Telomere G-strand structure and function analyzed by chemical protection, base analogue substitution, and utilization by telomerase in vitro

Eukaryotic telomeres have a 12-16 nucleotide long deoxyguanosine (dG) rich single-stranded overhang at their molecular termini. Some of the unique features of telomeres are probably attributable to a specialized structure formed by this overhang. In the ciliated protozoan Tetrahymena thermophila, the dG-rich overhang is comprised of approximately two repeats of the sequence d(TTGGGG). Previous work has shown that the synthetic oligonucleotide d(TTGGGG)4 can form an unusual non-Watson-Crick base-paired structure (the "G-strand structure") containing G-G base pairs and syn-guanines. We have tested the susceptibility of various dGs in this structure to methylation by DMS. At 0-10 degrees C one dG residue is hypersensitive to methylation while others are particularly resistant. By systematically substituting deoxyinosine (dI) for dG in d(TTGGGG)4 we identify N2 groups of guanine essential for formation of the G-strand structure. We show that dI-substituted molecules that cannot form the G-strand structure nonetheless function as substrates for telomere repeat addition in vitro by the telomere lengthening enzyme, telomerase. The implications of these data are discussed.     

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.     

G-overhang dynamics at Tetrahymena telomeres

To learn more about the structure of the DNA terminus at Tetrahymena thermophila telomeres, we have devised a ligation-mediated primer extension protocol to accurately measure the length of the G-strand overhang. We show that overhang length and the identity of the 3'-terminal nucleotide are tightly regulated. The majority of overhangs terminate in the sequence 5'-TTGGGGT and >80% are either 14-15 or 20-21 nucleotides in length. No significant changes in overhang length were detected as cells traversed the cell cycle. However, changes in length distribution were observed when cells exited the cell cycle, indicating an altered balance between DNA synthesis and degradation or end protection. We also provide evidence that rDNA molecules have overhangs on both telomeres. Full-length rDNA could be cloned by a strategy that depends on overhangs being present at both ends. Moreover, analysis of leading strand telomeres revealed that a significant fraction have overhangs > or =5 nucleotides. Our results indicate that generation of the terminal telomeric DNA structure is highly regulated and requires several distinct DNA-processing events.     

Effects of double-strand break repair proteins on vertebrate telomere structure

Although telomeres are not recognized as double-strand breaks (DSBs), some DSB repair proteins are present at telomeres and are required for telomere maintenance. To learn more about the telomeric function of proteins from the homologous recombination (HR) and non-homologous end joining pathways (NHEJ), we have screened a panel of chicken DT40 knockout cell lines for changes in telomere structure. In contrast to what has been observed in Ku-deficient mice, we found that Ku70 disruption did not result in telomere–telomere fusions and had no effect on telomere length or the structure of the telomeric G-strand overhang. G-overhang length was increased by Rad51 disruption but unchanged by disruption of DNA-PKcs, Mre11, Rad52, Rad54, XRCC2 or XRCC3. The effect of Rad51 depletion was unexpected because gross alterations in telomere structure have not been detected in yeast HR mutants. Thus, our results indicate that Rad51 has a previously undiscovered function at vertebrate telomeres. They also indicate that Mre11 is not required to generate G-overhangs. Although Mre11 has been implicated in overhang generation, overhang structure had not previously been examined in Mre11-deficient cells. Overall our findings indicate that there are significant species-specific differences in the telomeric function of DSB repair proteins.     

Mammalian Ku86 protein prevents telomeric fusions independently of the length of TTAGGG repeats and the G-strand overhang

Ku86 together with Ku70, DNA-PKcs, XRCC4 and DNA ligase IV forms a complex involved in repairing DNA double-strand breaks (DSB) in mammals. Yeast Ku has an essential role at the telomere; in particular, Ku deficiency leads to telomere shortening, loss of telomere clustering, loss of telomeric silencing and deregulation of the telomeric G-overhang. In mammals, Ku proteins associate to telomeric repeats; however, the possible role of Ku in regulating telomere length has not yet been addressed. We have measured telomere length in different cell types from wild-type and Ku86-deficient mice. In contrast to yeast, Ku86 deficiency does not result in telomere shortening or deregulation of the G-strand overhang. Interestingly, Ku86^–/– cells show telomeric fusions with long telomeres (>81 kb) at the fusion point. These results indicate that mammalian Ku86 plays a fundamental role at the telomere by preventing telomeric fusions independently of the length of TTAGGG repeats and the integrity of the G-strand overhang.     

The absence of the dna-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang

The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs(-/-) cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.     

G-strand overhangs on telomeres in telomerase-deficient mouse cells.

Telomeres of eukaryotic chromosomes contain 3' overhangs which are thought to be essential for the maintenance of proper chromosome end structure and function. We examined the requirement for telomerase activity for the generation of these G-strand overhangs in mammalian cells. Using non-denaturing in-gel hybridization to both tissue and cultured cells from mice deficient for the telomerase RNA component, we found that G-strand overhangs exist in the absence of telomerase activity. Quantitation of overhang signal intensity showed no significant reduction in telomerase-deficient cells relative to wild-type. These results support a telomerase-independent mechanism for generating G-strand overhangs.     

ERCC1/XPF removes the 3' overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes

Human telomeres are protected by TRF2. Inhibition of this telomeric protein results in partial loss of the telomeric 3' overhang and chromosome end fusions formed through nonhomologous end-joining (NHEJ). Here we report that ERCC1/XPF-deficient cells retained the telomeric overhang after TRF2 inhibition, identifying this nucleotide excision repair endonuclease as the culprit in overhang removal. Furthermore, these cells did not accumulate telomere fusions, suggesting that overhang processing is a prerequisite for NHEJ of telomeres. ERCC1/XPF was also identified as a component of the telomeric TRF2 complex. ERCC1/XPF-deficient mouse cells had a novel telomere phenotype, characterized by Telomeric DNA-containing Double Minute chromosomes (TDMs). We speculate that TDMs are formed through the recombination of telomeres with interstitial telomere-related sequences and that ERCC1/XPF functions to repress this process. Collectively, these data reveal an unanticipated involvement of the ERCC1/XPF NER endonuclease in the regulation of telomere integrity and establish that TRF2 prevents NHEJ at telomeres through protection of the telomeric overhang from ERCC1/XPF.     

Vertebrate POT1 restricts G-overhang length and prevents activation of a telomeric DNA damage checkpoint but is dispensable for overhang protection

Although vertebrate POT1 is thought to play a role in both telomere capping and length regulation, its function has proved difficult to analyze. We therefore generated a conditional cell line that lacks wild-type POT1 but expresses an estrogen receptor-POT1 fusion. The cells grow normally in tamoxifen, but drug removal causes loss of POT1 from the telomere, rapid cell cycle arrest, and eventual cell death. The arrested cells have a 4N DNA content, and addition of caffeine causes immediate entry into mitosis, suggesting a G(2) arrest due to an ATM- and/or ATR-mediated checkpoint. gammaH2AX accumulates at telomeres, indicating a telomeric DNA damage response, the likely cause of the checkpoint. However, POT1 loss does not cause degradation of the G-strand overhang. Instead, the amount of G overhang increases two- to threefold. Some cells eventually escape the cell cycle arrest and enter mitosis. They rarely exhibit telomere fusions but show severe chromosome segregation defects due to centrosome amplification. Our data indicate that vertebrate POT1 is required for telomere capping but that it functions quite differently from TRF2. Instead of being required for G-overhang protection, POT1 is required to suppress a telomeric DNA damage response. Our results also indicate significant functional similarities between POT1 and Cdc13 from budding yeast (Saccharomyces cerevisiae).     

Telomeric DNA sequence and structure following de novo telomere synthesis in Euplotes crassus.

To learn more about the mechanism of de novo telomere synthesis, we have characterized the sequence and structure of newly synthesized telomeres from Euplotes crassus. E. crassus is a particularly useful organism for studying telomere synthesis because millions of telomeres are made in each cell at a well-defined time during the sexual stage of the life cycle. These newly synthesized telomeres are approximately 50 bp longer than mature macronuclear telomeres. We have investigated the structure of the newly synthesized telomeres and have found that they are much more heterogeneous in length than mature telomeres. Most of the heterogeneity is present on the G-rich strand, indicating that the length of this strand is rather loosely controlled. In contrast, the length of the C-rich strand is much less variable, suggesting that synthesis of this strand is the more precisely regulated step in telomere addition. The G-rich strand exhibits variability both in the total number of G4T4 repeats and in the identity of the terminal nucleotide. In most cases, the G-rich strnd extends beyond the C-rich strand to leave a 3' overhang. While the size of this overhang is variable, the median length is 10 nucleotides. This research provides the first detailed picture of a newly synthesized telomere and has allowed us to formulate a model to describe the various steps involved in de novo telomere synthesis.     

Telomeres: the molecular events driving end-to-end fusions

Recent data indicate that loss of the protective telomeric capping function leads to active degradation of the telomeric G-strand overhang and DNA ligase IV-mediated non-homologous end joining. These molecular events may contribute to genomic instability early in tumorigenesis.     

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.     

Molecular steps of G‐overhang generation at human telomeres and its function in chromosome end protection

Telomeric G‐overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G‐overhang generation at human telomeres is poorly understood. Here, we show that G‐overhangs can undergo cell cycle‐regulated changes independent of telomerase activity. G‐overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C‐strand fill‐in, whereas the sizes of G‐overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C‐strands are precisely defined throughout the cell cycle, indicating that C‐strand resection is strictly regulated. We demonstrate that C‐strand fill‐in is mediated by DNA polymerase α (polα) and controlled by cyclin‐dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G‐overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G‐overhangs and an increase in telomeric DNA damage. Our results suggest that G‐overhang generation at human telomeres is regulated by multiple tightly controlled processes and C‐strand fill‐in is under the control of polα and CDK1.     

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.     

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.     

TRF2 promotes dynamic and stepwise looping of POT1 bound telomeric overhang

Human telomeres are protected by shelterin proteins, but how telomeres maintain a dynamic structure remains elusive. Here, we report an unexpected activity of POT1 in imparting conformational dynamics of the telomere overhang, even at a monomer level. Strikingly, such POT1-induced overhang dynamics is greatly enhanced when TRF2 engages with the telomere duplex. Interestingly, TRF2, but not TRF2ΔB, recruits POT1-bound overhangs to the telomere ds/ss junction and induces a discrete stepwise movement up and down the axis of telomere duplex. The same steps are observed regardless of the length of the POT1-bound overhang, suggesting a tightly regulated conformational dynamic coordinated by TRF2 and POT1. TPP1 and TIN2 which physically connect POT1 and TRF2 act to generate a smooth movement along the axis of the telomere duplex. Our results suggest a plausible mechanism wherein telomeres maintain a dynamic structure orchestrated by shelterin.     

C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3' G-strand overhangs as well as 5' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5' and 3' single-stranded termini.     

CDK1 differentially regulates G-overhang generation at leading- and lagging-strand telomeres in telomerase-negative cells in G2 phase

Human telomeres contain single-stranded 3' G-overhangs that function in telomere end protection and telomerase action. Previously we have demonstrated that multiple steps involving C-strand end resection, telomerase elongation and C-strand fill-in contribute to G-overhang generation in telomerase-positive cancer cells. However, how G-overhangs are generated in telomerase-negative human somatic cells is unknown. Here, we report that C-strand fill-in is present at lagging-strand telomeres in telomerase-negative human cells but not at leading-strand telomeres, suggesting that C-strand fill-in is independent of telomerase extension of G-strand. We further show that while cyclin-dependent kinase 1 (CDK1) positively regulates C-strand fill-in, CDK1 unlikely regulates G-overhang generation at leading-strand telomeres. In addition, DNA polymerase α (Polα) association with telomeres is not altered upon CDK1 inhibition, suggesting that CDK1 does not control the loading of Polα to telomeres during fill-in. In summary, our results reveal that G-overhang generation at leading- and lagging-strand telomeres are regulated by distinct mechanisms in human cells.