Repair of DNA broken ends is similar in embryonic fibroblasts with and without telomerase

Telomeres cap the ends of chromosomes, preventing end-to-end fusions and subsequent chromosome instability. Here we used a telomerase knockout model to investigate whether telomerase participates in the processes of DNA break repair by de novo synthesis of telomere repeats at broken chromosome ends (chromosome healing). Chromosome healing giving rise to new detectable telomeric signals has not been observed in embryonic fibroblasts of telomerase-proficient mice exposed to ionizing radiation. Since the synthesis of telomeric sequences to broken DNA ends would make them refractory to rejoining events, the efficiency of rejoining of broken chromosomes in cell environments with and without telomerase has also been investigated. We conclude that the efficiency of rejoining broken chromosomes is not significantly different in the two cell environments. All together, our results indicate that there is no significant involvement of telomerase in the healing of broken DNA ends by synthesizing new telomeres in mouse embryo fibroblasts after exposure to ionizing radiation.     

AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus densiflora

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8–76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.     

Dynamics of telomere length in captive Siamese cobra (Naja kaouthia) related to age and sex

Telomeres comprise tandem repeated DNA sequences that protect the ends of chromosomes from deterioration or fusion with neighboring chromosomes, and their lengths might vary with sex and age. Here, age‐ and sex‐related telomere lengths in male and female captive Siamese cobras (Naja kaouthia) were investigated using quantitative real‐time polymerase chain reaction based on cross‐sectional data. A negative correlation was shown between telomere length and body size in males but not in females. Age‐related sex differences were also recorded. Juvenile female snakes have shorter telomeres relative to males at up to 5 years of age, while body size also rapidly increases during this period. This suggests that an accelerated increase in telomere length of female cobra results from sex hormone stimulation to telomerase activity, reflecting sexually dimorphic phenotypic traits. This might also result from amplification of telomeric repeats on sex chromosomes. By contrast, female Siamese cobras older than 5 years had longer telomeres than males. Diverse sex hormone levels and oxidative stress parameters between sexes may affect telomere length.     

The role of HeT-A and TART retrotransposons in Drosophila telomere capping

Drosophila telomeres contain multiple copies of HeT-A and TART retrotransposons. These elements specifically transpose to chromosomal ends, compensating for loss of terminal nucleotides that occurs at each cycle of DNA replication. We have investigated the role of these sequences in the formation of telomere–telomere attachments induced by mutations in the UbcD1 gene. We have constructed UbcD1 mutant males carrying terminally deleted X chromosomes devoid of both HeT-A and TART sequences. Cytological analysis of larval neuroblasts from these males revealed that telomeres lacking HeT-A and TART and normal telomeres that contain these sequences participate in telomeric fusions with comparable frequencies. These results indicate that the UbcD1 substrate(s) binds chromosomal termini in a sequence-independent manner. Previous studies have shown that the telomere-capping protein HP1 also binds telomeres lacking HeT-A and TART. Taken together, these findings strongly suggest that the assembly of DNA–protein complexes that protect chromosome ends from fusions do not require specific terminal sequences.     

Dynamics of telomere turnover inPlasmodium berghei

Non-uniform composition in telomeric repeats at the extremities ofPlasmodium chromosomes was exploited in order to obtain data on intraclonal diversification of telomeric sequences, relevant for the study of telomere regeneration dynamics. Families of sibling telomeric clones were obtained from several chromosomal ends ofPlasmodium berghei, and analysed so as to determine the exact points from which individual clones start to diverge. As much as 90% of the telomeric tract appears to be subject to events causing abrupt changes in the sequence of telomeric repeats. The results are compatible with the hypothesis that breakpoint probability is a continuously increasing function over the entire telomeric tract.     

Repetitive sequences associated with differentiation of W chromosome in Semaprochilodus taeniurus

The possible origins and differentiation of a ZZ/ZW sex chromosome system in Semaprochilodus taeniurus, the only species of the family Prochilodontidae known to possess heteromorphic sex chromosomes, were examined by conventional (C-banding) and molecular (cross-species hybridization of W-specific WCP, Fluorescence in situ hybridization (FISH) with telomere (TTAGGG)n, and Rex1 probes) cytogenetic protocols. Several segments obtained by W-specific probe were cloned, and the sequences localized on the W chromosome were identified by DNA sequencing and search of nucleotide collections of the NCBI and GIRI using BLAST and CENSOR, respectively. Blocks of constitutive heterochromatin in chromosomes of S. taeniurus were observed in the centromere of all autosomal chromosomes and in the terminal, interstitial, and pericentromeric regions of the W chromosome, which did not demonstrate interstitial telomeric sites with FISH of the telomere probe. The Rex1 probe displayed a compartmentalized distribution pattern in some chromosomes and showed signs of invasion of the pericentromeric region in the W chromosome. Chromosomal painting with the W-specific WCP of S. taeniurus onto its own chromosomes showed complete staining of the W chromosome, centromeric sites, and the ends of the Z chromosome, as well as other autosomes. However, cross-species painting using this WCP on chromosomes of S. insignis, Prochilodus lineatus, and P. nigricans did not reveal a proto-W element, but instead demonstrated scattered positive signals of repetitive DNAs. Identification of the W-specific repetitive sequences showed high similarity to microsatellites and transposable elements. Classes of repetitive DNA identified in the W chromosome suggested that the genetic degeneration of this chromosome in S. taeniurus occurred through accumulation of these repetitive DNAs.     

De novo synthesis of telomere sequences at the healed breakpoints of wheat deletion chromosomes

When chromosomes are broken, the breakpoints become highly unstable and acquire the ability to fuse with other broken ends. The breakpoints are, however, eventually stabilized, and, therefore, the broken chromosomes are transmitted to the daughter cells without further morphological change. This phenomenon, known as “healing of breakpoints”, involves the addition of repetitive telomere sequences at the breakpoints by telomerase, the enzyme that normally synthesizes the telomere sequence at normal chromosome terminals. In many higher organisms, however, this property has not been well investigated. In this study, we examined the telomere sequences in wheat deletion lines with breakpoints on chromosome 1B. Lines that had breakpoints around the nucleolar organizer region were first selected on the basis of cytological observations, and the precise breakpoints were determined by mapping a fragment of rDNA and RFLP markers. In three lines – in addition to one previously reported – the DNA fragments encompassing the breakpoints were amplified by PCR using primers located in the rDNA and in telomere sequences. The DNA sequences provide insight into the properties of the telomerase activity at the breakpoints. The telomere sequences initiated from 2- to 4-nucleotide motifs in the original ribosomal DNA sequence which are also found in the repeat unit characteristic of telomere sequences. No specific sequences or structures were observed at or around the breakpoints. At all of the four breakpoints investigated, the newly synthesized telomere sequences contained considerable numbers of atypical telomere sequence units, particularly TTAGGG, which is the common unit of mammalian telomere sequences. Based on these results, we discuss the ability of plant telomerase to initiate the de novo synthesis of telomere sequences at internal breakpoints. Received: 15 June 1999 / Accepted: 6 August 1999     

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.     

Telomeric localization of the Arabidopsis‐type heptamer repeat, (TTTAGGG)n,at the chromosome ends in Saccharina japonica (Phaeophyta)

Telomeres generally consist of short repeats of minisatellite DNA sequences and are useful in chromosome identification and karyotype analysis. To date, telomeres have not been characterized in the economically important brown seaweed Saccharina japonica, thus its full cytogenetic research and genetic breeding potential has not been realized. Herein, the tentative sequence of telomeres in S. japonica was identified by PCR amplification with primers designed based on the Arabidopsis‐type telomere sequence (TTTAGGG)_n, which was chosen out of three possible telomeric repeat DNA sequences typically present in plants and algae. After PCR optimization and cloning, sequence analysis of the amplified products from S. japonica genomic DNA showed that they were composed of repeat units, (TTTAGGG)_n, in which the repeat number ranged from 15 to 63 (n = 46). This type of repeat sequence was verified by a Southern blot assay with the Arabidopsis‐type telomere sequence as a probe. The digestion of S. japonica genomic DNA with the exonuclease Bal31 illustrated that the target sequence corresponding to the Arabidopsis‐type telomere sequence was susceptible to Bal31 digestion, suggesting that the repeat sequence was likely located at the outermost ends of the kelp chromosomes. Fluorescence in situ hybridizations with the aforementioned probe provided the initial cytogenetic evidence that the hybridization signals were principally localized at both ends of S. japonica chromosomes. This study indicates that the telomeric repeat of the kelp chromosomes is (TTTAGGG)_n which differs from the previously reported (TTAGGG)_n sequence in Ectocarpus siliculosus through genome sequencing, thereby suggesting distinct telomeres in brown seaweeds.     

Interaction of an Arabidopsis RNA-binding protein with plant single-stranded telomeric DNA modulates telomerase activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3' overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.     

Telomeres: more than chromosomal non-sticking ends

Telomeres are specialized natural ends of eukaryotic chromosomes that, contrary to the ends of broken chromosomes, are stable and do not fuse with the ends of other chromosomes. In addition, telomeres protect chromosomal ends from degradation, facilitate completion of chromosomal DNA replication, and contribute to chromosome positioning within nuclei. Telomeric DNA consists of repetitive sequences and specific associated proteins, including the telomere repeat-binding factors TRF1 and TRF2. A lack of TRF2 enables end-to-end chromosome fusion. A structural disruption of telomeres not only causes chromosomal mechanical instability but also activates a programmed cell death cascade.     

Mapping of rRNA genes and telomeric sequences in Danube salmon (<Emphasis Type="Italic">Hucho hucho</Emphasis>) chromosomes using primed in situ labeling technique (PRINS)

In the current paper we described the application of primed in situ (PRINS) labeling approach for the chromosomal mapping of repetitive DNA sequences in Danube salmon (Hucho hucho) (2n = 82, NF = 112). PRINS was successfully performed with primers enabling amplification of 5S rRNA genes (minor rDNAs), NOR building DNA sequences (major rDNAs), and telomeric sequences. Two loci of 5S rRNA were observed on distinct chromosome pairs; the minor arrays were located interstitially on the long (q) arms of two large metacentrics (chromosomes No. 3) and the large clusters of 5S rDNAs were assigned to the short (p) arms of two subtelocentric chromosomes No. 18. Major rDNA clusters were observed on the p-arms of two submeta-subtelocentric chromosomes No. 10. These chromosomal areas were built with GC-rich chromatin what was proved in the course of chromomycin A(3) (CMA(3)) staining performed sequentially. Major and minor rDNA families were not co-localized in the Danube salmon chromosomes.The distinct hybridization signals at the ends of all the chromosomes were provided in the course of PRINS with (CCCTAA)( n ) primer. The chromosomal localization of rRNA genes and telomeric DNA sequences was discussed in the context of Salmonidae karyotype evolution.     

Telomeric signals in robertsonian fusion and fission chromosomes: implications for the origin of pseudoaneuploidy.

In situ hybridization with synthetic plant telomeric sequences resulted in labeling of all broad bean (Vicia faba) chromosomes at their ends only. Telocentric chromosomes derived by fission of the metacentric satellite chromosome of V. faba also showed signals at both of their ends, whereas the ancestral metacentric did not display signals at its primary constriction, the point of fission. As in V. faba, all acrocentric mouse chromosomes were labeled by in situ hybridization with a vertebrate telomeric probe at both ends of each chromatid exclusively. However, different metacentric Robertsonian chromosomes derived by fusion of defined acrocentrics did not show signals at their primary constrictions. The mechanism of Robertsonian rearrangement leading to a pseudoaneuploid increase or decrease in chromosome number therefore cannot consist solely of a simple fission or fusion of chromosomes without a concomitant gain or loss of chromatin material. The additional assumption of a subdetectable deletion of telomeric sequences after fusion and amplification of these sequences following fission is necessary to explain the present observations.     

Progressive rearrangement of telomeric sequences added to both the ITR ends of the yeast linear pGKL plasmid

Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. InSaccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats TG_1–3 of 300–350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG_1–3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids. Published: February 17, 2003     

Telomere-related sequences at interstitial sites in the human genome

The ends (telomeres) of eukaryotic chromosomes are protected from degradation and from loss during DNA replication by buffers of simple tandem repetitive sequence. The nucleotide sequence of these telomeric arrays is fundamental to telomere function as a site for protein and ribonucleoprotein binding and varies only slightly in a wide range of organisms. We present evidence that arrays of this human telomeric sequence, TTAGGG, are present not only at the ends of human chromosomes but also at numerous interstitial sites. These interstitial loci share nucleotide sequence similarity outside the repetitive array, suggesting that they are related functionally or have evolved from a common progenitor locus.     

Searching for telomeric sequences in two Allium species.

Some Alliaceae species have no tandemly repeated TTTAGGG sequences. Instead, at the very end of their chromosomes, there are highly repetitive satellite and (or) rDNA sequences. These sequences apparently replace the canonical plant telomeric sequences in these species. A method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs), combined with fluorescent in situ hybridization, has revealed that telomeric chromatin is tightly condensed at the ends of SCs in plants and animals. Using this method, we have tested the organization and location of those sequences postulated to cap the chromosomes in two species of the genus Allium: A. cepa and A. altaicum. We have also extended this study to other putative telomere candidates, such as LTR (long terminal repeat) and non-LTR retrotransposons. None of the DNA sequences analyzed showed the characteristic telomeric organization at pachytene.     

In vitro and in vivo reconstitution and stability of vertebrate chromosome ends.

Telomeres are essential repetitive sequences at the ends of chromosomes that prevent chromosome fusion and degradation. We have successfully recapitulated these two protective functions in vivo and in vitro by incubating blunt-end DNA constructs having vertebrate telomeric ends in Xenopus eggs and egg extracts. Constructs with telomeric ends are stable as linear molecules; constructs with non-telomeric ends undergo intramolecular fusion. In extracts, 99.8% of the telomeric constructs from 78 to 700 bp in length are assembled into 'model telomeres' in <5 min and have an extra-polated half-life of >3.5 years. Non-telomeric constructs circularize with first order kinetics and a half-life of 4 h. In living eggs the telomeric constructs are protected from fusion and degradation. The stability of the telomeric constructs is not due to covalent processing. Extract can protect approximately 100 pM telomeric ends (equivalent to 1.7 x 10(7) ends/egg) even in the presence of a 20-fold excess of double-stranded telomeric DNA, suggesting that protection requires end-specific factors. Constructs with (TTGGGG) n repeats are unstable, suggesting that short tracts of this and other telomere-like sequences found within human telomeres could lead to genome instability if exposed by partial telomere erosion during aging.     

HOT1 is a mammalian direct telomere repeat‐binding protein contributing to telomerase recruitment

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC‐based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere‐binding protein 1 (HOT1). HOT1 directly and specifically binds double‐stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase‐dependent telomere elongation.     

Importance of TRF1 for functional telomere structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.