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

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

Function, replication and structure of the mammalian telomere

Telomeres are specialized structures at the ends of linear chromosomes that were originally defined functionally based on observations first by Muller (1938) and subsequently by McClintock (1941) that naturally occurring chromosome ends do not behave as double-stranded DNA breaks, in spite of the fact that they are the physical end of a linear, duplex DNA molecule. Double-stranded DNA breaks are highly unstable entities, being susceptible to nucleolytic attack and giving rise to chromosome rearrangements through end-to-end fusions and recombination events. In contrast, telomeres confer stability upon chromosome termini, as evidenced by the fact that chromosomes are extraordinarily stable through multiple cell divisions and even across evolutionary time. This protective function of telomeres is due to the formation of a nucleoprotein complex that sequesters the end of the DNA molecule, rendering it inaccessible to nucleases and recombinases as well as preventing the telomere from activating the DNA damage checkpoint pathways. The capacity of a functional end-protective complex to form is dependent upon maintenance of sufficient telomeric DNA. We have learned a great deal about telomere structure and how this specialized nucleoprotein complex confers stability on chromosome ends since the original observations that defined telomeres were made. This review summarizes our current understanding of mammalian telomere replication, structure and function.     

POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end

The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining.     

An interlocked dimer of the protelomerase TelK distorts DNA structure for the formation of hairpin telomeres

The termini of linear chromosomes are protected by specialized DNA structures known as telomeres that also facilitate the complete replication of DNA ends. The simplest type of telomere is a covalently closed DNA hairpin structure found in linear chromosomes of prokaryotes and viruses. Bidirectional replication of a chromosome with hairpin telomeres produces a catenated circular dimer that is subsequently resolved into unit-length chromosomes by a dedicated DNA cleavage-rejoining enzyme known as a hairpin telomere resolvase (protelomerase). Here we report a crystal structure of the protelomerase TelK from Klebsiella oxytoca phage varphiKO2, in complex with the palindromic target DNA. The structure shows the TelK dimer destabilizes base pairing interactions to promote the refolding of cleaved DNA ends into two hairpin ends. We propose that the hairpinning reaction is made effectively irreversible by a unique protein-induced distortion of the DNA substrate that prevents religation of the cleaved DNA substrate.     

Telomere higher-order structure and genomic instability

Telomeres, nucleoprotein complexes at the end of eukaryotic chromosomes, have vital roles in chromosome integrity. Telomere chromatin structure is both intricate and dynamic allowing for a variety of responses to several stimuli. A critical determinant in telomere structure is the G-strand overhang. Facilitated by telomeric proteins, the G-strand overhang stabilizes telomere higher-order assemblies most likely by adopting unusual DNA structures. These structures influence activities that occur at the chromosome end. Dysfunctional telomeres induce signals resulting in cell growth arrest or death. To overcome telomere dysfunction, cancer cells activate the DNA polymerase, telomerase. The presence of telomerase at the telomere may establish a particular telomeric state. If the chromosome ends of cancer and normal cells exist in different states, cancer-specific telomere structures would offer a unique chemotherapeutic target.     

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, telomerase and senescence

Eukaryotic chromosomes end with tandem repeats of simple sequences. These GC rich repeats allow telomere replication and stabilize chromosome ends. Telomere replication involves an equilibrium of sequence loss and addition at the ends of chromosomes. Repeats are added de novo by telomerase, an unusual DNA polymerase. Telomerase is an RNP in which an essential RNA component provides the template for the added telomere repeats. Telomere length maintenance plays an essential role in cell viability.     

De novo telomere addition to spacer sequences prior to their developmental degradation in Euplotes crassus

During sexual reproduction, Euplotes crassus precisely fragments its micronuclear chromosomes and synthesizes new telomeres onto the resulting DNA ends to generate functional macronuclear minichromosomes. In the micronuclear chromosomes, the macronuclear-destined sequences are typically separated from each other by spacer DNA segments, which are eliminated following chromosome fragmentation. Recently, in vivo chromosome fragmentation intermediates that had not yet undergone telomere addition have been characterized. The ends of both the macronuclear-destined and eliminated spacers were found to consist of six-base, 3′ overhangs. As this terminal structure on the macronuclear-destined sequences serves as the substrate for de novo telomere addition, we sought to determine if the spacer DNAs might also undergo telomere addition prior to their elimination. Using a polymerase chain reaction approach, we found that at least some spacer DNAs undergo de novo telomere addition. In contrast to macronuclear-destined sequences, heterogeneity could be observed in the position of telomeric repeat addition. The observation of spacer DNAs with telomeric repeats makes it unlikely that differential telomere addition is responsible for differentiating between retained and eliminated DNA. The heterogeneity in telomere addition sites for spacer DNA also resembles the situation found for telomeric repeat addition to macronuclear-destined sequences in other ciliate species.     

Predicted elements of telomere organization and function in Ustilago maydis

Telomeres are specialized caps of nucleoprotein complexes located at the chromosome termini. They consist of short DNA repeats and of an assortment of associated proteins whose function is currently under intense investigation in model systems. These specialized structures protect the linear ends of eukaryotic chromosomes against DNA repair and degradation activities, and serve as the substrate for telomerase, the ribonucleoprotein complex that synthesises the telomere repeats. The pivotal role of the telomeres in the maintenance of cell viability in several model eukaryotes, including humans, greatly promoted research in telomere biology. Studies on telomere structure and function in fungi other than model systems are limited to providing information on the telomeric repeat sequences. Here, we have summarized the current knowledge on the organization of chromosome ends and on the proteins participating in telomere function in model systems including recent information obtained for filamentous fungi. We also describe Ustilago maydis genes that are potential homologs of proteins known from other systems to participate in telomere biology.     

高等植物端粒和端粒酶的研究进展

端粒是构成真核生物线状染色体末端重要的DNA-蛋白质复合结构,DNA由简单的串联重复序列组成。它的合成由一个特殊的具有反转录活性的核糖核蛋白-端粒酶完成。端粒对染色体、整个生物基因组,甚至对细胞的稳定都具有重要意义。本文就植物端粒、端粒酶、端粒结合蛋白,以及端粒变化、端粒酶在植物生长发育中的调节作一概述。     

Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

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.     

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

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

DNA-dependent protein kinase catalytic subunit is not required for dysfunctional telomere fusion and checkpoint response in the telomerase-deficient mouse

Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerase-deficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc(-/-) DNA-PKcs(-/-) cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)--an additional crucial NHEJ component--was also permissive for chromosome fusions in mTerc(-/-) cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNA-PKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc(-/-) tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.     

Structural anatomy of telomere OB proteins

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.     

Structural anatomy of telomere OB proteins

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.