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The Est3 protein associates with yeast telomerase through an OB-fold domain.

ABSTRACT: The Ever shorter telomeres 3 (Est3) protein is a small regulatory subunit of yeast telomerase which is dispensable for enzyme catalysis but essential for telomere replication in vivo. Using structure prediction combined with in vivo characterization, we show here that Est3 consists of a predicted OB (oligosaccharide/oligonucleotide binding)-fold. We used mutagenesis of predicted surface residues to generate a functional map of one surface of Est3, identifying a site that mediates association with the telomerase complex. Unexpectedly, the predicted OB-fold of Est3 is structurally similar to the OB-fold of the human TPP1 protein, despite the fact that Est3 and TPP1, as components of telomerase and a telomere-capping complex, respectively, perform functionally distinct tasks at chromosome ends. Our analysis of Est3 may be instructive in generating comparable missense mutations on the surface of the OB-fold domain of TPP1.

SUBMITTER: Lee J

PROVIDER: S-EPMC2669685 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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The Est3 protein associates with yeast telomerase through an OB-fold domain.

Lee Jaesung J Mandell Edward K EK Tucey Timothy M TM Morris Danna K DK Lundblad Victoria V

Nature structural & molecular biology 20080901 9

The Ever shorter telomeres 3 (Est3) protein is a small regulatory subunit of yeast telomerase which is dispensable for enzyme catalysis but essential for telomere replication in vivo. Using structure prediction combined with in vivo characterization, we show here that Est3 consists of a predicted OB (oligosaccharide/oligonucleotide binding)-fold. We used mutagenesis of predicted surface residues to generate a functional map of one surface of Est3, identifying a site that mediates association wit ...[more]

PMID: 19172754

Similar Datasets

A proposed OB-fold with a protein-interaction surface in Candida albicans telomerase protein Est3.

Project description:Ever shorter telomeres 3 (Est3) is an essential telomerase regulatory subunit thought to be unique to budding yeasts. Here we use multiple sequence alignment and hidden Markov model-hidden Markov model (HMM-HMM) comparison to uncover potential similarities between Est3 and the mammalian telomeric protein Tpp1. Analysis of site-specific mutants of Candida albicans Est3 revealed functional distinctions between residues that are conserved between Est3 and Tpp1 and those that are unique to Est3. Although both types of residues are important for telomere maintenance in vivo, only the former contributes to telomerase activity in vitro and facilitates the association of Est3 with telomerase core components. Consistent with a function in protein-protein interaction, the residues common to Est3 and Tpp1 map to one face of an OB-fold model structure, away from the canonical nucleic acid binding surface. We propose that Est3 and the OB-fold domain of Tpp1 mediate a conserved function in telomerase regulation.

| S-EPMC2656765 | biostudies-literature

TPP1 OB-fold domain controls telomere maintenance by recruiting telomerase to chromosome ends.

Project description:Telomere synthesis in cancer cells and stem cells involves trafficking of telomerase to Cajal bodies, and telomerase is thought to be recruited to telomeres through interactions with telomere-binding proteins. Here, we show that the OB-fold domain of the telomere-binding protein TPP1 recruits telomerase to telomeres through an association with the telomerase reverse transcriptase TERT. When tethered away from telomeres and other telomere-binding proteins, the TPP1 OB-fold domain is sufficient to recruit telomerase to a heterologous chromatin locus. Expression of a minimal TPP1 OB-fold inhibits telomere maintenance by blocking access of telomerase to its cognate binding site at telomeres. We identify amino acids required for the TPP1-telomerase interaction, including specific loop residues within the TPP1 OB-fold domain and individual residues within TERT, some of which are mutated in a subset of pulmonary fibrosis patients. These data define a potential interface for telomerase-TPP1 interaction required for telomere maintenance and implicate defective telomerase recruitment in telomerase-related disease.

| S-EPMC3516183 | biostudies-literature

The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain.

Project description:Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions.

| S-EPMC3874162 | biostudies-literature

The N Terminus of the OB Domain of Telomere Protein TPP1 Is Critical for Telomerase Action.

Project description:Telomerase recruitment to telomeres and enzymatic processivity are mediated by TPP1, an essential component of telomere integrity and telomerase function. A surface on the OB domain of TPP1 called the TEL patch is critical for TPP1's telomerase-associated functions. Here, we identify a separate region in the N terminus of the OB domain (termed NOB) of TPP1 that, like the TEL patch, is essential for telomerase repeat addition processivity in vitro as well as telomerase recruitment to telomeres and telomere lengthening in cells. Although well-conserved among most mammalian TPP1 homologs, the NOB region in mice is distinct. Swapping the sequence of human NOB into mouse TPP1 allows it to stimulate human telomerase, qualifying NOB as an important determinant of species specificity for TPP1-telomerase interaction. Our studies show that TPP1 NOB is critical for telomerase function and demonstrate that the telomerase interaction surface on TPP1 is more elaborate than previously appreciated.

| S-EPMC5815312 | biostudies-literature

Stimulation of yeast telomerase activity by the ever shorter telomere 3 (Est3) subunit is dependent on direct interaction with the catalytic protein Est2.

Project description:Telomerase is a multisubunit enzyme that maintains genome stability through its role in telomere replication. Although the Est3 protein is long recognized as an essential telomerase component, how it associates with and functions in the telomerase complex has remained enigmatic. Here we provide the first evidence of a direct interaction between Saccharomyces cerevisiae Est3p and the catalytic protein subunit (Est2p) by demonstrating that recombinant Est3p binds the purified telomerase essential N-terminal (TEN) domain of Est2p in vitro. Mutations in a small cluster of amino acids predicted to lie on the surface of Est3p disrupt this interaction with Est2p, reduce assembly of Est3p with telomerase in vivo, and cause telomere shortening and senescence. We also show that recombinant Est3p stimulates telomerase activity above basal levels in vitro in a manner dependent on the Est2p TEN domain interaction. Together, these results define a direct binding interaction between Est3p and Est2p and reconcile the effect of S. cerevisiae Est3p with previous experiments showing that Est3p homologs in related yeast species influence telomerase activity. Additionally, it contributes functional support to the idea that Est3p is structurally related to the mammalian shelterin protein, TPP1, which also influences telomerase activity through interaction with the Est2p homolog, TERT.

| S-EPMC3143607 | biostudies-literature

Investigating the role of the Est3 protein in yeast telomere replication.

Project description:The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Delta strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Delta telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity.

| S-EPMC2853109 | biostudies-literature

Telomerase regulatory subunit Est3 in two Candida species physically interacts with the TEN domain of TERT and telomeric DNA.

Project description:The yeast telomerase regulatory protein Est3 is required for telomere maintenance in vivo, and shares intriguing structural and functional similarities with the mammalian telomeric protein TPP1. Here we report our physical and functional characterizations of Est3 homologues from Candida parapsilosis and Lodderomyces elongisporus, which bear unique N- and C-terminal tails in addition to a conserved central OB fold. We show that these Est3 homologues form stable complexes with the TEN domain of telomerase reverse transcriptase. Efficient complex formation requires both the N- and C-terminal tails, as well as conserved OB fold residues of Est3. Other Est3 homologues devoid of the tails failed to interact strongly with the cognate TEN domains. Remarkably, the C. parapsilosis Est3 alone exhibits no appreciable DNA-binding activity, but can be crosslinked to telomeric DNA in the presence of the TEN domain. A conserved basic residue on the putative DNA-binding surface of CpEst3 is required for efficient crosslinking. Mutating the equivalent residue in Candida albicans Est3 caused telomere attrition. We propose that interaction with the TEN domain unmasks a functionally important nucleic acid-binding activity in Est3. Our findings provide insights on the mechanisms and evolution of a widely conserved and functionally critical telomeric/telomerase component.

| S-EPMC3251127 | biostudies-literature

Bioinformatics analysis identify novel OB fold protein coding genes in C. elegans.

Project description:BackgroundThe C. elegans genome has been extensively annotated by the WormBase consortium that uses state of the art bioinformatics pipelines, functional genomics and manual curation approaches. As a result, the identification of novel genes in silico in this model organism is becoming more challenging requiring new approaches. The Oligonucleotide-oligosaccharide binding (OB) fold is a highly divergent protein family, in which protein sequences, in spite of having the same fold, share very little sequence identity (5-25%). Therefore, evidence from sequence-based annotation may not be sufficient to identify all the members of this family. In C. elegans, the number of OB-fold proteins reported is remarkably low (n=46) compared to other evolutionary-related eukaryotes, such as yeast S. cerevisiae (n=344) or fruit fly D. melanogaster (n=84). Gene loss during evolution or differences in the level of annotation for this protein family, may explain these discrepancies.Methodology/principal findingsThis study examines the possibility that novel OB-fold coding genes exist in the worm. We developed a bioinformatics approach that uses the most sensitive sequence-sequence, sequence-profile and profile-profile similarity search methods followed by 3D-structure prediction as a filtering step to eliminate false positive candidate sequences. We have predicted 18 coding genes containing the OB-fold that have remarkably partially been characterized in C. elegans.Conclusions/significanceThis study raises the possibility that the annotation of highly divergent protein fold families can be improved in C. elegans. Similar strategies could be implemented for large scale analysis by the WormBase consortium when novel versions of the genome sequence of C. elegans, or other evolutionary related species are being released. This approach is of general interest to the scientific community since it can be used to annotate any genome.

| S-EPMC3636199 | biostudies-literature

Nucleic acid recognition by OB-fold proteins.

Project description:The OB-fold domain is a compact structural motif frequently used for nucleic acid recognition. Structural comparison of all OB-fold/nucleic acid complexes solved to date confirms the low degree of sequence similarity among members of this family while highlighting several structural sequence determinants common to most of these OB-folds. Loops connecting the secondary structural elements in the OB-fold core are extremely variable in length and in functional detail. However, certain features of ligand binding are conserved among OB-fold complexes, including the location of the binding surface, the polarity of the nucleic acid with respect to the OB-fold, and particular nucleic acid-protein interactions commonly used for recognition of single-stranded and unusually structured nucleic acids. Intriguingly, the observation of shared nucleic acid polarity may shed light on the longstanding question concerning OB-fold origins, indicating that it is unlikely that members of this family arose via convergent evolution.

| S-EPMC1564333 | biostudies-literature

Insights into the structure and function of Est3 from the Hansenula polymorpha telomerase.

Project description:Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.

| S-EPMC7338525 | biostudies-literature

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