Normal Human Telomeres Are Not Late Replicating

Telomeres in yeast are late replicating. Genes placed next to telomeres in yeast can be repressed (telomere positional effects), leading to the hypothesis that telomeres may be heterochromatic and may control the expression of subtelomeric genes. In addition, yeast telomeres are processed to have a transient long overhang at the end of S phase. The applicability of the yeast data to human biology was examined by determining the timing of telomere replication and processing in normal human diploid fibroblasts. Telomeres were purified from synchronized cells that had been labeled with 5-bromodeoxyuridine (BrdU) at hourly intervals, and the fraction of labeled telomeres was analyzed by retrieval with anti-BrdU antibodies. We determined that normal human telomeres replicate throughout S phase rather than being very late replicating. Furthermore, the overall timing of replication was unaffected by telomere length in young versus old cells or cells whose telomeres had been elongated following transfection with the catalytic subunit of telomerase. Finally, the asymmetry in the length of the G-rich overhang in daughter telomeres produced by leading versus lagging strand synthesis was shown to be established within 1 h of telomere replication, indicating there is no significant delay between synthesis and the processing events that contribute to the establishment of asymmetric overhangs. Therefore, the timings of replication and processing of human telomeres are very different from those of yeast.     

At Short Telomeres Tel1 Directs Early Replication and Phosphorylates Rif1

The replication time of Saccharomyces cerevisiae telomeres responds to TG_1–3 repeat length, with telomeres of normal length replicating late during S phase and short telomeres replicating early. Here we show that Tel1 kinase, which is recruited to short telomeres, specifies their early replication, because we find a tel1Δ mutant has short telomeres that nonetheless replicate late. Consistent with a role for Tel1 in driving early telomere replication, initiation at a replication origin close to an induced short telomere was reduced in tel1Δ cells, in an S phase blocked by hydroxyurea. The telomeric chromatin component Rif1 mediates late replication of normal telomeres and is a potential substrate of Tel1 phosphorylation, so we tested whether Tel1 directs early replication of short telomeres by inactivating Rif1. A strain lacking both Rif1 and Tel1 behaves like a rif1Δ mutant by replicating its telomeres early, implying that Tel1 can counteract the delaying effect of Rif1 to control telomere replication time. Proteomic analyses reveals that in yku70Δ cells that have short telomeres, Rif1 is phosphorylated at Tel1 consensus sequences (S/TQ sites), with phosphorylation of Serine-1308 being completely dependent on Tel1. Replication timing analysis of a strain mutated at these phosphorylation sites, however, suggested that Tel1-mediated phosphorylation of Rif1 is not the sole mechanism of replication timing control at telomeres. Overall, our results reveal two new functions of Tel1 at shortened telomeres: phosphorylation of Rif1, and specification of early replication by counteracting the Rif1-mediated delay in initiation at nearby replication origins.     

Human telomeres replicate using chromosome-specific, rather than universal, replication programs

Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs-i.e., origin distribution, termination site location, and fork rate and direction-of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.     

Processing of telomeric DNA ends requires the passage of a replication fork.

During telomere replication in yeast, chromosome ends acquire a long single-stranded extension of the strand making the 3' end. Previous work showed that these 3' tails are generated late in S-phase, when conventional replication is virtually complete. In addition, the extensions were also observed in cells that lacked telomerase. Therefore, a model was proposed that predicted an activity that recessed the 5' ends at yeast telomeres after conventional replication was complete. Here, we demonstrate that this processing activity is dependent on the passage of a replication fork through yeast telomeres. A non-replicating linear plasmid with telomeres at each end does not acquire single-stranded extensions, while an identical construct containing an origin of replication does. Thus, the processing activity could be associated with the enzymes at the replication fork itself, or the passage of the fork through the telomeric sequences allows a transient access for the activity to the telomeres. We therefore propose that there is a mechanistic link between the conventional replication machinery and telomere maintenance.     

Organization of DNA sequences and replication origins at yeast telomeres

We have shown that the DNA sequences adjacent to the telomeres of Saccharomyces cerevisiae chromosomes are highly conserved and contain a high density of replication origins. The salient features of these telomeres can be summarized as follows. There are three moderately repetitive elements present at the telomeres: the 131 sequence (1 to 1.5 kb), the highly conserved Y sequence (5.2 kb), and the less conserved X sequence (0.3 to 3.75 kb). There is a high density of replication origins spaced about 6.7 kb apart at the telomeres. These replication origins are part of the X or the Y sequences. Some of the 131-Y repetitive units are tandemly arranged. The terminal sequence T (about 0.33 to 0.6 kb) is different from the 131, X, or Y sequences and is heterogeneous in length. The order of these sequences from the telomeric end towards the centromere is T-(Y-131)n-X-, where n ranges from 1 to no more than 4. Although these telomeric sequences are conserved among S. cerevisiae strains, they show striking divergence in certain closely related yeast species.     

Replication Timing of Human Telomeres Is Chromosome Arm–Specific,Influenced by Subtelomeric Structures and Connected to Nuclear Localization

The mechanisms governing telomere replication in humans are still poorly understood. To fill this gap, we investigated the timing of replication of single telomeres in human cells. Using in situ hybridization techniques, we have found that specific telomeres have preferential time windows for replication during the S-phase and that these intervals do not depend upon telomere length and are largely conserved between homologous chromosomes and between individuals, even in the presence of large subtelomeric segmental polymorphisms. Importantly, we show that one copy of the 3.3 kb macrosatellite repeat D4Z4, present in the subtelomeric region of the late replicating 4q35 telomere, is sufficient to confer both a more peripheral localization and a later-replicating property to a de novo formed telomere. Also, the presence of β-satellite repeats next to a newly created telomere is sufficient to delay its replication timing. Remarkably, several native, non-D4Z4–associated, late-replicating telomeres show a preferential localization toward the nuclear periphery, while several early-replicating telomeres are associated with the inner nuclear volume. We propose that, in humans, chromosome arm–specific subtelomeric sequences may influence both the spatial distribution of telomeres in the nucleus and their replication timing.     

Length-dependent processing of telomeres in the absence of telomerase

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5′-3′ resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5′-3′ resection at the shortest telomeres.     

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.     

Replication at the telomeres of the Streptomyces linear plasmid pSLA2

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non-displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging-strand fill-in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full-length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading-strand 3' overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome-mediated leading-strand fold-back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5' DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3' end of the leading-strand overhang to a site near the overhang's base — providing a recognition site for terminal-protein-primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.     

The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres

Telomeres protect chromosome ends from being detected as lesions and from triggering DNA damage checkpoints. Paradoxically, telomere function depends on checkpoint proteins such as ATM and ATR, but a molecular model explaining this seemingly contradictory relationship has been missing so far. Here we show that the DNA damage machinery acts on telomeres in at least two independent steps. First, the ATR-dependent machinery is recruited to telomeres before telomere replication is completed, likely in response to single-stranded DNA resulting from replication fork stalling. Second, after replication, telomeres attract ATM and the homologous recombination (HR) machinery. In vivo and in vitro results suggest that the HR machinery is required for formation of a telomere-specific structure at chromosome ends after replication. Our results suggest that telomere ends need to be recognized as DNA damage to complete end replication and to acquire a structure that is essential for function.     

Early replication of short telomeres in budding yeast

The maintenance of an appropriate number of telomere repeats by telomerase is essential for proper chromosome protection. The action of telomerase at the telomere terminus is regulated by opposing activities that either recruit/activate the enzyme at shorter telomeres or inhibit it at longer ones, thus achieving a stable average telomere length. To elucidate the mechanistic details of telomerase regulation we engineered specific chromosome ends in yeast so that a single telomere could be suddenly shortened and, as a consequence of its reduced length, elongated by telomerase. We show that shortened telomeres replicate early in S phase, unlike normal-length telomeres, due to the early firing of origins of DNA replication in subtelomeric regions. Early telomere replication correlates with increased telomere length and telomerase activity. These data reveal an epigenetic effect of telomere length on the activity of nearby replication origins and an unanticipated link between telomere replication timing and telomerase action.     

Time of replication of yeast centromeres and telomeres

The time of replication of centromeres and telomeres of the yeast S. cerevisiae was determined by performing Meselson-Stahl experiments with synchronized cells. The nine centromeres examined become hybrid in density early in S phase, eliminating the possibility that a delay in the replication of centromeres until mitosis is responsible for sister chromatid adherence and proper chromosome segregation at anaphase. The conserved sequence element Y', present at most telomeres, replicates late in S phase, as do the unique sequences adjacent to five specific telomeres. The early and late replication times of these structural elements may be either essential for their proper function or a consequence of some architectural feature of the chromosome.     

Telomeric replication stress: the beginning and the end for alternative lengthening of telomeres cancers

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomeric DNA comprises terminal tracts of G-rich tandem repeats, which are inherently difficult for the replication machinery to navigate. Structural aberrations that promote activation of the alternative lengthening of telomeres (ALT) pathway of telomere maintenance exacerbate replication stress at ALT telomeres, driving fork stalling and fork collapse. This form of telomeric DNA damage perpetuates recombination-mediated repair pathways and break-induced telomere synthesis. The relationship between replication stress and DNA repair is tightly coordinated for the purpose of regulating telomere length in ALT cells, but has been shown to be experimentally manipulatable. This raises the intriguing possibility that induction of replication stress can be used as a means to cause toxic levels of DNA damage at ALT telomeres, thereby selectively disrupting the viability of ALT cancers.     

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.     

Fanconi anemia proteins in telomere maintenance

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell.     

Replication timing of large <Emphasis Type="Italic">Sorex granarius</Emphasis> (<Emphasis Type="Italic">Soricidae</Emphasis>, <Emphasis Type="Italic">Eulipotyphla</Emphasis>) telomeres

Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ?-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.     

端粒结合蛋白在端粒复制与损伤修复中的作用

端粒位于真核细胞线性染色体末端，正常的端粒长度与结构对于细胞基因组稳定的维持有重要作用. 端粒DNA序列的高度重复性使其容易形成一些特殊的二级结构，相比染色体其他位置更难复制. 结合在端粒上的Shelterin蛋白复合体由六个端粒结合蛋白组成，该复合体可以通过抑制端粒处异常DNA损伤修复途径的激活维持端粒的稳定. 此外，近几年的研究显示，Shelterin蛋白复合体还具有调控功能异常端粒的DNA修复途径选择，参与端粒的复制功能. 因此，本文就最近发现的Shelterin蛋白复合体成员调控的端粒处DNA修复及参与的端粒复制过程进行综述.     

TopoIIα prevents telomere fragility and formation of ultra thin DNA bridges during mitosis through TRF1-dependent binding to telomeres

Telomeres are repetitive nucleoprotein structures at the ends of chromosomes. Like most genomic regions consisting of repetitive DNA, telomeres are fragile sites prone to replication fork stalling and generation of chromosomal instability. In particular, abrogation of the TRF1 telomere binding protein leads to stalled replication forks and aberrant telomere structures known as “multitelomeric signals”. Here, we report that TRF1 deficiency also leads to the formation of “ultra-fine bridges” (UFB) during mitosis, and to an increased time to complete mitosis mediated by the spindle assembly checkpoint proteins (SAC). We find that topoisomerase IIα (TopoIIα), an enzyme essential for resolution of DNA replication intermediates, binds telomeres in a TRF1-mediated manner. Indeed, similar to TRF1 abrogation, TopoIIα downregulation leads to telomere fragility and UFB, suggesting that these phenotypes are due to decreased TopoIIα at telomeres. We find that SAC proteins bind telomeres in vivo, and that this is disrupted upon TRF1 deletion. These findings suggest that TRF1 links TopoIIα and SAC proteins in a pathway that ensures correct telomere replication and mitotic segregation, unveiling how TRF1 protects from telomere fragility and mitotic defects.