Telomeric 3' overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST

A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.     

SNMIB/Apollo protects leading‐strand telomeres against NHEJ‐mediated repair

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell''s inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo''s conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo^−/−ATM^−/− MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5′–3′ exonuclease function of SNM1B/Apollo in the generation of 3′ single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.     

The Apollo 5' exonuclease functions together with TRF2 to protect telomeres from DNA repair

A major issue in telomere research is to understand how the integrity of chromosome ends is preserved . The human telomeric protein TRF2 coordinates several pathways that prevent checkpoint activation and chromosome fusions. In this work, we identified hSNM1B, here named Apollo, as a novel TRF2-interacting factor. Interestingly, the N-terminal domain of Apollo is closely related to that of Artemis, a factor involved in V(D)J recombination and DNA repair. Both proteins belong to the beta-CASP metallo-beta-lactamase family of DNA caretaker proteins. Apollo appears preferentially localized at telomeres in a TRF2-dependent manner. Reduced levels of Apollo exacerbate the sensitivity of cells to TRF2 inhibition, resulting in severe growth defects and an increased number of telomere-induced DNA-damage foci and telomere fusions. Purified Apollo protein exhibits a 5'-to-3' DNA exonuclease activity. We conclude that Apollo is a novel component of the human telomeric complex and works together with TRF2 to protect chromosome termini from being recognized and processed as DNA damage. These findings unveil a previously undescribed telomere-protection mechanism involving a DNA 5'-to-3' exonuclease.     

Requirement of DDX39 DEAD box RNA helicase for genome integrity and telomere protection

Human chromosome ends associate with shelterin, a six-protein complex that protects telomeric DNA from being recognized as sites of DNA damage. The shelterin subunit TRF2 has been implicated in the protection of chromosome ends by facilitating their organization into the protective capping structure and by associating with several accessory proteins involved in various DNA transactions. Here we describe the characterization of DDX39 DEAD-box RNA helicase as a novel TRF2-interacting protein. DDX39 directly interacts with the telomeric repeat binding factor homology domain of TRF2 via the FXLXP motif (where X is any amino acid). DDX39 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT but has no effect on telomerase activity. Whereas overexpression of DDX39 in telomerase-positive human cancer cells led to progressive telomere elongation, depletion of endogenous DDX39 by small hairpin RNA (shRNA) resulted in telomere shortening. Furthermore, depletion of DDX39 induced DNA-damage response foci at internal genome as well as telomeres as evidenced by telomere dysfunction-induced foci. Some of the metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an aberrant telomere structure. Our findings suggest that DDX39, in addition to its role in mRNA splicing and nuclear export, is required for global genome integrity as well as telomere protection and represents a new pathway for telomere maintenance by modulating telomere length homeostasis.     

Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.     

Distinct roles of TRF1 in the regulation of telomere structure and lengthening

The telomere is a functional chromatin structure that consists of G-rich repetitive sequences and various associated proteins. Telomeres protect chromosomal ends from degradation, provide escape from the DNA damage response, and regulate telomere lengthening by telomerase. Multiple proteins that localize at telomeres form a complex called shelterin/telosome. One component, TRF1, is a double-stranded telomeric DNA binding protein. Inactivation of TRF1 disrupts telomeric localization of other shelterin components and induces chromosomal instability. Here, we examined how the telomeric localization of shelterin components is crucial for TRF1-mediated telomere-associated functions. We found that many of the mTRF1 deficient phenotypes, including chromosomal instability, growth defects, and dysfunctional telomere damage response, were suppressed by the telomere localization of shelterin components in the absence of functional mTRF1. However, abnormal telomere signals and telomere elongation phenotypes were either not rescued or only partially rescued, respectively. These data suggest that TRF1 regulates telomere length and function by at least two mechanisms; in one TRF1 acts through the recruiting/tethering of other shelterin components to telomeres, and in the other TRF1 seems to play a more direct role.     

RECQL4, the protein mutated in Rothmund-Thomson syndrome, functions in telomere maintenance

Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance.     

In Vivo Stoichiometry of Shelterin Components

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1·POT1 heterodimer was present 50–100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1·POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ∼10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1·POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.     

p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

The human telomeric protein TRF2 is required to protect chromosome ends by facilitating their organization into the protective capping structure. Post-translational modifications of TRF2 such as phosphorylation, ubiquitination, SUMOylation, methylation and poly(ADP-ribosyl)ation have been shown to play important roles in telomere function. Here we show that TRF2 specifically interacts with the histone acetyltransferase p300, and that p300 acetylates the lysine residue at position 293 of TRF2. We also report that p300-mediated acetylation stabilizes the TRF2 protein by inhibiting its ubiquitin-dependent proteolysis and is required for efficient telomere binding of TRF2. Furthermore, overexpression of the acetylation-deficient mutant, K293R, induces DNA-damage response foci at telomeres, thereby leading to induction of impaired cell growth, cellular senescence and altered cell cycle distribution. A small but significant number of metaphase chromosomes show no telomeric signals at chromatid ends, suggesting an aberrant telomere structure. These findings demonstrate that acetylation of TRF2 by p300 plays a crucial role in the maintenance of functional telomeres as well as in the regulation of the telomere-associated DNA-damage response, thus providing a new route for modulating telomere protection function.     

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.     

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.     

Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination

Ku70-Ku80 heterodimers promote the non-homologous end-joining (NHEJ) of DNA breaks and, as shown here, the fusion of dysfunctional telomeres. Paradoxically, this heterodimer is also located at functional mammalian telomeres and interacts with components of shelterin, the protein complex that protects telomeres. To determine whether Ku contributes to telomere protection, we analysed Ku70(-/-) mouse cells. Telomeres of Ku70(-/-) cells had a normal DNA structure and did not activate a DNA damage signal. However, Ku70 repressed exchanges between sister telomeres - a form of homologous recombination implicated in the alternative lengthening of telomeres (ALT) pathway. Sister telomere exchanges occurred at approximately 15% of the chromosome ends when Ku70 and the telomeric protein TRF2 were absent. Combined deficiency of TRF2 and another NHEJ factor, DNA ligase IV, did not elicit this phenotype. Sister telomere exchanges were not elevated at telomeres with functional TRF2, indicating that TRF2 and Ku70 act in parallel to repress recombination. We conclude that mammalian chromosome ends are highly susceptible to homologous recombination, which can endanger cell viability if an unequal exchange generates a critically shortened telomere. Therefore, Ku- and TRF2-mediated repression of homologous recombination is an important aspect of telomere protection.     

TRIP6 and LPP,but not Zyxin,are present at a subset of telomeres in human cells

The protection of chromosome ends requires the inhibition of DNA damage responses at telomeres. This inhibition is exerted in great part by the shelterin complex, known to prevent inappropriate ATM and ATR activation. The molecular mechanisms by which shelterin protects telomeres are incompletely understood. Recently, we have implicated for the first time a class of molecules, LIM domain proteins, in telomere protection. This protection occurred through interaction with shelterin, possibly through POT1, and required the pair of LIM proteins TRIP6 and LPP, themselves part of the Zyxin family. The domain similarity between TRIP6, LPP and Zyxin led us to ask whether the latter also interacted with telomeres. Here, we show that there is specificity in the association of LIM proteins with telomeres: Zyxin, despite a high degree of similarity with TRIP6 and LPP, was not detected at telomeres, nor found in a complex with shelterin. TRIP6 and LPP, however, were detected by immunofluorescence at a small subset of telomeres, perhaps those that are critically short. We speculate that specific LIM proteins are part of complex events occurring in the context of the telomere dysfunction response and are possibly at play during the induction of senescence.Key words: telomere, LIM domain, shelterin, POT1, TRIP6, LPP, zyxin, DNA damage     

AKTIP/Ft1, a New Shelterin-Interacting Factor Required for Telomere Maintenance

Telomeres are nucleoprotein complexes that protect the ends of linear chromosomes from incomplete replication, degradation and detection as DNA breaks. Mammalian telomeres are protected by shelterin, a multiprotein complex that binds the TTAGGG telomeric repeats and recruits a series of additional factors that are essential for telomere function. Although many shelterin-associated proteins have been so far identified, the inventory of shelterin-interacting factors required for telomere maintenance is still largely incomplete. Here, we characterize AKTIP/Ft1 (human AKTIP and mouse Ft1 are orthologous), a novel mammalian shelterin-bound factor identified on the basis of its homology with the Drosophila telomere protein Pendolino. AKTIP/Ft1 shares homology with the E2 variant ubiquitin-conjugating (UEV) enzymes and has been previously implicated in the control of apoptosis and in vesicle trafficking. RNAi-mediated depletion of AKTIP results in formation of telomere dysfunction foci (TIFs). Consistent with these results, AKTIP interacts with telomeric DNA and binds the shelterin components TRF1 and TRF2 both in vivo and in vitro. Analysis of AKTIP- depleted human primary fibroblasts showed that they are defective in PCNA recruiting and arrest in the S phase due to the activation of the intra S checkpoint. Accordingly, AKTIP physically interacts with PCNA and the RPA70 DNA replication factor. Ft1-depleted p53^-/- MEFs did not arrest in the S phase but displayed significant increases in multiple telomeric signals (MTS) and sister telomere associations (STAs), two hallmarks of defective telomere replication. In addition, we found an epistatic relation for MST formation between Ft1 and TRF1, which has been previously shown to be required for replication fork progression through telomeric DNA. Ch-IP experiments further suggested that in AKTIP-depleted cells undergoing the S phase, TRF1 is less tightly bound to telomeric DNA than in controls. Thus, our results collectively suggest that AKTIP/Ft1 works in concert with TRF1 to facilitate telomeric DNA replication.     

TRIP6 and LPP,but not Zyxin,are present at a subset of telomeres in human cells

The protection of chromosome ends requires the inhibition of DNA damage responses at telomeres. This inhibition is exerted in great part by the shelterin complex, known to prevent inappropriate ATM and ATR activation. The molecular mechanisms by which shelterin protects telomeres are incompletely understood. Recently, we have implicated for the first time a class of molecules, LIM domain proteins, in telomere protection. This protection occurred through interaction with shelterin, possibly through POT1, and required the pair of LIM proteins TRIP6 and LPP, themselves part of the Zyxin family. The domain similarity between TRIP6, LPP and Zyxin led us to ask whether the latter also interacted with telomeres. Here, we show that there is specificity in the association of LIM proteins with telomeres: Zyxin, despite a high degree of similarity with TRIP6 and LPP, was not detected at telomeres, nor found in a complex with shelterin. TRIP6 and LPP, however, were detected by immunofluorescence at a small subset of telomeres, perhaps those that are critically short. We speculate that specific LIM proteins are part of complex events occurring in the context of the telomere dysfunction response, and possibly at play during the induction of senescence.     

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.     

Recent expansion of the telomeric complex in rodents: Two distinct POT1 proteins protect mouse telomeres

Human telomeres are protected by shelterin, a complex that includes the POT1 single-stranded DNA binding protein. We found that mouse telomeres contain two POT1 paralogs, POT1a and POT1b, and we used conditional deletion to determine their function. Double-knockout cells showed that POT1a/b are required to prevent a DNA damage signal at chromosome ends, endoreduplication, and senescence. In contrast, POT1a/b were largely dispensable for repression of telomere fusions. Single knockouts and complementation experiments revealed that POT1a and POT1b have distinct functions. POT1a, but not POT1b, was required to repress a DNA damage signal at telomeres. Conversely, POT1b, but not POT1a, had the ability to regulate the amount of single-stranded DNA at the telomere terminus. We conclude that mouse telomeres require two distinct POT1 proteins whereas human telomeres have one. Such divergence is unprecedented in mammalian chromosome biology and has implications for modeling human telomere biology in mice.     

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.     

Give me a break: How telomeres suppress the DNA damage response

Linear organization of the genome requires mechanisms to protect and replicate chromosome ends. To this end eukaryotic cells evolved telomeres, specialized nucleoproteic complexes, and telomerase, the enzyme that maintains the telomeric DNA. Telomeres allow cells to distinguish chromosome ends from sites of DNA damage. In mammalian cells this is accomplished by a protein complex, termed shelterin, that binds to telomeric DNA and is able to shield chromosome ends from the DNA damage machinery. In recent years, we have seen major advances in our understanding of how this protein complex works due to the generation of mouse models carrying mutations of individual shelterin components. This review will focus on our current understanding of how the shelterin complex is able to suppress the DNA damage response pathways, and on the cellular and organismal outcomes of telomere dysfunction.