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The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The CST (CTC1-STN1-TEN1) complex mediates critical functions in maintaining telomere DNA and overcoming genome-wide replication stress. A conserved biochemical function of the CST complex is its primase-Pol α (PP) stimulatory activity. In this report, we demonstrate the ability of purified human STN1 alone to promote PP activity in vitro. We show that this regulation is mediated primarily by the N-terminal OB fold of STN1, but does not require the DNA-binding activity of this domain. Rather, we observed a strong correlation between the PP-stimulatory activity of STN1 variants and their abilities to bind POLA2. Remarkably, the main binding target of STN1 in POLA2 is the latter's central OB fold domain. In the substrate-free structure of PP, this domain is positioned so as to block nucleic acid entry to the Pol α active site. Thus the STN1-POLA2 interaction may promote the necessary conformational change for nucleic acid delivery to Pol α and subsequent DNA synthesis. A disease-causing mutation in human STN1 engenders a selective defect in POLA2-binding and PP stimulation, indicating that these activities are critical for the in vivo function of STN1. Our findings have implications for the molecular mechanisms of PP, STN1 and STN1-related molecular pathology., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Ku heterodimer serves in the initial step in repairing DNA double-strand breaks by the non-homologous end-joining pathway. Besides this key function, Ku also plays a role in other cellular processes including telomere maintenance. Inactivation of Ku can lead to DNA repair defects and telomere aberrations. In model organisms where Ku has been studied, inactivation can lead to DNA repair defects and telomere aberrations. In general Ku deficient mutants are viable, but a notable exception to this is human where Ku has been found to be essential. Here we report that similar to the situation in human Ku is required for cell proliferation in the fungus Ustilago maydis. Using conditional strains for Ku expression, we found that cells arrest permanently in G2 phase when Ku expression is turned off. Arrest results from cell cycle checkpoint activation due to persistent signaling via the DNA damage response (DDR). Our results point to the telomeres as the most likely source of the DNA damage signal. Inactivation of the DDR makes the Ku complex dispensable for proliferation in this organism. Our findings suggest that in U. maydis, unprotected telomeres arising from Ku depletion are the source of the signal that activates the DDR leading to cell cycle arrest., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The telomere repeat units of Candida species are substantially longer and more complex than those in other organisms, raising interesting questions concerning the recognition mechanisms of telomere-binding proteins. Herein we characterized the properties of Candida parapsilosis Cdc13A and Cdc13B, two paralogs that are responsible for binding and protecting the telomere G-strand tails. We found that Cdc13A and Cdc13B can each form complexes with itself and a heterodimeric complex with each other. However, only the heterodimer exhibits high-affinity and sequence-specific binding to the telomere G-tail. EMSA and crosslinking analysis revealed a combinatorial mechanism of DNA recognition, which entails the A and B subunit making contacts to the 3' and 5' region of the repeat unit. While both the DBD and OB4 domain of Cdc13A can bind to the equivalent domain in Cdc13B, only the OB4 complex behaves as a stable heterodimer. The unstable Cdc13AB(DBD) complex binds G-strand with greatly reduced affinity but the same sequence specificity. Thus the OB4 domains evidently contribute to binding by promoting dimerization of the DBDs. Our investigation reveals a rare example of combinatorial recognition of single-stranded DNA and offers insights into the co-evolution of telomere DNA and cognate binding proteins.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for extending one strand of the telomere terminal repeats. Unique among reverse transcriptases, telomerase is thought to possess a DNA-binding domain (known as anchor site) that allows the enzyme to add telomere repeats processively. Previous crosslinking and mutagenesis studies have mapped the anchor site to an N-terminal region of TERT, and the structure of this region of Tetrahymena TERT was recently determined at atomic resolutions. Here we use a combination of homology modeling, electrostatic calculation and site-specific mutagenesis analysis to identify a positively charged, functionally important surface patch on yeast TERT. This patch is lined by both conserved and non-conserved residues, which when mutated, caused loss of telomerase processivity in vitro and telomere shortening in vivo. In addition, we demonstrate that a point mutation in this domain of yeast TERT simultaneously enhanced the repeat addition processivity of telomerase and caused telomere elongation. Our data argue that telomerase anchor site has evolved species-specific residues to interact with species-specific telomere repeats. The data also reinforce the importance of telomerase processivity in regulating telomere length.

The telomerase ribonucleoprotein in Candida albicans is presumed to contain at least three Est proteins: CaEst1p, CaEst2p/TERT and CaEst3p. We constructed mutants missing each of the protein subunit of telomerase and analyzed overall telomere dynamics and single-stranded telomere overhangs over the course of many generations. The est1-DeltaDelta mutant manifested abrupt telomere loss and recovery, consistent with heightened recombination. Both the est2-DeltaDelta and est3-DeltaDelta mutant exhibited progressive telomere loss, followed by the gradual emergence of survivors with long telomeres. In no case was telomere loss accompanied by severe growth defects, suggesting that cells with short telomeres can continue to proliferate. Furthermore, the amount of G-strand terminal overhangs was greatly increased in the est2-DeltaDelta mutant, but not others. Our results suggest that in addition to their well-characterized function in telomere elongation, both CaEst1p and CaEst2p mediate some aspects of telomere protection in Candida, with the former suppressing excessive recombination, and the latter preventing excessive C-strand degradation.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Telomerase-mediated sequence addition is dictated by a short 'template' region of the RNA component. Despite the short template segment, telomerases from many organisms have been shown to mediate the synthesis of long extension products. This synthesis presumably depends on two types of translocation events: simultaneous translocation of the RNA-DNA duplex relative to the active site after each nucleotide incorporation (type I or nucleotide addition processivity), and translocation of the RNA relative to the DNA product after each round of repeat synthesis (type II or repeat addition processivity). In contrast, telomerases from yeasts have been shown to synthesize mostly short products, implying a defect in one or both types of translocation. In this report, we analyzed the processivity of yeast telomerase in vitro, and identified two position-specific elongation barriers within the 5' region of the RNA template that can account for the synthesis of incomplete first round products. These barriers respond differently to variations in nucleotide concentration, primer sequence and mutations in the catalytic protein subunit, consistent with their having distinct mechanistic bases. In addition, by using optimal primers and high concentrations of dGTP, we were able to detect significant type II translocation by the yeast enzyme. Thus, the difference between the elongation property of yeast and other telomerases appears to be quantitative rather than qualitative. Our results suggest that yeast may be a useful system for investigating the physiologic significance of repeat addition processivity.

Telomerase is a ribonucleoprotein reverse trans-criptase responsible for the maintenance of one strand of telomere terminal repeats. The mechanisms whereby telomerase recognizes chromosomal ends are not fully characterized. Earlier studies showed that the yeast telomerase RNP could bind the dG-rich strand of yeast telomeres with high affinity and sequence specificity. Further analysis of telomerase-telomere complex formation in vitro as described in this report led to the following conclusions. First, telomerase binding to short DNAs is magnesium-dependent, while binding to long DNAs is magnesium-independent, consistent with the existence of more than one interaction site. Second, binding is likely to be mediated largely through the RNA subunit of telomerase (TLC1), because de-proteinated TLC1 RNA also binds telomeres with high affinity and sequence specificity, and exhibits the same length and divalent cation dependence as telomerase RNP. The crucial role of RNA in binding is further supported by the ability of TLC1 transcripts synthesized in vitro to form stable complexes with telomeric DNA. Finally, results from deletion analysis and RNase H-mediated cleavage suggest that a specific conformation(s) of the RNA is required for stable binding, and that non-template regions of the TLC1 RNA may contribute directly or indirectly to the stability of the RNA-DNA complex.

A number of published studies indicate that telomerase may interact with oligonucleotide primers in a bipartite manner, with the 3'-end of the primer positioned at the catalytic site of the enzyme and a more 5' region of the primer binding to a second or 'anchor' site of the enzyme. We systematically investigated the effects of mutations in the DNA primer on overall binding and polymerization by yeast telomerase. Our studies indicate that there is sequence-specific interaction between telomerase and a substantial region of the DNA primer. Mutations in the 3'-most positions of the primer reduced polymerization, yet had little effect on overall binding affinity. In contrast, mutations around the -20 position reduced binding affinity but had no effect on polymerization. Most strikingly, mutations centered around the -12 position of the DNA primer reduced overall binding affinity but dramatically enhanced primer extension, as well as primer cleavage. This finding suggests that reduced interaction with the -12 region of the DNA primer can facilitate a step in the catalytic region of yeast telomerase that leads to greater polymerization. A tripartite model of interaction between primer and telomerase is proposed to account for the distinct effects of mutations in different regions of the DNA primer.

A gel mobility shift assay was developed to examine recognition of yeast telomeres by telomerase. An RNase-sensitive G-rich strand-specific binding activity can be detected in partially purified yeast telomerase fractions. The binding activity was attributed to telomerase, because it co-purifies with TLC1 RNA and telomerase activity over three different chromatographic steps and because the complex co-migrates with TLC1 RNA when subjected to electrophoresis through native gels. Analysis of the binding specificity of yeast telomerase indicates that it recognizes the G-rich strand of yeast telomeres with high affinity and specificity. The K d for the interaction is approximately 3 nM. Single-stranded G-rich telomeres from other species, such as human and Tetrahymena, though capable of being extended by yeast telomerase in polymerization assays at high concentrations, bind the enzyme with at least 100-fold lower affinities. The ability of a sequence to be bound tightly by yeast telomerase in vitro correlates with its ability to seed telomere formation in vivo. The implications of these findings for regulation of telomerase activity are discussed.

A telomerase-like primer extension activity has been detected in chromatographic fractions derived from Schizosaccharomyces pombe extracts. This primer extension activity acts preferentially on dG-rich oligodeoxynucleotides, is sensitive to RNase A pretreatment and requires all four deoxynucleotides for optimal polymerization. The extension products are also truncated by the inclusion of any one of the four dideoxynucleotides, consistent with the presence of all four bases in the S.pombe telomeric repeats. The intensity distribution of the extension products and the dideoxynucleotide termination pattern suggest that nucleotide addition is template directed, and that telomere-like sequences are added to the primers. In particular, the sequence d(CGGTTA), a variant of the S.pombe telomeric repeat, can be added directly by the in vitro activity. Partially purified S.pombe telomerase sediments as a 35S particle, suggesting that it exists in vivo as part of a large multi-protein complex.