T-loop assembly in vitro involves binding of TRF2 near the 3' telomeric overhang

Mammalian telomeres contain a duplex TTAGGG-repeat tract terminating in a 3' single-stranded overhang. TRF2 protein has been implicated in remodeling telomeres into duplex lariats, termed t-loops, in vitro and t-loops have been isolated from cells in vivo. To examine the features of the telomeric DNA essential for TRF2-promoted looping, model templates containing a 500 bp double-stranded TTAGGG tract and ending in different single-stranded overhangs were constructed. As assayed by electron microscopy, looped molecules containing most of the telomeric tract are observed with TRF2 at the loop junction. A TTAGGG-3' overhang of at least six nucleotides is required for loop formation. Termini with 5' overhangs, blunt ends or 3' termini with non-telomeric sequences at the junction are deficient in loop formation. Addition of non-telomeric sequences to the distal portion of a 3' overhang beginning with TTAGGG repeats only modestly diminishes looping. TRF2 preferentially localizes to the junction between the duplex repeats and the single-stranded overhang. Based on these findings we suggest a model for the mechanism by which TRF2 remodels telomeres into t-loops.     

Mammalian telomeres end in a large duplex loop.

Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat-binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3' overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.     

Taz1 binding to a fission yeast model telomere: formation of telomeric loops and higher order structures

Similar to its human homologues TRF1 and TRF2, fission yeast Taz1 protein is a component of telomeric chromatin regulating proper telomere maintenance. As mammalian TRF1 and TRF2 proteins have been shown to directly bind telomeric DNA to form protein arrays and looped structures, termed t-loops, the ability of Taz1p to act on fission yeast telomeric DNA in similar ways was examined using purified protein and model DNA templates. When incubated with Taz1p, model telomeres containing 3' single-stranded telomeric overhangs formed t-loops at a frequency approaching 13%. Termini with blunt ends and non-telomeric overhangs were deficient in t-loop formation. In addition, we observed arrays of multiple Taz1p molecules bound to the telomeric regions, resembling the pattern of TRF1 binding. The presence of t-loops larger than the telomeric tract, a high frequency of end-bound DNAs and a donut shape of the Taz1p complex suggest that Taz1p binds the 3' overhang then extrudes a loop that grows in size as the donut slides along the duplex DNA. Based on these in vitro results we discuss possible general implications for fission yeast telomere dynamics.     

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.     

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.     

DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion

Telomere attrition and other forms of telomere damage can activate the ATM kinase pathway. What generates the DNA damage signal at mammalian chromosome ends or at other double-strand breaks is not known. Telomere dysfunction is often accompanied by disappearance of the 3' telomeric overhang, raising the possibility that DNA degradation could generate the structure that signals. Here we address these issues by studying telomere structure after conditional deletion of mouse TRF2, the protective factor at telomeres. Upon removal of TRF2 from TRF2(F/-) p53-/- mouse embryo fibroblasts, a telomere damage response is observed at most chromosome ends. As expected, the telomeres lose the 3' overhang and are processed by the non-homologous end-joining pathway. Non-homologous end joining of telomeres was abrogated in DNA ligase IV-deficient (Lig4-/-) cells. Unexpectedly, the telomeres of TRF2-/- Lig4-/- p53-/- cells persisted in a free state without undergoing detectable DNA degradation. Notably, the telomeres retained their 3' overhangs, but they were recognized as sites of DNA damage, accumulating the DNA damage response factors 53BP1 and gamma-H2AX, and activating the ATM kinase. Thus, activation of the ATM kinase pathway at chromosome ends does not require overhang degradation or other overt DNA processing.     

Control of human telomere length by TRF1 and TRF2

Telomere length in human cells is controlled by a homeostasis mechanism that involves telomerase and the negative regulator of telomere length, TRF1 (TTAGGG repeat binding factor 1). Here we report that TRF2, a TRF1-related protein previously implicated in protection of chromosome ends, is a second negative regulator of telomere length. Overexpression of TRF2 results in the progressive shortening of telomere length, similar to the phenotype observed with TRF1. However, while induction of TRF1 could be maintained over more than 300 population doublings and resulted in stable, short telomeres, the expression of exogenous TRF2 was extinguished and the telomeres eventually regained their original length. Consistent with their role in measuring telomere length, indirect immunofluorescence indicated that both TRF1 and TRF2 bind to duplex telomeric DNA in vivo and are more abundant on telomeres with long TTAGGG repeat tracts. Neither TRF1 nor TRF2 affected the expression level of telomerase. Furthermore, the presence of TRF1 or TRF2 on a short linear telomerase substrate did not inhibit the enzymatic activity of telomerase in vitro. These findings are consistent with the recently proposed t loop model of telomere length homeostasis in which telomerase-dependent telomere elongation is blocked by sequestration of the 3' telomere terminus in TRF1- and TRF2-induced telomeric loops.     

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.     

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.     

端粒及端粒酶的研究进展

端粒是真核细胞染色体末端的特有结构,是由端粒结合蛋白和一段重复序列的端粒DNA组成的一个高度精密的复合体,在维持染色体末端稳定性,避免染色体被核酸酶降解等方面起着重要的作用。端粒的长度、结构及组织形式受多种端粒结合因子的调控。由于端粒的重要性,在哺乳动物细胞里,端粒的长度或端粒结构变化与癌症发生及细胞衰老有密切的关系。由于末端复制问题的存在,随着细胞分裂次数的增加,端粒不断缩短,细胞不可避免的走向衰老或凋亡。由于在细胞分裂过程中端粒长度的不断缩短与细胞分裂代数增加具有相关性,即端粒长度反应了细胞的分裂次数,因此有人将端粒形象的比喻为生物时钟。在90%的癌细胞中,端粒酶被重新激活,以此来维持端粒的长度,使细胞走向永生化。简要综述了端粒、端粒酶及端粒酶结合蛋白的最新研究进展。     

Genetic and Physical Mapping of Telomeres in the Rice Blast Fungus, Magnaporthe Grisea

Telomeric restriction fragments were genetically mapped to a previously described linkage map of Magnaporthe grisea, using RFLPs identified by a synthetic probe, (TTAGGG)(3). Frequent rearrangement of telomeric sequences was observed in progeny isolates creating a potential for misinterpretation of data. Therefore a consensus segregation data set was used to minimize mapping errors. Twelve of the 14 telomeres were found to be genetically linked to existing RFLP markers. Second-dimensional electrophoresis of restricted chromosomes confirmed these linkage assignments and revealed the chromosomal location of the two unlinked telomeres. We were thus able to assign all 14 M. grisea telomeres to their respective chromosome ends. The Achilles' cleavage (AC) technique was employed to determine that chromosome 1 markers 11 and CH5-120H were ~1.8 Mb and 1.28 Mb, respectively, from their nearest telomeres. RecA-AC was also used to determine that unlinked telomere 6 was ~530 kb from marker CH5-176H in strain 2539 and 580 kb in Guy11. These experiments indicated that large portions of some chromosome ends are unrepresented by genetic markers and provided estimates of the relationship of genetic to physical distance in these regions of the genome.     

Rap1 binds single-stranded DNA at telomeric double- and single-stranded junctions and competes with Cdc13 protein

The ends of eukaryotic chromosomes are protected by specialized telomere chromatin structures. Rap1 and Cdc13 are essential for the formation of functional telomere chromatin in budding yeast by binding to the double-stranded part and the single-stranded 3' overhang, respectively. We analyzed the binding properties of Saccharomyces castellii Rap1 and Cdc13 to partially single-stranded oligonucleotides, mimicking the junction of the double- and single-stranded DNA (ds-ss junction) at telomeres. We determined the optimal and the minimal DNA setup for a simultaneous binding of Rap1 and Cdc13 at the ds-ss junction. Remarkably, Rap1 is able to bind to a partially single-stranded binding site spanning the ds-ss junction. The binding over the ds-ss junction is anchored in a single double-stranded hemi-site and is stabilized by a sequence-independent interaction of Rap1 with the single-stranded 3' overhang. Thus, Rap1 is able to switch between a sequence-specific and a nonspecific binding mode of one hemi-site. At a ds-ss junction configuration where the two binding sites partially overlap, Rap1 and Cdc13 are competing for the binding. These results shed light on the end protection mechanisms and suggest that Rap1 and Cdc13 act together to ensure the protection of both the 3' and the 5' DNA ends at telomeres.     

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.     

端粒结合蛋白TRF2的研究进展

端粒DNA结合蛋白TRF2(TTAGGG repeat binding factor-2)以二聚体形式通过Myb结构域与端粒重复序列TTAGGG结合,并与TRF1、TIN2、Rap1、TINT1及POT1蛋白组成Shelterin蛋白复合物,协同在端粒动态平衡维持过程中起关键作用,进而影响整个基因组的稳定性。此外,TRF2在细胞DNA损伤应答过程中可能发挥重要作用。本文将对TRF2结构和功能研究的最新进展进行综述。     

Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres

The terminal t-loop structure adopted by mammalian telomeres is thought to prevent telomeres from being recognized as double-stranded DNA breaks by sequestering the 3' single-stranded G-rich overhang from exposure to the DNA damage machinery. The POT1 (protection of telomeres) protein binds the single-stranded overhang and is required for both chromosomal end protection and telomere length regulation. The mouse genome contains two POT1 orthologs, Pot1a and Pot1b. Here we show that conditional deletion of Pot1a elicits a DNA damage response at telomeres, resulting in p53-dependent replicative senescence. Pot1a-deficient cells exhibit overall telomere length and 3' overhang elongation as well as aberrant homologous recombination (HR) at telomeres, manifested as increased telomere sister chromatid exchanges and formation of telomere circles. Telomeric HR following Pot1a loss requires NBS1. Pot1a deletion also results in chromosomal instability. Our results suggest that POT1a is crucial for the maintenance of both telomere integrity and overall genomic stability.     

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.     

Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops

A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway. In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy. Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography. Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb. t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb. The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis. The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis. These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species. Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.     

Human POT1 facilitates telomere elongation by telomerase

Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.