Functional reconstitution of wild-type and mutant Tetrahymena telomerase.

Abstract

Telomerase is a ribonucleoprotein that catalyzes telomere elongation in vitro and in vivo. The 159-nucleotide RNA component of Tetrahymena telomerase contains the sequence 5'-CAACCCCAA-3' ("template region"), which serves as a template for the addition of the sequence d(TTGGGG)" to Tetrahymena telomeres. To dissect the Tetrahymena telomerase enzyme mechanism, we developed a functional in vitro reconstitution assay. After removal of the essential telomerase RNA by micrococcal nuclease digestion of partially purified telomerase, the addition of in vitro-transcribed telomerase RNA reconstituted telomerase activity. The reconstituted activity was processive and showed the same primer specificities as native telomerase. Mutants in the RNA template region were tested in reconstitution assays to determine the role of the residues in this region in primer recognition and elongation. Two template mutants, encoding the sequences 5'-UAACCCCAA-3' and 5'-UAACCCUAA-3', specified the incorporation of dATP into the sequence d(TTAGGG). Telomerase reconstituted with a template mutant encoding the sequence 5'-CAACCCUAA-3' did not specify dATP incorporation and elongation by this mutant was not terminated by the addition of ddATP. In addition, a template mutant encoding the sequence 5'-CGGCCCCAA-3' specified the incorporation of ddCTP but not ddTTP while a mutant encoding the sequence 5'-CAACCCCGG-3' specified the incorporation of ddTTP but not ddCTP. These data suggest that only the most 5' six residues of the template region dictate the addition of telomeric repeats.

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

“…In contrast, the data leading to these conclusions (21) can be explained by using our model involving poor processivity, multiple annealingsynthesis-dissociation cycles, and a crucial role for annealing in controlling TLC1 registration. If the 3Ј end were available for synthesis and the central regions of the template were not available for annealing, then S. cerevisiae telomerase would be substantially different from ciliate telomerases (29). By contrast, our results suggest that yeast telomerase is much more similar to other telomerases.…”

“…Furthermore, these groups are consistent, with only 50% of the TGGGTGT sequences in bulk telomeres being followed by GGT (21,22). Second, if the 50% termination rate holds for all junction sequences, then registrations before T 7 are mostly likely caused by multiple annealing-synthesis-dissociation cycles involving other parts of TLC1, suggesting that (TG) n sequences require multiple cycles and that the first half of the TLC1 homology (the 3Ј end of the template) is used in annealing and not commonly for copying, similar to ciliate telomerases (29). Third, common addition sequences (Fig.…”

Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo

“…1; Greider and Blackburn 1989;Singer and Gottschling 1994;Dandjinou et al 2004;Zappulla and Cech 2004). Together, Est2 and TLC1 form the minimal telomerase catalytic core required for activity in vitro (Zappulla et al 2005), demonstrating that the yeast core enzyme composition is conserved with telomerase of ciliates and mammals (Autexier and Greider 1994;Weinrich et al 1 1997). In vivo, however, several accessory proteins/protein complexes, in addition to Est2, bind to telomerase RNA to form the RNP holoenzyme.…”

Polymerization Defects within Human Telomerase Are Distinct from Telomerase RNA and TEP1 Binding

“…The first telomerase reconstitution assay was accomplished in Tetrahymena, where the functional requirements for the telomerase RNA were delineated by the addition of recombinant telomerase RNA to micrococcal nucleasetreated extracts (Autexier and Greider, 1994;Gilley et al, 1995;Gilley and Blackburn, 1996;Autexier and Greider, 1998;Autexier and Triki, 1999). With a similar assay to study human telomerase, all 5Ј nucleotides leading up to the telomerase RNA template, or the last 240 nucleotides of the telomerase RNA are dispensable for telomerase activity (Autexier et al, 1996) (Figure 1B).…”

“…The mammalian telomerase reverse transcriptase (TERT) shares amino acid sequence similarity with the catalytic telomerase subunit previously identified in ciliates and yeast (Harrington et al, 1997b;Kilian et al, 1997;Lingner et al, 1997;Meyerson et al, 1997;Nakamura et al, 1997). Human TERT is a limiting component for telomerase activity: the hTERT mRNA is often up-regulated in cells containing telomerase activity, and the introduction of hTERT confers telomerase activity to primary human cells Weinrich et al, 1997;Bodnar et al, 1998;Counter et al, 1998;Nakayama et al, 1998;Vaziri and Benchimol, 1998).The first telomerase reconstitution assay was accomplished in Tetrahymena, where the functional requirements for the telomerase RNA were delineated by the addition of recombinant telomerase RNA to micrococcal nucleasetreated extracts (Autexier and Greider, 1994;Gilley et al, 1995;Gilley and Blackburn, 1996;Autexier and Greider, 1998;Autexier and Triki, 1999). With a similar assay to study human telomerase, all 5Ј nucleotides leading up to the telomerase RNA template, or the last 240 nucleotides of the telomerase RNA are dispensable for telomerase activity (Autexier et al, 1996) (Figure 1B).…”