Cytological and functional aspects of telomere maintenance

The fact that eukaryotic chromosomes are linear poses a special problem for their maintenance: the natural ends of chromosomes must be distinguished from ends generated by chromosomal breakage and somehow, the chromosome ends must also be fully replicated to maintain their integrity. Telomeres, the complex structures at the ends of chromosomes are thought to be instrumental for both of these functions. However, recent insights in telomere biology suggest that these terminal structures do much more than just fulfill these two basic functions. Cytological data demonstrate that telomeres may play leading roles in chromatin organization and nuclear architecture during mitosis and meiosis. Moreover, non-functional telomeres may lead to genetic instability, a common prelude to cancer. Here, we review the basic functions of telomeres during chromosome replication and discuss the cytological aspects of telomere function during mitosis and meiosis.     

Does a Sentinel or a Subset of Short Telomeres Determine Replicative Senescence?

The proliferative life span of human cells is limited by telomere shortening, but the specific telomeres responsible for determining the onset of senescence have not been adequately determined. We here identify the shortest telomeres by the frequency of signal-free ends after in situ hybridization with telomeric probes and demonstrate that probes adjacent to the shortest ends colocalize with gammaH2AX-positive DNA damage foci in senescent cells. Normal BJ cells growth arrest at senescence before developing significant karyotypic abnormalities. We also identify all of the telomeres involved in end-associations in BJ fibroblasts whose cell-cycle arrest at the time of replicative senescence has been blocked and demonstrate that the 10% of the telomeres with the shortest ends are involved in >90% of all end-associations. The failure to find telomeric end-associations in near-senescent normal BJ metaphases, the presence of signal-free ends in 90% of near-senescent metaphases, and the colocalization of short telomeres with DNA damage foci in senescent interphase cells suggests that end-associations rather than damage signals from short telomeres per se may be the proximate cause of growth arrest. These results demonstrate that a specific group of chromosomes with the shortest telomeres rather than either all or only one or two sentinel telomeres is responsible for the induction of replicative senescence.     

Role of the TRF2 Telomeric Protein in Cancer and Aging

Telomeres are the special heterochromatin that forms the ends of chromosomes, consisting of TTAGGG repeats and associated proteins. Telomeres protect the ends from degradation and recombination, and are essential for chromosomal stability. Both a minimal length of telomere repeats and the telomere-binding proteins are required for telomere protection. Telomerase is a DNA polymerase that specifically elongates telomeres, in this way regulating telomere length and function. A minimal telomere length is required to maintain tissue homeostasis. On one hand, critically short telomeres trigger loss of cell viability and premature death in mice deficient for telomerase activity. Furthermore, altered functioning of telomerase and telomere-interacting proteins is present in some human premature ageing syndromes and cancer. A new mouse model with critically short telomeres has been generated by over-expressing the TRF2 telomere-binding protein, K5-TRF2 mice. These mice show short telomeres in the presence of telomerase activity, leading to premature aging and increased cancer. Short telomeres in TRF2 mice can be rescued in the absence of the XPF nuclease, indicating that this enzyme rapidly degrades telomeres in the presence of increased TRF2 expression. K5-TRF2 mice represent a new tool to understand the consequences of critical telomere shortening a telomerase-proficient genetic background, more closely resembling human cancer and aging pathologies.     

Genetic instability of the Streptomyces chromosome

The Streptomyces wild-type chromosome is linear in all examples studied. The ends of the chromosome or telomeres consist of terminal inverted repeats of various sizes with proteins covalently bound to their 5' ends. The chromosome is very unstable and undergoes very large deletions spontaneously at rates higher than 0.1% of spores. Frequently, the telomeres are included in the deletions. Loss of both telomeres leads to circularization of the chromosome. The wild-type chromosome can also be circularized artificially by targeted recombination. Spontaneously or artificially circularized chromosomes are even more unstable than the linear ones. High-copy-number tandem amplifications of specific chromosomal regions are frequently associated with the deletions. RecA seems to be involved in the amplification mechanism and control of genetic instability.     

A family of complex tandem DNA repeats in the telomeres of Chironomus pallidivittatus.

A family of 340-bp tandem telomere-associated DNA repeats is present in 50- to 200-kb blocks in seven of the eight paired chromosome ends in Chironomus pallidivittatus. It consists of four main subfamilies, differing from each other by small clusters of mutations. This differentiation may reflect different functional roles for the repeats. Here we find that one subfamily, D3, is consistently localized most peripherally and extends close to the ends of the chromosomes, as shown by its sensitivity to the exonuclease Bal 31. The amounts of D3 are highly variable between individuals. The repeat characteristic for D3 forms a segment with pronounced dyad symmetry, which in single-strand form would give rise to a hairpin. Evidence from an interspecies comparison suggests that a similar structure is the result of selective forces. Another subfamily, M1, is present more proximally in a subgroup of telomeres characterized by a special kind of repeat variability. Thus, a complex block with three kinds of subfamilies may occupy different M1 telomeres depending on the stock of animals. We conclude that subfamilies are differentially distributed between and within telomeres and are likely to serve different functions.     

Telomeres and double-strand breaks - all's well that "ends" well. ..

Bailey, S. Telomeres and Double-Strand Breaks - All's Well that "Ends" Well. ... Radiat. Res. 169, 1-7 (2008). Sometimes one's life (including one's science) makes a lot more sense when viewed from the perspective of time, reflected back on over a number of years. That has indeed been the case for me. Strangely enough, the story begins with chromosomes and "ends" with telomeres, both at Colorado State University. And, just as with chromosomes, a lot happened in between. Telomeres were first identified based on their function-they protected the physical ends of chromosomes from interaction with broken DNA ends created by ionizing radiation. While I was at Los Alamos National Laboratory, the sequence of human telomeres was discovered, making probes available that allowed us to re-examine and provide direct support of these early observations; thus began my fascination with telomeres. Chromosome orientation in situ hybridization (CO-FISH) also came onto the scene while I was in Los Alamos. This strand-specific modification of standard FISH, especially when combined with telomeric sequence probes, has proven to be a powerful approach that provides information not available by any other means. Applications have included pericentric inversion detection, distinction between leading- and lagging-strand telomeres, and identification of telomere-double-strand break (DSB) fusions. We also provided the first direct evidence that DSB repair proteins (DNA-PK in particular) are required for mammalian telomeric end capping, and we have been characterizing telomere dysfunction in NHEJ and HR repair-deficient backgrounds ever since. Cells must correctly distinguish between DNA ends represented by telomeres and DNA ends produced by DSBs if all is to end well. Just as these studies have provided new insight into the complex, often surprising, interactions at DNA ends, they also provoke new questions. Whereas it is now well established that DSB repair proteins associate with telomeres, most recently we've been asking whether the reverse scenario holds: Do telomere proteins interact with DSBs? We find that DSBs induced by ionizing radiations are not sufficient to recruit the essential telomere protein TRF2 as an early damage response, so perhaps this interplay is a one-way street. The rest of the story waits to unfold.     

An in situ study of variant telomeric repeats in human chromosomes.

Variant telomeric repeats are selectively detected in human telomeres in situ by the novel approach of dideoxy-PRINS, displaying their organization in a format where all the individual chromosome ends can be viewed individually and simultaneously. All human chromosome ends are found to contain variant repeats, though not all types of repeats can be detected on all chromosome ends. Although the staining frequency at particular chromosome ends seems polymorphic among individuals, some chromosome ends are more commonly stained with a given probe than others. A few chromosome ends also appear with particularly strong signals. With a probe for one type of variant repeat ((AGGGTG)n), peculiar patterns with more than two signals per chromosome end are observed.     

The structures at the termini of chromosomes: what is there hidden under the cap ?

The telomeres are the nucleoproteic structures present at the ends of eukaryotic chromosomes. One can compare them to the protective ends of a shoelace; when the ends get eroded, the shoelace disintegrates and we dispose of it. The same thus applies to the chromosomes; when telomeres reach a critical threshold for function, the genome becomes unstable and the cell senesces. Therefore, telomeres, and particularly their terminal DNA structures, are critical for the integrity of the genome.     

Subtelomere-binding protein Tbf1 and telomere-binding protein Rap1 collaborate to inhibit localization of the Mre11 complex to DNA ends in budding yeast

Chromosome ends, known as telomeres, have to be distinguished from DNA double-strand breaks that activate DNA damage checkpoints. In budding yeast, the Mre11-Rad50-Xrs2 (MRX) complex associates with DNA ends and promotes checkpoint activation. Rap1 binds to double-stranded telomeric regions and recruits Rif1 and Rif2 to telomeres. Rap1 collaborates with Rif1 and Rif2 and inhibits MRX localization to DNA ends. This Rap1-Rif1-Rif2 function becomes attenuated at shortened telomeres. Here we show that Rap1 acts together with the subtelomere-binding protein Tbf1 and inhibits MRX localization to DNA ends. The placement of a subtelomeric sequence or TTAGGG repeats together with a short telomeric TG repeat sequence inhibits MRX accumulation at nearby DNA ends in a Tbf1-dependent manner. Moreover, tethering of both Tbf1 and Rap1 proteins decreases MRX and Tel1 accumulation at nearby DNA ends. This Tbf1- and Rap1-dependent pathway operates independently of Rif1 or Rif2 function. Depletion of Tbf1 protein stimulates checkpoint activation in cells containing short telomeres but not in cells containing normal-length telomeres. These data support a model in which Tbf1 and Rap1 collaborate to maintain genomic stability of short telomeres.     

Short telomeres in yeast are highly recombinogenic

We report that recombination rates specifically increase by up to 10(3) near shortened telomeres in K. lactis cells. This occurs in cells lacking telomerase that undergo growth senescence as well as in cells with stably shortened telomeres that cause little effect on cell growth. The high rates of gene conversion allowed a subtelomeric marker, initially present at a single telomere, to efficiently spread to most or all other telomeres in the cell. We propose that short telomeres in K. lactis are not fully competent at capping chromosome ends and hence are occasionally processed by proteins that normally act to repair broken DNA ends through recombination. This helps explain how recombination can be frequent enough to permit maintenance of telomeres in yeast cells lacking telomerase.     

Manipulating mouse telomeres: models of tumorigenesis and aging

Telomeres are capping structures at the ends of chromosomes, composed of a repetitive DNA sequence and associated proteins. Both a minimal length of telomeric repeats and telomere-associated binding proteins are necessary for proper telomere function. Functional telomeres are essential for maintaining the integrity and stability of eukaryotic genomes. The capping structure enables cells to distinguish chromosome ends from double strand breaks (DSBs) in the genome. Uncapped chromosome ends are at great risk for degradation, recombination, or chromosome fusion by cellular DNA repair systems. Dysfunctional telomeres have been proposed to contribute to tumorigenesis and some aging phenotypes. The analysis of mice deficient in telomerase activity and other telomere-associated proteins has allowed the roles of dysfunctional telomeres in tumorigenesis and aging to be directly tested. Here we will focus on the analysis of different mouse models disrupted for proteins that are important for telomere functions and discuss known and proposed consequences of telomere dysfunction in tumorigenesis and aging.     

Telomeres: more than chromosomal non-sticking ends

Telomeres are specialized natural ends of eukaryotic chromosomes that, contrary to the ends of broken chromosomes, are stable and do not fuse with the ends of other chromosomes. In addition, telomeres protect chromosomal ends from degradation, facilitate completion of chromosomal DNA replication, and contribute to chromosome positioning within nuclei. Telomeric DNA consists of repetitive sequences and specific associated proteins, including the telomere repeat-binding factors TRF1 and TRF2. A lack of TRF2 enables end-to-end chromosome fusion. A structural disruption of telomeres not only causes chromosomal mechanical instability but also activates a programmed cell death cascade.     

Unusually large telomeric repeats in the yeast Candida albicans.

We have identified sequences at the telomeres of the yeast Candida albicans and have found that they are composed of tandem copies of a 23-bp sequence. Through the cloning of native telomeric ends and the characterization and cloning of a "healed" end, we demonstrate that these repeated sequences are sufficient to function as a telomere. All copies of the 23-bp repeat that have been sequenced from a number of C. albicans strains are identical. In contrast, adjacent subtelomeric sequences are variable both between strains and within the WO-1 strain. In the WO-1 strain, the lengths of the telomeres are dependent upon growth temperature and are substantially longer at higher temperatures. Telomere growth is accompanied by increases in the number of the 23-bp repeats present on the telomeric fragments. These results suggest that either telomerase-maintained telomeres can be more complex in structure than was previously imagined or that Candida telomeres are maintained via a telomerase-independent mechanism.     

TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends

Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends.     

Within the genome,long telomeres are more informative than short telomeres with respect to fitness components in a long‐lived seabird

Telomeres, DNA‐protein structures at chromosome ends, shorten with age, and telomere length has been linked to age‐related diseases and survival. In vitro studies revealed that the shortest telomeres trigger cell senescence, but whether the shortest telomeres are also the best biomarker of ageing is not known. We measured telomeres in erythrocytes of wild common terns Sterna hirundo using terminal restriction fragment analysis. This yields a distribution of telomere lengths for each sample, and we investigated how different telomere subpopulations (percentiles) varied in their relation to age and fitness proxies. Longer telomeres within a genome lost more base pairs with age and were better predictors of survival than shorter telomeres. Likewise, fitness proxies such as arrival date at the breeding grounds and reproductive success were best predicted by telomere length at the higher percentiles. Our finding that longer telomeres within a genome predict fitness components better than the shorter telomeres indicates that they are a more informative ageing biomarker. This finding contrasts with the fact that cell senescence is triggered by the shortest telomeres. We suggest that this paradox arises, because longer telomeres lose more base pairs per unit time and thus better reflect the various forms of stress that accelerate telomere shortening, and that telomeres primarily function as biomarker because their shortening reflects cumulative effects of various stressors rather than reflecting telomere‐induced cell senescence.     

Telomeric repeat organization--a comparative in situ study between man and rodent

Human, hamster, and mouse chromosomes show both similarities and differences in telomeric organization, detectable with a novel version of the PRINS technique. The differences allowed us to investigate the fate of the telomeres on a chromosome from one species when this chromosome was introduced into the cells of another species. For this purpose, we tested telomeres in cell lines of somatic cell hybrids containing human chromosomes on a rodent background, finding that the telomeres on human chromosomes could not be discriminated from the telomeres on rodent chromosomes. All telomeres in the cell lines were much shorter than the telomeres in normal cells. In the mouse-derived cell lines, half of the mouse chromosomes were fused to other mouse chromosomes at the ends of their short arms. At the points of fusion we were generally unable to detect telomeric signals. In these cell lines, we also found a fraction of chromosomes ends with only one telomeric signal. In chromosomes where both ends showed only one signal, the relative orientation of the signals appeared to be nonrandom with respect to sister chromatids.     

Structure of the growing telomeres of Trypanosomes

We have developed a method for the molecular cloning of DNA adjacent to chromosome ends (telomeres). A recombinant DNA clone obtained from the telomeres of the protozoan Trypanosoma brucei contains large stretches of the repeat (CCCTAA)n. This repeat is flanked by a larger subtelomeric repeat (29 bp in one case). These repeats account for the presence of large DNA stretches not cut by restriction enzymes downstream of telomeric VSG genes. All telomeres analyzed thus far (more than 30) grow by approximately 6 bp per trypanosomal division and contract by occasional large deletions. Our results suggest that growth is due mainly to addition of CCCTAA units.     

MRX-dependent DNA damage response to short telomeres

Telomere structure allows cells to distinguish the natural chromosome ends from double-strand breaks (DSBs). However, DNA damage response proteins are intimately involved in telomere metabolism, suggesting that functional telomeres may be recognized as DNA damage during a time window. Here we show by two different systems that short telomeres are recognized as DSBs during the time of their replication, because they induce a transient MRX-dependent DNA damage checkpoint response during their prolonged elongation. The MRX complex, which is recruited at telomeres under these conditions, dissociates from telomeres concomitantly with checkpoint switch off when telomeres reach a new equilibrium length. We also show that MRX recruitment to telomeres is sufficient to activate the checkpoint independently of telomere elongation. We propose that MRX can signal checkpoint activation by binding to short telomeres only when they become competent for elongation. Because full-length telomeres are refractory to MRX binding and the shortest telomeres are elongated of only a few base pairs per generation, this limitation may prevent unscheduled checkpoint activation during an unperturbed S phase.