Processing of Nontelomeric 3′ Ends by Telomerase: Default Template Alignment and Endonucleolytic Cleavage

Broken chromosomes healed by de novo addition of a telomere are a major class of genome rearrangements seen in Saccharomyces cerevisiae and similar to rearrangements seen in human tumors. We have analyzed the sequences of 534 independent de novo telomere additions within a 12-kb region of chromosome V. The distribution of events mirrored that of four-base sequences consisting of the GG, GT, and TG dinucleotides, suggesting that de novo telomere additions occur at short regions of homology to the telomerase guide RNA. These chromosomal sequences restrict potential registrations of the added telomere sequence. The first 11 nucleotides of the addition sequences fell into common families that included 91% of the breakpoints. The observed registrations suggest that the 3 end of the TLC1 guide RNA is involved in annealing but not as a template for synthesis. Some families of added sequences can be accounted for by one cycle of annealing and extension, whereas others require a minimum of two. The same pattern emerges for sequences added onto the most common addition sequence, indicating that de novo telomeres are added and extended by the same process. Together, these data indicate that annealing is central to telomerase registration, which limits telomere heterogeneity and resolves the problem of synthesizing Rap1 binding sites by a nonprocessive telomerase with a lowcomplexity guide RNA sequence.

Section: Discussionmentioning

confidence: 87%

Chromosome healing through terminal deletions generated by de novo telomere additions in Saccharomyces cerevisiae

Putnam

Pennaneach

Kolodner

2004

“…Ciliate telomerases can extend nontelomeric substrates, both in vitro and in vivo, but always do so from a specific position at the 3Ј end of the template (24)(25)(26)(27). Likewise, after synthesis to the 5Ј end of the template and product dissociation, the RNP returns the 3Ј end of the template to the default position, allowing for repeat-addition processivity (28).…”

Section: Discussionmentioning

confidence: 99%

Telomerase recognizes its template by using an adjacent RNA motif

Miller

Collins

2002

Telomerase adds telomeric repeats to chromosome 3 ends, forestalling the cellular senescence, apoptosis, and genomic instability that result from telomere loss caused by incomplete DNA replication. The telomerase ribonucleoprotein is dedicated to synthesis of tandem, simple-sequence repeats by virtue of its specialization for copying only a specific template region within the integral RNA. Here, using circularly permuted variants of Tetrahymena thermophila telomerase RNA, we identify the features that allow recognition of the template region within the RNA. We engineered a template-less telomerase ribonucleoprotein that can position and reverse transcribe an exchangeable RNA oligonucleotide template accurately. Only a short ''template-recognition'' element sequence tag is required to direct efficient use of adjacent 5 residues as a template for telomeric repeat synthesis. Our findings reveal molecular requirements for template selection by telomerase and physically resolve templating from other RNA functions in catalysis.T elomerase, a specialized reverse transcriptase (RT), copies a small template region within its RNA subunit to extend chromosome 3Ј ends (1). A minimal, catalytically active recombinant telomerase ribonucleoprotein (RNP) can be produced in rabbit reticulocyte lysate by expressing the telomerase RNA and the telomerase RT protein (TERT) from mammals (2-4) or the ciliate Tetrahymena thermophila (5). Telomerase RNAs vary widely in length and primary sequence, but elements of conserved secondary structure have been predicted for ciliate and vertebrate RNAs by phylogenetic analysis (6, 7). The primary sequences of TERTs also are highly divergent, sharing only RT active site motifs and a few telomerase-specific motifs common to TERTs from all or phylogenetically related subsets of species (8-11).Unlike viral RTs, which copy a variety of single-stranded nucleic acid templates, telomerase can copy only the template region of its stably associated RNA. The molecular mechanism underlying one aspect of internal template use, templateboundary definition, has been examined previously by using telomerases from Kluyveromyces lactis and Tetrahymena. Different mechanisms establish the template 5Ј boundary in these organisms: RNA secondary structure is required in K. lactis (12) and protein-RNA interaction is required in Tetrahymena (10, 13). Left remaining is the broader question of how a telomerase RNP recognizes and accurately positions the template in the first place. The correct template region must be selected from within the larger telomerase RNA and delivered to the RT active site, an aspect of template selection that we refer to as ''template recognition.'' What features of the RNP could accomplish template recognition? One possibility is that the template is recognized in a sequence-specific manner. This simple mechanism seems unlikely given the relatively high accessibility of template bases to chemical modification (14) and the tolerance of endogenous Tetrahymena telomerase RNP catalytic activity for ...

“…4C, lanes 6 and 8, arrows and schematic). Such cleavage and 3Ј replacement reactions have been reported for wild-type Tetrahymena and Euplotes telomerases with certain primers but have not been reported for (T 2 G 4 ) 3 or comparable primers (21,22).…”

Section: Fig 3 Southern Blot Analysis Of Genomic Dna From T Thermomentioning

confidence: 99%

A functional telomerase RNA swap in vivo reveals the importance of nontemplate RNA domains

Bhattacharyya

Blackburn

1997

The ribonucleoprotein (RNP) enzyme telomerase is required for replication of eukaryotic chromosomal termini. The RNA moiety of telomerase is essential for enzyme function and provides the template for telomeric DNA synthesis. However, the roles of its nontemplate domains have not been explored. Here we demonstrate that a novel interspecies telomerase RNA swap in vivo creates a functional but aberrant telomerase. Telomerase RNA from the ciliate Glaucoma chattoni was expressed in Tetrahymena thermophila cells. The telomerase RNAs from these two species have almost superimposable secondary structures. The template region base sequence is identical in the two RNAs, but elsewhere their sequences differ by 49%. This hybrid telomerase RNP was enzymatically active but added only short stretches of telomeric repeat tracts in vivo and in vitro. This new enzyme also had a strong, aberrant DNA cleavage activity in vitro. Thus, molecular interactions in the RNP involving nontemplate RNA domains affect specific aspects of telomerase enzyme function, raising the possibility that they may regulate telomerase activity.The G-rich telomeric DNA tracts found at most eukaryotic chromosomal termini are added by a specialized component of the cellular DNA replication machinery, the ribonucleoprotein (RNP) reverse transcriptase telomerase. Telomeric DNA has numerous important functions and properties, including allowing completion of DNA replication, mediating chromosome stability (1), and affecting mitotic chromosome separation (2). Therefore, telomere synthesis and maintenance are crucial aspects of stable genomic inheritance in eukaryotes. The RNA moiety of the telomerase RNP is essential for enzymatic function and contains an internal sequence that templates telomeric DNA polymerization (3). Studies of template point mutations also have revealed other critical roles for specific template residues in enzyme action (4, 5). However, little is known about the function of other portions of telomerase RNA.Telomerase RNAs in ciliated protozoa (3, 6, 7), yeasts (8, 9), and mammals (10, 11) have diverged rapidly in primary sequence. Phylogenetic (12-14) and RNA conformational analyses (15, 16) of the small (Ϸ150-200 nt) ciliate telomerase RNAs have shown that their secondary structures are highly conserved. Comparison of 13 telomerase RNAs from one group of ciliates has shown a strikingly bipartite sequence arrangement; they all contain a 17-nt stretch of identical sequence centered on the template domain, and the primary sequence of the rest of the RNA differs by up to 76% yet preserves similar structural domains (12,14). To investigate directly how this nontemplate portion of telomerase RNA affects enzymatic function, we tested whether the Ϸ160-nt telomerase RNAs in the ciliates Tetrahymena thermophila and Glaucoma chattoni were functionally interchangeable in vivo. These two telomerase RNAs share an identical 23-base sequence in and around the template domain. In contrast, the rest of the RNA sequence, which has an almost identic...