Specific vulnerability of long telomeres to undergo end fusions revealed by mutational analysis of Rap1

The conserved Rap1 protein is part of the shelterin complex that plays critical roles in chromosome end protection and telomere length homeostasis. Previous studies addressed how fission yeast Rap1 contributes to telomere length maintenance, but the mechanism by which the protein inhibits end fusions has remained elusive. Here, we use a genetic screen in combination with high throughput sequencing to identify several amino acid positions in Rap1 that have a key role in end protection. Interestingly, mutations at these sites render cells susceptible to genome instability in a conditional manner with longer telomeres being prone to undergoing end fusions, while short telomeres are sufficiently protected. The protection of long telomeres requires their nuclear envelope attachment mediated by the Rap1-Bqt4 interaction. Our data demonstrates that longer telomeres pose an additional challenge for the maintenance of genome integrity and provides an explanation for a species-specific upper limit in telomere length.

Telomere Targeting Approaches in Cancer: Over the Length Maintenance.

Telomeres are crucial structures that preserve genome stability. Their progressive erosion over rounds of DNA duplication determines senescence of cells and organisms. Telomere length homeostasis is critical for cancer development then telomere maintenance mechanisms are established targets in cancer treatment. Besides telomere elongation, telomere’s dysfunction impinges on intracellular signalling pathways, in particular DNA damage signalling and repair affecting cancer cell survival and proliferation. This review summarizes and discusses about the recent findings in anti-cancer drug development targeting different “telosome” components.

Mechanistic Model of Telomere Length Homeostasis in Yeast

Cells maintain homeostatic telomere length, and this homeostatic disruption leads to various types of diseases. Presently, it is not clear how telomeres achieve homeostasis. One of the prevailing hypotheses is a protein-counting model with a built-in sensor mechanism that counts proteins that directly regulate the telomeric length. However, it does not explain telomere length regulation at the mechanistic level. Here, we present a mathematical model based on the underlying molecular mechanisms of length regulation needed to establish telomere length homeostasis in yeast. We perform both deterministic and stochastic simulations to validate the models with the experimental data of Teixeira et al., rate-balance plot, and phase plane analysis to understand the nature of dynamics exhibited by the models. For global analysis, we constructed bifurcation diagrams. The model explains the role of negative and positive feedback loops and a delay between telomerase and telomere-bound proteins, leading to oscillations in telomere length. We map these in-silico results to proposition by Teixeira of telomeres making a transition between extendible and non-extendible states.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment

The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214–218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

Spontaneous replication fork collapse regulates telomere length homeostasis in wild type cells

Telomeres present unique challenges for genomes with linear chromosomes, including the inability of the semi-conservative DNA replication machinery to fully duplicate the ends of linear molecules. This is solved in virtually all eukaryotes by the enzyme telomerase, through the addition of telomeric repeats onto chromosome ends. It is widely assumed that the primary site of action for telomerase is the single-stranded G-rich overhang at the ends of chromosomes, formed after DNA replication is complete. We show here that the preferred substrate for telomerase in wild type yeast is instead a collapsed fork generated during replication of duplex telomeric DNA. Furthermore, newly collapsed forks are extensively elongated by telomerase by as much as ∼200 nucleotides in a single cell division, indicating that a major source of newly synthesized telomeric repeats in wild type cells occurs at collapsed forks. Fork collapse and the subsequent response by telomerase are coordinated by the dual activities of a telomere-dedicated RPA-like complex, which facilitates replication of duplex telomeric DNA and also recruits telomerase to the fork, thereby ensuring a high probability of re-elongation if DNA replication fails. We further show that the ability of telomerase to elongate newly collapsed forks is dependent on the Rad51 protein, indicating that telomerase activity in response to fork collapse proceeds through a regulatory pathway distinct from how telomerase engages fully replicated chromosome termini. We propose a new model in which spontaneous replication fork collapse and the subsequent response by telomerase is a major determinant of telomere length homeostasis.

The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis

Shelterin is a six-proteins complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

Telomere maintenance in interplay with DNA repair in pathogenesis and treatment of colorectal cancer

Colorectal cancer (CRC) continues to be one of the leading malignancies and causes of tumour-related deaths worldwide. Both impaired DNA repair mechanisms and disrupted telomere length homeostasis represent key culprits in CRC initiation, progression and prognosis. Mechanistically, altered DNA repair results in the accumulation of mutations in the genome and, ultimately, in genomic instability. DNA repair also determines the response to chemotherapeutics in CRC treatment, suggesting its utilisation in the prediction of therapy response and individual approach to patients. Telomere attrition resulting in replicative senescence, simultaneously by-passing cell cycle checkpoints, is a hallmark of malignant transformation of the cell. Telomerase is almost ubiquitous in advanced solid cancers, including CRC, and its expression is fundamental to cell immortalisation. Therefore, there is a persistent effort to develop therapeutics, which are telomerase-specific and gentle to non-malignant tissues. However, in practice, we are still at the level of clinical trials. The current state of knowledge and the route, which the research takes, gives us a positive perspective that the problem of molecular models of telomerase activation and telomere length stabilisation will finally be solved. We summarise the current literature herein, by pointing out the crosstalk between proteins involved in DNA repair and telomere length homeostasis in relation to CRC.

Loss of Ku’s DNA end binding activity affects telomere length via destabilizing telomere-bound Est1 rather than altering TLC1 homeostasis

ABSTRACTSaccharomyces cerevisiae telomerase, which maintains telomere length, is comprised of an RNA component, TLC1, the reverse transcriptase, Est2, and regulatory subunits, including Est1. The Yku70/Yku80 (Ku) heterodimer, a DNA end binding (DEB) protein, also contributes to telomere length maintenance. Ku binds TLC1 and telomere ends in a mutually exclusive fashion, and is required to maintain levels and nuclear localization of TLC1. Ku also interacts with Sir4, which localizes to telomeres. Here we sought to determine the role of Ku’s DEB activity in telomere length maintenance by utilizing yku70-R456E mutant strains, in which Ku has reduced DEB and telomere association but proficiency in TLC1 and Sir4 binding, and TLC1 nuclear retention. Telomere lengths in a yku70-R456E strain were nearly as short as those in ykuΔ strains and shorter than in strains lacking either Sir4, Ku:Sir4 interaction, or Ku:TLC1 interaction. TLC1 levels were decreased in the yku70-R456E mutant, yet overexpression of TLC1 failed to restore telomere length. Reduced DEB activity did not impact Est1’s ability to associate with telomerase but did result in decreased association of Est1 with the telomere. These findings suggest Ku’s DEB activity maintains telomere length homeostasis by preserving Est1’s interaction at the telomere rather than altering TLC1 levels.