Looping‐out mechanism for resolution of replicative stress at telomeres

Repetitive DNA is prone to replication fork stalling, which can lead to genome instability. Here, we find that replication fork stalling at telomeres leads to the formation of t‐circle‐tails, a new extrachromosomal structure that consists of circular telomeric DNA with a single‐stranded tail. Structurally, the t‐circle‐tail resembles cyclized leading or lagging replication intermediates that are excised from the genome by topoisomerase II‐mediated cleavage. We also show that the DNA damage repair machinery NHEJ is required for the formation of t‐circle‐tails and for the resolution of stalled replication forks, suggesting that NHEJ, which is normally constitutively suppressed at telomeres, is activated in the context of replication stress. Inhibition of NHEJ or knockout of DNA‐PKcs impairs telomere replication, leading to multiple‐telomere sites (MTS) and telomere shortening. Collectively, our results support a “looping‐out” mechanism, in which the stalled replication fork is cut out and cyclized to form t‐circle‐tails, and broken DNA is religated. The telomere loss induced by replication stress may serve as a new factor that drives replicative senescence and cell aging.     

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.     

FEN1 Ensures Telomere Stability by Facilitating Replication Fork Re-initiation

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.     

Telomeres in drag: Dressing as DNA damage to engage telomerase

The telomere field concentrates both on mechanisms of telomere synthesis and the mechanisms by which telomeres protect chromosome termini from fusion and degradation. Recent studies show that the DNA damage response (DDR) machinery, formerly thought to be the culprit in deleterious telomeric fusion and degradation reactions, plays an active role not only in telomere protection but also in regulating telomere synthesis. Conversely, semi-conservative DNA replication, responsible for the bulk of telomere synthesis, now appears to be a pivotal event on the road to telomere de-protection. These advances prompt the notion that the two guises of telomere function are intricately entangled. Indeed, telomeres appear to expose themselves to the DDR upon passage of the replication fork, in turn attracting telomerase.     

Stabilization of quadruplex DNA perturbs telomere replication leading to the activation of an ATR-dependent ATM signaling pathway

Functional telomeres are required to maintain the replicative ability of cancer cells and represent putative targets for G-quadruplex (G4) ligands. Here, we show that the pentacyclic acridinium salt RHPS4, one of the most effective and selective G4 ligands, triggers damages in cells traversing S phase by interfering with telomere replication. Indeed, we found that RHPS4 markedly reduced BrdU incorporation at telomeres and altered the dynamic association of the telomeric proteins TRF1, TRF2 and POT1, leading to chromosome aberrations such as telomere fusions and telomere doublets. Analysis of the molecular damage pathway revealed that RHPS4 induced an ATR-dependent ATM signaling that plays a functional role in the cellular response to RHPS4 treatment. We propose that RHPS4, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing resulting in telomere dysfunction and activation of a replication stress response pathway. The detailed understanding of the molecular mode of action of this class of compounds makes them attractive tools to understand telomere biology and provides the basis for a rational use of G4 ligands for the therapy of cancer.     

Mrc1 protects uncapped budding yeast telomeres from exonuclease EXO1

Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork machinery and is necessary for checkpoint activation after replication stress. In this study, we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1 contributes to the vitality of both cdc13-1 and yku70Delta telomere capping mutants. Cells with telomere capping defects containing MRC1 or mrc1(AQ), a checkpoint defective allele, exhibit similar growth, suggesting growth defects of cdc13-1 mrc1Delta are not due to checkpoint defects. This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping. Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding a nuclease that contributes to single stranded DNA accumulation after telomere uncapping, improves growth of cdc13-1 mrc1Delta strains and decreases ssDNA production. Our observations show that Mrc1, a core component of the replication fork, plays an important role in telomere capping, protecting from nucleases and checkpoint pathways.     

Timeless tunes: Replicating happy endings

Comment on: Leman AR, et al. Cell Cycle 2012; 11:2337-47.DNA replication is at the heart of the inheritance of genetic material. A single replication fork can progress through hundreds of kilobases of DNA, melting parental double-stranded DNA and leaving newly synthesized strands in its wake. A beautiful illustration showing how the replication machinery accomplishes this complex task is one of the triumphs of molecular biology. However, it is known that DNA replication is not always as processive as the textbooks suggest. Specifically, the rate of fork progression varies depending on the regions being replicated, and the replication fork even stalls in some circumstances, during replication of heterochromatin or damaged DNA, for example. A stalled replication fork has two fates. It may restart DNA replication, or it may collapse after prolonged stalling. A collapsed replication fork is particularly dangerous for the genome, because the DNA intermediate left by the collapsed fork may form a double-stranded break, a highly mutagenic lesion that can undergo illegitimate recombination. To circumvent replication fork collapse, cells are equipped with specialized proteins that stabilize the stalled replication fork. Timeless and Tipin are highly conserved in eukaryotes. from yeast to humans, and form a complex to protect stalled replication forks.In a paper published in Cell Cycle, Noguchi and his group investigated how Timeless plays a role in telomere replication in human cells.¹ Telomeres consist of tandem arrays of short repetitive DNA (TTAGGG/CCCTAA in mammals) at the ends of chromosomes and numerous associated proteins. Telomeres are essential for the stable maintenance of genomic DNA, because they protect the DNA termini from undergoing accidental recombination and exonuclease attack. Dysfunctional telomeres lead to genetic instability that eventually results in senescence and cancer development. Because of the heterochromatic nature of telomeres, it has been recognized that telomere DNA is one of the genomic regions that impede replication fork progression. Indeed, in vitro DNA replication experiments using SV40 DNA, and cell extracts demonstrated that telomere DNA is replicated less efficiently and incurs more fork stalling than non-telomeric DNA.² Moreover, overexpression of telomere-DNA binding protein TRF1 in HeLa cells led to an accumulation of replicating telomeres, consistent with a slower replication rate of telomeres under those circumstance. Furthermore, experiments using TRF1-deleted murine cells showed that TRF1 is essential for efficient telomere DNA replication.³ Collectively, these results confirm that the telomere is a difficult-to-replicate region.There is an apparent contradiction between two earlier studies, however, with TRF1 described as an anti-replication protein in one report² and a pro-replication protein in the other.³ One potential explanation for the inconsistency might be that TRF1 requires other protein(s) to perform its pro-replication function, and the second factor was missing in the TRF1-overexpression experiments. Noguchi and his colleagues investigated this possibility by testing whether Timeless is required for proficient telomere DNA replication.¹ They found that Timeless-knockdown cells displayed telomere length shortening and an increased frequency of dysfunctional telomeres. In vitro replication assays of SV40 DNA revealed that Timeless-depleted extracts supported non-telomere replication proficiently, while telomere replication was inefficient. They then demonstrated that addition of recombinant TRF1 to the replication system slowed telomere replication. Importantly, Timeless depletion and TRF1 addition did not produce additive effects on telomere replication, suggesting that Timeless and TRF1 function in the same pathway. These results suggest a model as described in Figure 1. A replication fork frequently stalls at telomeres because of the molecularly crowded nature of telomeric chromatin. Timeless presumably encounters TRF1 at telomeres and protects the stalled fork from undergoing collapse. In the absence of Timeless, the stalled forks easily collapse, leading to an abrupt shortening of telomeres. Several questions remain to be answered. Given that Timeless moves along the genomic DNA as a component of the replication machinery,⁴ it will be particularly interesting to see how Timeless (or the replication machinery) interacts with telomeric chromatin. In such studies, a dynamic transaction between the regional chromatin at telomeres and the replication machinery may be revealed.Open in a separate windowFigure 1. Hard life at telomeres. (A) Mammalian telomeres consist of repetitive DNA that potentially forms higher-ordered structures [G-quartet(G4)-DNA] and numerous proteins, including telomere DNA-binding protein TRF1. (B) Replication fork is frequently stalled at telomeres. Overexpressed TRF1 slows down fork progression at the telomere, while endogenous TRF1 together with Timeless protein facilitates it. Timeless protects the stalled replication fork from collapse. (C) Telomeres are unique in that the most distal replication fork is not coupled with another fork progressing inversely. (D) Prolonged fork stalling may lead to the formation of a DNA double-strand break. Because of the lack of another fork compensating the telomere replication (C), the break immediately results in the abrupt single-step shortening of telomere DNAs.     

The Werner Syndrome Protein Suppresses Telomeric Instability Caused by Chromium (VI) Induced DNA Replication Stress

Telomeres protect the chromosome ends and consist of guanine-rich repeats coated by specialized proteins. Critically short telomeres are associated with disease, aging and cancer. Defects in telomere replication can lead to telomere loss, which can be prevented by telomerase-mediated telomere elongation or activities of the Werner syndrome helicase/exonuclease protein (WRN). Both telomerase and WRN attenuate cytotoxicity induced by the environmental carcinogen hexavalent chromium (Cr(VI)), which promotes replication stress and DNA polymerase arrest. However, it is not known whether Cr(VI)-induced replication stress impacts telomere integrity. Here we report that Cr(VI) exposure of human fibroblasts induced telomeric damage as indicated by phosphorylated H2AX (γH2AX) at telomeric foci. The induced γH2AX foci occurred in S-phase cells, which is indicative of replication fork stalling or collapse. Telomere fluorescence in situ hybridization (FISH) of metaphase chromosomes revealed that Cr(VI) exposure induced an increase in telomere loss and sister chromatid fusions that were rescued by telomerase activity. Human cells depleted for WRN protein exhibited a delayed reduction in telomeric and non-telomeric damage, indicated by γH2AX foci, during recovery from Cr(VI) exposure, consistent with WRN roles in repairing damaged replication forks. Telomere FISH of chromosome spreads revealed that WRN protects against Cr(VI)-induced telomere loss and downstream chromosome fusions, but does not prevent chromosome fusions that retain telomere sequence at the fusion point. Our studies indicate that environmentally induced replication stress leads to telomere loss and aberrations that are suppressed by telomerase-mediated telomere elongation or WRN functions in replication fork restoration.     

Variant repeats are interspersed throughout the telomeres and recruit nuclear receptors in ALT cells

Telomeres in cells that use the recombination-mediated alternative lengthening of telomeres (ALT) pathway elicit a DNA damage response that is partly independent of telomere length. We therefore investigated whether ALT telomeres contain structural abnormalities that contribute to ALT activity. Here we used next generation sequencing to analyze the DNA content of ALT telomeres. We discovered that variant repeats were interspersed throughout the telomeres of ALT cells. We found that the C-type (TCAGGG) variant repeat predominated and created a high-affinity binding site for the nuclear receptors COUP-TF2 and TR4. Nuclear receptors were directly recruited to telomeres and ALT-associated characteristics were induced after incorporation of the C-type variant repeat by a mutant telomerase. We propose that the presence of variant repeats throughout ALT telomeres results from recombination-mediated telomere replication and spreading of variant repeats from the proximal regions of the telomeres and that the consequent binding of nuclear receptors alters the architecture of telomeres to facilitate further recombination.     

Polymerase η suppresses telomere defects induced by DNA damaging agents

Telomeres at chromosome ends are normally masked from proteins that signal and repair DNA double strand breaks (DSBs). Bulky DNA lesions can cause DSBs if they block DNA replication, unless they are bypassed by translesion (TLS) DNA polymerases. Here, we investigated roles for TLS polymerase η, (polη) in preserving telomeres following acute physical UVC exposure and chronic chemical Cr(VI) exposure, which both induce blocking lesions. We report that polη protects against cytotoxicity and replication stress caused by Cr(VI), similar to results with ultraviolet C light (UVC). Both exposures induce ataxia telangiectasia and Rad3-related (ATR) kinase and polη accumulation into nuclear foci and localization to individual telomeres, consistent with replication fork stalling at DNA lesions. Polη-deficient cells exhibited greater numbers of telomeres that co-localized with DSB response proteins after exposures. Furthermore, the genotoxic exposures induced telomere aberrations associated with failures in telomere replication that were suppressed by polη. We propose that polη''s ability to bypass bulky DNA lesions at telomeres is critical for proper telomere replication following genotoxic exposures.     

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.     

Telomerase is required to protect chromosomes with vertebrate-type T2AG3 3' ends in Saccharomyces cerevisiae

Telomeres containing vertebrate-type DNA repeats can be stably maintained in Saccharomyces cerevisiae cells. We show here that telomerase is required for growth of yeast cells containing these vertebrate-type telomeres. When present at the chromosome termini, these heterologous repeats elicit a DNA damage response and a certain deprotection of telomeres. The data also show that these phenotypes are due only to the terminal localization of the vertebrate repeats because if they are sandwiched between native yeast repeats, no phenotype is observed. Indeed and quite surprisingly, in this latter situation, telomeres are of virtually normal lengths, despite the presence of up to 50% of heterologous repeats. Furthermore, the presence of the distal vertebrate-type repeats can cause increased problems of the replication fork. These results show that in budding yeast the integrity of the 3' overhang is required for proper termination of telomere replication as well as protection.     

Telomere dysfunction puts the brakes on oncogene-induced cancers

EMBO J 31 13, 2839–2851 (2012); published online May082012Senescence represents a major tumour suppressor checkpoint activated by telomere dysfunction or cellular stress factors such as oncogene activation. In this issue of The EMBO Journal, Suram et al (2012) reveal a surprising interconnection between oncogene activation and telomere dysfunction induced senescence. The study supports an alternative model of tumour suppression, indicating that oncogene-induced accumulation of telomeric DNA damage contributes to the induction of senescence in telomerase-negative tumours.Telomere shortening limits the proliferative capacity of primary human cells after 50–70 cell divisions by induction of replicative senescence activated by critically short, dysfunctional telomeres. Different mechanisms were thought to initiate senescence in response to oncogene activation, which occurs abruptly within a few cell doublings (Serrano et al, 1997). Oncogene-induced senescence (OIS) involves an activation of DNA damage signals at stalled replication forks induced by DNA replication stress (Bartkova et al, 2006; Di Micco et al, 2006). Replication fork stalling in response to oncogene activation preferentially affects common fragile sites of the DNA (Tsantoulis et al, 2008). The ends of eukaryotic chromosomes—the telomeres–represent common fragile sites that are sensitive to replication fork stalling (Sfeir et al, 2009). These data made it tempting to speculate whether replication fork stalling at telomeres was causatively involved in OIS. Studies on replicative senescence in human fibroblast also supported this possibility showing that mitogenic signals amplify DNA damage responses in senescent cells (Satyanarayana et al, 2004).Multiple studies revealed experimental evidences that senescence suppresses tumour progression in mouse models and early human tumours (for review see Collado and Serrano, 2010). The relative contribution of OIS and telomere dysfunction induced senescence (TDIS) to tumour suppression and possible interconnections between the two pathways at the level of checkpoint induction were not investigated in previous studies. In this issue of The EMBO Journal, Suram et al (2012) describe the presence of TDIS in human precursor lesions but not in the corresponding malignant tumours. Mechanistically, the study shows that oncogenic signals cause replication fork stalling, resulting in telomeric DNA damage accumulation and activation of DNA damage checkpoints reminiscent to TDIS. Telomerase expression does not rescue replication fork stalling but prevents the accumulation of DNA damage at telomeres allowing a bypass of OIS.The study has several important implications for molecular pathways and therapeutic approaches in cancer that need to be further explored (Figure 1):Open in a separate windowFigure 1Traditional and new models of senescence in tumour suppression. (A) Traditional model of replicative senescence: Telomerase-negative tumour cell clones experience telomere shortening as a consequence of cell division. After a lack period depending on the initial telomere length, tumour cells accumulate telomere dysfunction and activation of senescence impairs tumour growth. Telomerase activation represents a late event allowing tumour progression. (B) New model of oncogene induced, telomere-dependent senescence: Oncogene activation leads to abrupt accumulation of DNA damage at telomeres resulting in senescence and tumour suppression. Telomerase-positive stem cells could be resistant to OIS and may be selected as the cell type of origin of tumour development.(i) Telomere length independent roles of telomeres in tumour suppressionThe classical model of telomere-dependent tumour suppression indicates that proliferation-dependent telomere shortening leads to telomere dysfunction, activation of DNA damage checkpoints, and induction of senescence suppressing the growth of telomerase-negative tumour clones. Studies on mouse models supported this concept showing that telomere shortening impairs the progression of initiated tumours in a telomere length-dependent manner (Feldser and Greider, 2007). The new data from Suram et al (2012) indicate that oncogene-induced replication fork stalling activates a telomere-dependent senescence checkpoint, which is independent of telomere length. The study shows that replication forks stall in response to oncogene activation throughout the genome. However, stalled replication forks are resolved in non-telomeric regions, whereas fork stalling inside telomeres leads to un-repairable DNA damage in telomerase-negative cells. These findings are in line with recent publication showing accumulation of un-repairable DNA damage in telomeric DNA in response to aging and stress-induced DNA damage (Fumagalli et al, 2012).(ii) Telomere length independent roles of telomerase in tumour progressionFollowing the classical model telomeres in tumour suppression (Figure 1A), telomerase re-activation is required for tumour progression by limiting telomere dysfunction and the induction of DNA damage checkpoints in response to telomere shortening. The new data from Suram et al (2012) indicate that telomerase has an additional telomere length independent role in tumour progression. The study shows that catalytically active telomerase prevents the activation of DNA damage signals originating from stalled replication forks inside telomeres in response to oncogene activation (Figure 1B). The exact mechanisms of telomerase-dependent healing of stalled replication forks at telomeres remain to be elucidated. It is also unclear whether telomerase activity can prevent any type of DNA damage at telomeres as an over-expression of TERT could not suppress irradiation-induced cellular senescence or the persistence of telomeric DDR following irradiation, H₂O₂, or chemotherapy induced DNA damage (Hewitt et al, 2012).The data could provide a plausible explanation for the increased tumorigenesis in telomerase transgenic mice—a finding which is difficult to explain by telomere length dependent effects of telomerase given the long telomere reserves in mouse tissues (Gonzalez-Suarez et al, 2001). According to the findings of Suram et al (2012), anti-telomerase therapies could have immediate anti-cancer effects in tumours depending on telomerase-mediated healing of stalled replication forks at telomeres. Specific markers for this dependency could be of clinical value. In addition, the data support the concept that somatic stem cells could represent the cell type of origin of cancers. In contrast to differentiated somatic cells, tissues stem cells are often telomerase-positive, indicating that stem cells might be less sensitive to OIS.     

Functional human telomeres are recognized as DNA damage in G2 of the cell cycle

Telomeres have to be distinguished from DNA breaks that initiate a DNA damage response. Proteins involved in the DNA damage response have previously been found at telomeres in transformed cells; however, the importance of these factors for telomere function has not been understood. Here, we show that telomeres of telomerase-negative primary cells recruit Mre11, phosphorylated NBS1, and ATM in every G2 phase of the cell cycle. This recruitment correlates with a partial release of telomeric POT1; moreover, telomeres were found to be accessible to modifying enzymes at this time in the cell cycle, suggesting that they are unprotected. Degradation of the MRN complex, as well as inhibition of ATM, led to telomere dysfunction. Consequentially, we propose that a localized DNA damage response at telomeres after replication is essential for recruiting the processing machinery that promotes formation of a chromosome end protection complex.     

Mammalian DNA2 helicase/nuclease cleaves G‐quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G‐quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2‐deficient mice develop aneuploidy‐associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.     

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.     

Checkpoint-dependent phosphorylation of Exo1 modulates the DNA damage response

Exo1 is a nuclease involved in mismatch repair, DSB repair, stalled replication fork processing and in the DNA damage response triggered by dysfunctional telomeres. In budding yeast and mice, Exo1 creates single-stranded DNA (ssDNA) at uncapped telomeres. This ssDNA accumulation activates the checkpoint response resulting in cell cycle arrest. Here, we demonstrate that Exo1 is phosphorylated when telomeres are uncapped in cdc13-1 and yku70Delta yeast cells, and in response to the induction of DNA damage. After telomere uncapping, Exo1 phosphorylation depends on components of the checkpoint machinery such as Rad24, Rad17, Rad9, Rad53 and Mec1, but is largely independent of Chk1, Tel1 and Dun1. Serines S372, S567, S587 and S692 of Exo1 were identified as targets for phosphorylation. Furthermore, mutation of these Exo1 residues altered the DNA damage response to uncapped telomeres and camptothecin treatment, in a manner that suggests Exo1 phosphorylation inhibits its activity. We propose that Rad53-dependent Exo1 phosphorylation is involved in a negative feedback loop to limit ssDNA accumulation and DNA damage checkpoint activation.     

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.     

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.