Regulation of Telomere Length by an N-Terminal Region of the Yeast Telomerase Reverse Transcriptase

Abstract

Telomerase is a reverse transcriptase that maintains chromosome integrity through synthesis of repetitive telomeric sequences on the ends of eukaryotic chromosomes. In the yeast Saccharomyces cerevisiae, telomere length homeostasis is achieved through negative regulation of telomerase access to the chromosome terminus by telomere-bound Rap1 protein and its binding partners, Rif1p and Rif2p, and positive regulation by factors such as Ku70/80, Tel1p, and Cdc13p. Here we report the identification of mutations within an N-terminal region (region I) of the yeast telomerase catalytic subunit (Est2p) that cause telomere lengthening without altering measurable catalytic properties of the enzyme in vitro. These telomerase mutations affect telomere length through a Ku-independent mechanism and do not alter chromosome end structure. While Tel1p is required for expression of the telomere-lengthening phenotype, Rif1p and Rif2p are not, suggesting that telomere overextension is independent of Rap1p. Taken together, these data suggest that specific amino acids within region I of the catalytic subunit of yeast telomerase play a previously unanticipated role in the response to Tel1p regulation at the telomere.Telomeres are nucleoprotein structures that cap the ends of linear eukaryotic chromosomes. In most species, these termini are composed of tandem, short G-rich repeats and associated protein complexes (reviewed in reference 56). Telomeres play an essential role in genome stability by preventing recognition of the normal chromosome terminus as a DNA double-strand break. However, the inability of the conventional DNA polymerase machinery to fully replicate terminal sequences results in gradual erosion of telomeric repeats, leading to checkpoint activation and cellular senescence. In cells that maintain proliferative potential, this end replication problem is counteracted by telomerase, a reverse transcriptase capable of synthesizing telomeric repeats onto chromosome ends using an intrinsic RNA template (18,19). In budding yeast, TLC1 and EST2 encode the template RNA and reverse transcriptase subunit, respectively, of the catalytic core of the telomerase holoenzyme (27,31,50). As predicted, strains lacking these genes undergo progressive telomere shortening and senescence (referred to as the EST [ever shorter telomere] phenotype). Mutations of three additional genes (EST1, EST3, and EST4 [CDC13]) result in the same phenotype (27,34,41). However, unlike strains lacking EST2 or TLC1, strains bearing these mutations retain telomerase catalytic activity, suggesting that these proteins are essential regulators of telomere replication in vivo (30,41).Est2p belongs to a family of proteins (TERT; telomerase reverse transcriptase) that contains a domain characteristic of reverse transcriptases in its C-terminal half (31). Sequence alignment among numerous TERT proteins as well as functional analysis in ciliate, yeast, and human TERT (1,12,13,36,38,39,59) TERT region I contributes several functions to the telomerase holoenzyme, many of ...

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

“…This result, combined with biochemical evidence, suggests that mutations in the finger subdomain (including est2-up34) render telomerase at least partially resistant to negative regulation by Pif1p (15). Mutations in three distinct regions cause longer-than-normal telomeres: est2-LT in the TEN domain, est2-up in motifs 1 and 2, and est2-MotifE in the RT domain (7,16,30). The est2-up34 allele utilized in this study (D460N) is indicated with an asterisk.…”

“…This mutation mimics the viral consensus sequence within motif E of the RT domain and increases the nucleotide addition processivity in vitro (30), a phenotype not shared by est2-LT E76K or est2-LT N95A (16). Despite this difference, est2-motifE decreases Rap1p association with telomeres in vivo in a manner indistinguishable from that of est2-LT E76K (Fig.…”

“…Telomerase negatively influences telomere length through three distinct mechanisms corresponding to the three classes of est2 mutations that overelongate telomeres (7,16,30, and this work). Unexpectedly, some of these mutants (est2-LT and est2- FIG.…”

“…PIF1 gene disruption was accomplished in YKF501 (containing pKF410 [proA-EST2 CEN URA3] [16]) by transforming a PCR fragment (the primers were 5Ј-GTAGTTTTGTGATGCTGTTCAG-3Ј and 5Ј-GTGAGTTAGTCTCCTTT GGC-3Ј, and the template was genomic DNA from the pif1⌬::Kan r strain [Open Biosystems, Huntsville, AL]) to generate YKF503. TLC1 was replaced with tlc1-human as described previously (14) in strain YKF501 (containing pKF417 [EST2 CEN LEU2]) to create YKF504.…”

“…In contrast, the est2-up mutants in the finger subdomain (motifs 1 and 2) allow telomerase to escape inhibition by Pif1p helicase (7). We previously identified four est2-LT (for long telomere) mutations in the N-terminal TEN domain (16) that overelongate telomeres by an unknown mechanism. Here, we demonstrate that est2-LT strains do not increase telomeric Rap1p association or gene silencing, as has been observed upon telomere overelongation in other genetic backgrounds.…”