Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

Abstract

In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.Telomeres are nucleoprotein structures that protect chromosome ends from nucleolytic digestion and preclude the recognition of normal ends as double-strand breaks (6). In most eukaryotes, telomeres are maintained by telomerase, a ribonucleoprotein that uses a short region of its RNA subunit as a template for reverse transcription. In Saccharomyces cerevisiae, a reverse transcriptase (RT) (EST2) and RNA subunit (TLC1) form the catalytic core of telomerase (18,21,35). Est2p contains at least three functional domains: an N-terminal TEN domain that anchors Est2p on its DNA substrate (23,24) and that may interact with other protein components (10), an RNA binding (RBD) region associates with that TLC1 RNA and contributes to dimerization (20), and an RT domain conserved among other telomerases and viral RTs (21).While many organisms synthesize perfect TG-rich telomeric repeats, several protozoa, fungi, slime molds, and plants have heterogeneous telomere sequences (40). S. cerevisiae displays considerable degeneracy, with a consensus of 5Ј-[(TG) 0-6 TG GGTGTG(G)] n (9). Several models have been proposed to explain this heterogeneity. An analysis of wild-type (WT) telomeres and telomeres generated in the presence of template mutations suggests that the registration of the telomere terminus occurs preferentially at the 3Ј end of the template, with processive synthesis through a central core region and decreasing processivity at the 5Ј end of the template (9, 33). In contrast, telomere junction fragments generated during chromosome healing events are more consistent with nonprocessive synthesis and patterning driven by substrate/template alignment (32). In humanized yeast cells, in which the yeast RNA template is replaced with that of humans, Est2p generates perfect hexanucleotide repeats, suggesting that the...

A mutation in the catalytic subunit of yeast telomerase alters primer–template alignment while promoting processivity and protein–DNA binding

“…Our previous work showed that mutations in EST2 that cause telomere lengthening are associated with changes in the telomere sequence (Ji et al, 2008). Both est2-LT E76K and est2-up34 [D460N; located within the reverse transcriptase domain (Eugster et al, 2006)] cause the same telomere sequence changes despite requiring different genetic pathways for over-elongation (Ji et al, 2008).…”

“…Both est2-LT E76K and est2-up34 [D460N; located within the reverse transcriptase domain (Eugster et al, 2006)] cause the same telomere sequence changes despite requiring different genetic pathways for over-elongation (Ji et al, 2008). Based on these data, it remained possible that telomere sequence changes observed in these est2 mutant strains are an indirect consequence of disrupted telomere length homeostasis.…”

“…Because telomeres in both wild-type and mutant strains contain core motifs, we have analyzed two properties of 'inter-core regions' that lie between core motifs. First, the frequency of core motif overlap is calculated as the number of inter-core regions that consist of overlaps divided by the total number of inter-core regions (Ji et al, 2008). Second, the frequency with which the GG dinucleotide occurs within the telomeric sequence is calculated as the number of inter-core regions containing the GG dinucleotide divided by the total number of core motifs (Ji et al, 2008).…”

“…First, the frequency of core motif overlap is calculated as the number of inter-core regions that consist of overlaps divided by the total number of inter-core regions (Ji et al, 2008). Second, the frequency with which the GG dinucleotide occurs within the telomeric sequence is calculated as the number of inter-core regions containing the GG dinucleotide divided by the total number of core motifs (Ji et al, 2008). By analyzing the sequence of an ADE2-marked telomere lacking subtelomeric repeats in the GA426 EST2 strain, we previously showed that 53% of core motifs are followed by a GG dinucleotide, whereas 29% of the inter-core regions are overlaps.…”

“…Interestingly, although these mutations increase telomere length by about 30% (,100 base pairs), this telomere over-elongation does not appear to be directly related to the change in telomere sequence (Ji et al, 2008). The est2-LT mutations do not affect nucleotide addition processivity in vitro (Ji et al, 2005), but the est2-LT E76K allele increases repeat addition processivity under specific primer extension conditions (Lue and Li, 2007).…”