomerase''s interaction with the telomeres comes through base pairing between the telomeric overhang and the template region of the telomerase RNA. In all known telomerases, the template region of the RNA subunit contains a region of telomere homology that is longer than the unit telomeric repeat. This presence of short sequence identities at the 3′ and 5′ borders of the template allow a newly synthesized telomeric repeat to translocate back to the 3′ end of the template prior to a second round of telomeric repeat synthesis (38).Kluyveromyces lactis is an ascomycetous yeast species that is a valuable model organism for studying telomeres and telomerase. Unlike the better-studied yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, K. lactis has telomeres composed of repeats of uniform size (25 bp) and sequence (24). This indicates that the translocation step during a round of DNA synthesis by the telomerase enzyme normally occurs between precise positions at the two ends of the telomerase RNA template region. Point mutations at any of multiple positions within either of the two 5-nucleotide (nt)-long direct repeats that border the telomerase RNA template result in telomeric repeats of abnormal size (35). These appear to result from disruption of the normal base-pairing interactions between template and telomeric DNA during the translocation step.Here we present genetic data that argue strongly that three additional nucleotides 3′ of the template and outside the region of continuous homology with the telomeric repeat are involved in the base pairing between telomeric DNA and the telomerase RNA template in K. lactis. Sequence data suggest that similar extended base-pairing regions are widespread in other yeast species.     

Specific features of telomerase RNA from Hansenula polymorpha

Telomerase, a ribonucleoprotein, is responsible for the maintenance of eukaryotic genome integrity by replicating the ends of chromosomes. The core enzyme comprises the conserved protein TERT and an RNA subunit (TER) that, in contrast, displays large variations in size and structure. Here, we report the identification of the telomerase RNA from thermotolerant yeast Hansenula polymorpha (HpTER) and describe its structural features. We show further that the H. polymorpha telomerase reverse transcribes the template beyond the predicted boundary and adds a nontelomeric dT in vitro. Sequencing of the chromosomal ends revealed that this nucleotide is specifically present as a terminal nucleotide at the 3′ end of telomeres. Mutational analysis of HpTER confirmed that the incorporation of dT functions to limit telomere length in this species.