Evolution of CST function in telomere maintenance

Telomeres consist of an elaborate, higher-order DNA architecture, and a suite of proteins that provide protection for the chromosome terminus by blocking inappropriate recombination and nucleolytic attack. In addition, telomeres facilitate telomeric DNA replication by physical interactions with telomerase and the lagging strand replication machinery. The prevailing view has been that two distinct telomere capping complexes evolved, shelterin in vertebrates and a trimeric complex comprised of Cdc13, Stn1 and Ten1 (CST) in yeast. The recent discovery of a CST-like complex in plants and humans raises new questions about the composition of telomeres and their regulatory mechanisms in multicellular eukaryotes. In this review we discuss the evolving functions and interactions of CST components and their contributions to chromosome end protection and DNA replication.Key words: telomere, telomerase, telomere protein, CTC1, STN1, TEN1, OB-fold, arabidopsis, DNA polymerase alpha, RPA     

Evolution of CST function in telomere maintenance

Telomeres consist of an elaborate, higher-order DNA architecture, and a suite of proteins that provide protection for the chromosome terminus by blocking inappropriate recombination and nucleolytic attack, and facilitate telomeric DNA replication by physical interactions with telomerase and the lagging strand replication machinery. The prevailing view has been that two distinct telomere capping complexes evolved, shelterin in vertebrates and a trimeric complex comprised of Cdc13, Stn1 and Ten1 (CST) in yeast. The recent discovery of a CST-like complex in plants and humans raises new questions about the composition of telomeres and their regulatory mechanisms in multicellular eukaryotes. In this review we discuss the evolving functions and interactions of CST components and their contributions to chromosome end protection and DNA replication.     

DNA organization and polymorphism of a wild-type Drosophila telomere region

Telomeres at the ends of linear chromosomes of eukaryotes protect the chromosome termini from degradation and fusion. While telomeric replication/elongation mechanisms have been studied extensively, the functions of subterminal sequences are less well understood. In general, subterminal regions can be quite polymorphic, varying in size from organism to organism, and differing among chromosomes within an organism. The subterminal regions of Drosophila melanogaster are not well characterized today, and it is not known which and how many different components they contain. Here we present the molecular characterization of DNA components and their organization in the subterminal region of the left arm of chromosome 2 of the Oregon RC wildtype strain of D. melanogaster, including a minisatellite with a 457 bp repeat length. Two distinct polymorphic arrangements at 2L were found and analyzed, supporting the Drosophila telomere elongation model by retrotransposition. The high incidence of terminal chromosome deficiencies occurring in natural Drosophila populations is discussed in view of the telomere structure at 2L.     

Mammalian telomere biology

The review considers the function of the important chromosome regions telomeres in normal and immortal cells. Telomeres are dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and end-to-end fusion. The functional state of telomeres depends on many interrelated parameters such as telomerase activity, the status of the telomere safety complex shelterin, and telomere-associated proteins (replication, recombination, DNA break repair factors, etc.). Special attention is paid to the mechanisms that control the telomere length in normal and immortal cells as well as in cells containing or lacking active telomerase. The features attributed to an alternative telomere length control are analyzed, in particular, in view of a recently discovered additional mechanism of telomere shortening by t-cycle trimming. The possibility of expressing both telomerase-dependent and recombinational pathways of telomere length control in normal mammalian cells is considered, as well as the role of shelterin proteins in choosing one of them to be dominant. The review additionally discusses the role of telomeres in the spatial organization of the nucleus during mitosis and meiosis and specific telomere organizations in mammals, including Iberian shrews with their unusual or rare chromosome structures.     

HOT1 is a mammalian direct telomere repeat‐binding protein contributing to telomerase recruitment

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC‐based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere‐binding protein 1 (HOT1). HOT1 directly and specifically binds double‐stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase‐dependent telomere elongation.     

Cell-cycle-dependent telomere elongation by telomerase in budding yeast

Telomeres are essential for the stability and complete replication of linear chromosomes. Telomere elongation by telomerase counteracts the telomere shortening due to the incomplete replication of chromosome ends by DNA polymerase. Telomere elongation is cell-cycle-regulated and coupled to DNA replication during S-phase. However, the molecular mechanisms that underlie such cell-cycle-dependent telomere elongation by telomerase remain largely unknown. Several aspects of telomere replication in budding yeast, including the modulation of telomere chromatin structure, telomere end processing, recruitment of telomere-binding proteins and telomerase complex to telomere as well as the coupling of DNA replication to telomere elongation during cell cycle progression will be discussed, and the potential roles of Cdk (cyclin-dependent kinase) in these processes will be illustrated.     

Agents that target telomerase and telomeres

Telomeres are guanine-rich regions that are located at the ends of chromosomes and are essential for preventing aberrant recombination and protecting against exonucleolytic DNA degradation. Telomeres are maintained by telomerase, an RNA-dependent DNA polymerase. Because telomerase is known to be expressed in tumor cells, which concurrently have short telomeres, and not in most somatic cells, which usually have long telomeres, telomerase and telomere structures have been recently proposed as attractive targets for the discovery of new anticancer agents. The most exciting current strategies are aimed at specifically designing new drugs that target telomerase or telomeres and new models have been formulated to study the biological effects of inhibitors of telomerase and telomeres both in vitro and in vivo.     

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G‐rich sequence, and with noncoding RNA, Telomeric repeat‐containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2′‐OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G‐quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate – the 3′‐end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3′‐end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Chromosome end elongation by recombination in the mosquito Anopheles gambiae.

One of the functions of telomeres is to counteract the terminal nucleotide loss associated with DNA replication. While the vast majority of eukaryotic organisms maintain their chromosome ends via telomerase, an enzyme system that generates short, tandem repeats on the ends of chromosomes, other mechanisms such as the transposition of retrotransposons or recombination can also be used in some species. Chromosome end regression and extension were studied in a medically important mosquito, the malaria vector Anopheles gambiae, to determine how this dipteran insect maintains its chromosome ends. The insertion of a transgenic pUChsneo plasmid at the left end of chromosome 2 provided a unique marker for measuring the dynamics of the 2L telomere over a period of about 3 years. The terminal length was relatively uniform in the 1993 population with the chromosomes ending within the white gene sequence of the inserted transgene. Cloned terminal chromosome fragments did not end in short repeat sequences that could have been synthesized by telomerase. By late 1995, the chromosome ends had become heterogeneous: some had further shortened while other chromosomes had been elongated by regenerating part of the integrated pUChsneo plasmid. A model is presented for extension of the 2L chromosome by recombination between homologous 2L chromosome ends by using the partial plasmid duplication generated during its original integration. It is postulated that this mechanism is also important in wild-type telomere elongation.     

Interaction of an Arabidopsis RNA-binding protein with plant single-stranded telomeric DNA modulates telomerase activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3' overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.     

Budding yeast with human telomeres: a puzzling structure

Telomeres share some common features among eukaryotes, with few exceptions such as the fruit fly Drosophila that uses transposons as telomeres, they consist of G-rich repetitive DNA that is elongated by telomerase and/or alternative pathways depending on recombination. Telomere structure comprises both cis-acting satellite DNA (telomeric DNA) and proteins that interact directly and/or indirectly with the underlying DNA. Telomeric DNAs are surprisingly conserved among the vertebrates and very similar in most eukaryotes, but present some differences in yeast such as Saccharomyces cerevisiae. The telomeric proteins are more variable although the basic mechanisms which control telomere lengthening and capping are very similar, in fact orthologues of the yeast telomeric proteins, which have been studied first, have been identified in other organisms. Here we describe the structure of human telomeres in budding yeast as compared to canonical yeast and mammalian telomeres taking into consideration the more recent findings highlighting the mechanisms that are responsible for chromosome end protection and lengthening, and the role of chromatin organization in telomere function. This yeast represents a model for the study of mammalian telomeres that could be reconstituted step-by-step in all their components, moreover it could be useful for the assembly of mammalian artificial chromosome.     

Telomere maintenance in Drosophila: Rapid transposon evolution at chromosome ends

The maintenance of terminal sequences is an important role of the telomere, since it prevents the loss of internal regions that encode essential genes. In most eukaryotes, this is accomplished by the telomerase. However, telomere length can also be maintained by other mechanisms, such as homologous recombination and transposition of telomeric retrotransposons to the chromosome ends. A remarkable situation is the case of Drosophila, where telomerase was lost, and thus telomeres managed to be maintained by occasional retrotransposition of telomeric elements to the receding ends. In the recent analysis of 12 Drosophila genomes, ¬¬the multiplicity of autonomous and non-autonomous telomere-specific retrotransposons has revealed extensive and rapid evolution of telomeric DNA. The phylogenetic relationship among these telomeric retrotransposons is congruent with the species phylogeny, suggesting that they have been vertically transmitted from a common ancestor. In this review, we also suggest that the formation of a non-canonical DNA structure at Drosophila telomeres could be the way to protect the ends.     

Linear chromosome maintenance in the absence of essential telomere-capping proteins

Telomeres were defined by their ability to cap chromosome ends. Proteins with high affinity for the structure at chromosome ends, binding the G-rich, 3' single-stranded overhang at telomeres include Pot1 in humans and fission yeast, TEBP in Oxytricha nova and Cdc13 in budding yeast. Cdc13 is considered essential for telomere capping because budding yeast that lack Cdc13 rapidly accumulate excessive single-stranded DNA (ssDNA) at telomeres, arrest cell division and die. Cdc13 has a separate, critical role in telomerase recruitment to telomeres. Here, we show that neither Cdc13 nor its partner Stn1 are necessary for telomere capping if nuclease activities that are active at uncapped telomeres are attenuated. Recombination-dependent and -independent mechanisms permit maintenance of chromosomes without Cdc13. Our results indicate that the structure of the eukaryotic telomere cap is remarkably flexible and that changes in the DNA damage response allow alternative strategies for telomere capping to evolve.