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A yeast telomerase complex containing the Est1 recruitment protein is assembled early in the cell cycle.

ABSTRACT: In budding yeast, association of the Est1 regulatory protein with telomerase is thought to be limited to the late S phase, when telomere elongation occurs. By monitoring the stoichiometry of telomerase subunits, we show instead that a telomerase complex containing Est1 is assembled much earlier in the cell cycle. We also report a biochemical interaction between Est1 and the telomere binding protein Cdc13 that recapitulates the previously observed genetic relationship between EST1 and CDC13. This supports a model in which regulated binding of Cdc13 to chromosome termini dictates subsequent interaction of a recruitment-competent telomerase complex with telomeres.

SUBMITTER: Tucey TM

PROVIDER: S-EPMC4172482 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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A yeast telomerase complex containing the Est1 recruitment protein is assembled early in the cell cycle.

Tucey Timothy M TM Lundblad Victoria V

Biochemistry 20130207 7

In budding yeast, association of the Est1 regulatory protein with telomerase is thought to be limited to the late S phase, when telomere elongation occurs. By monitoring the stoichiometry of telomerase subunits, we show instead that a telomerase complex containing Est1 is assembled much earlier in the cell cycle. We also report a biochemical interaction between Est1 and the telomere binding protein Cdc13 that recapitulates the previously observed genetic relationship between EST1 and CDC13. This ...[more]

PMID: 23390975

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Modulation of yeast telomerase activity by Cdc13 and Est1 in vitro.

Project description:Telomerase is the enzyme involved in extending telomeric DNA. Control of telomerase activity by modulating its access to chromosome ends is one of the most important fundamental mechanisms. This study established an in vitro yeast telomerase reconstitution system that resembles telomere replication in vivo. In this system, a tailed-duplex DNA formed by telomeric DNA was employed to mimic the structure of telomeres. The core catalytic components of telomerase Est2/Tlc1 RNA were used as the telomeric DNA extension machinery. Using the reconstituted systems, this study found that binding of Cdc13 to telomeric DNA inhibited the access of telomerase to its substrate. The result was further confirmed by a single-molecule approach using the tethered-particle motion (TPM)-based telomerase assay. The findings also showed that the inhibitory effect can be relieved by telomerase-associated protein Est1, consistent with the role of Cdc13 and Est1 in regulating telomere extension in vivo. Significantly, this study found that the DNA binding property of Cdc13 was altered by Est1, providing the first mechanistic evidence of Est1 regulating the access of telomerase to its substrate. Thus, the roles of Cdc13 and Est1 in modulating telomerase activity were clearly defined using the in vitro reconstituted system.

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The conserved Est1 protein stimulates telomerase DNA extension activity.

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N-terminal domain of yeast telomerase reverse transcriptase: recruitment of Est3p to the telomerase complex.

Project description:Telomerase is a reverse transcriptase that maintains chromosome ends. The N-terminal half of the catalytic protein subunit (TERT) contains three functional domains (I, II, and III) that are conserved among TERTs but not found in other reverse transcriptases. Guided by an amino acid sequence alignment of nine TERT proteins, mutations were introduced into yeast TERT (Est2p). In support of the proposed alignment, mutation of virtually all conserved residues resulted in loss-of-function or temperature sensitivity, accompanied by telomere shortening. Overexpression of telomerase component Est3p led to allele-specific suppression of the temperature-sensitive mutations in region I, suggesting that Est3p interacts with this protein domain. As predicted by the genetic results, a lethal mutation in region I resulted in loss of Est3p from the telomerase complex. We conclude that Est2p region I is required for the recruitment of Est3p to yeast telomerase. Given the phylogenetic conservation of region I of TERT, this protein domain may provide the equivalent function in all telomerases.

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A second essential function of the Est1-binding arm of yeast telomerase RNA.

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Project description:Telomerase is a telomere dedicated reverse transcriptase that replicates the very ends of eukaryotic chromosomes. Saccharomyces cerevisiae telomerase consists of TLC1 (the RNA template), Est2 (the catalytic subunit), and two accessory proteins, Est1 and Est3, that are essential in vivo for telomerase activity but are dispensable for catalysis in vitro. Est1 functions in both recruitment and activation of telomerase. The association of Est3 with telomeres occurred largely in late S/G2 phase, the time when telomerase acts and Est1 telomere binding occurs. Est3 telomere binding was Est1-dependent. This dependence is likely due to a direct interaction between the two proteins, as purified recombinant Est1 and Est3 interacted in vitro. Est3 abundance was neither cell cycle-regulated nor Est1-dependent. Est3 was the most abundant of the three Est proteins (84.3±13.3 molecules per cell versus 71.1±19.2 for Est1 and 37.2±6.5 for Est2), so its telomere association and/or activity is unlikely to be limited by its relative abundance. Est2 and Est1 telomere binding was unaffected by the absence of Est3. Taken together, these data indicate that Est3 acts downstream of both Est2 and Est1 and that the putative activation function of Est1 can be explained by its role in recruiting Est3 to telomeres.

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Telomerase activation after recruitment in fission yeast.

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Structural Insights into Yeast Telomerase Recruitment to Telomeres.

Project description:Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.

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Proteomics of yeast telomerase identified Cdc48-Npl4-Ufd1 and Ufd4 as regulators of Est1 and telomere length.

Project description:Almost 400 genes affect yeast telomere length, including Est1, which is critical for recruitment and activation of telomerase. Here we use mass spectrometry to identify novel telomerase regulators by their co-purification with the telomerase holoenzyme. In addition to all known subunits, over 100 proteins are telomerase associated, including all three subunits of the essential Cdc48-Npl4-Ufd1 complex as well as three E3 ubiquitin ligases. The Cdc48 complex is evolutionarily conserved and targets ubiquitinated proteins for degradation. Est1 levels are ∼40-fold higher in cells with reduced Cdc48, yet, paradoxically, telomeres are shorter. Furthermore, Est1 is ubiquitinated and its cell cycle-regulated abundance is lost in Cdc48-deficient cells. Deletion of the telomerase-associated E3 ligase, Ufd4, in cdc48-3 cells further increases Est1 abundance but suppresses the telomere length phenotype of the single mutant. These data argue that, in concert with Ufd4, the Cdc48 complex regulates telomerase by controlling the level and activity of Est1.

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Phosphorylation of TPP1 regulates cell cycle-dependent telomerase recruitment.

Project description:Telomere maintenance is essential for organisms with linear chromosomes and is carried out by telomerase during cell cycle. The precise mechanism by which cell cycle controls telomeric access of telomerase and telomere elongation in mammals remains largely unknown. Previous work has established oligonucleotide/oligosaccharide binding (OB) fold-containing telomeric protein TPP1, formerly known as TINT1, PTOP, and PIP1, as a key factor that regulates telomerase recruitment and activity. However, the role of TPP1 in cell cycle-dependent telomerase recruitment is unclear. Here, we report that human TPP1 is phosphorylated at multiple sites during cell cycle progression and associates with higher telomerase activity at late S/G2/M. Phosphorylation of Ser111 (S111) within the TPP1 OB fold appears important for cell cycle-dependent telomerase recruitment. Structural analysis indicates that phosphorylated S111 resides in the telomerase-interacting domain within the TPP1 OB fold. Mutations that disrupt S111 phosphorylation led to decreased telomerase activity in the TPP1 complex and telomere shortening. Our findings provide insight into the regulatory pathways and structural basis that control cell cycle-dependent telomerase recruitment and telomere elongation through phosphorylation of TPP1.

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The TPR-containing domain within Est1 homologs exhibits species-specific roles in telomerase interaction and telomere length homeostasis.

Project description:The first telomerase-associated protein (Est1) was isolated in yeast due to its essential role in telomere maintenance. The human counterparts EST1A, EST1B, and EST1C perform diverse functions in nonsense-mediated mRNA decay (NMD), telomere length homeostasis, and telomere transcription. Although Est1 and EST1A/B interact with the catalytic subunit of yeast and human telomerase (Est2 and TERT, respectively), the molecular determinants of these interactions have not been elaborated fully.To investigate the functional conservation of the EST1 protein family, we performed protein-protein interaction mapping and structure-function analysis. The domain in hEST1A most conserved between species, containing a TPR (tricotetrapeptide repeat), was sufficient for interaction of hEST1A with multiple fragments of hTERT including the N-terminus. Two mutations within the hTERT N-terminus that perturb in vivo function (NAAIRS(92), NAAIRS(122)) did not affect this protein interaction. ScEst1 hybrids containing the TPR of hEST1A, hEST1B, or hEST1C were expressed in yeast strains lacking EST1, yet they failed to complement senescence. Point mutations within and outside the cognate ScEst1 TPR, chosen to disrupt a putative protein interaction surface, resulted in telomere lengthening or shortening without affecting recruitment to telomeres.These results identify a domain encompassing the TPR of hEST1A as an hTERT interaction module. The TPR of S. cerevisiae Est1 is required for telomerase-mediated telomere length maintenance in a manner that appears separable from telomere recruitment. Discrete residues in or adjacent to the TPR of Est1 also regulate telomere length homeostasis.

| S-EPMC3215184 | biostudies-literature

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