End Replication Problem Research Articles

Age and Heat Stress as Determinants of Telomere Length in a Long-Lived Fish, the Siberian Sturgeon.

Telomeres shorten at each cell division due to the end-replication problem but also in response to oxidative stress. Consequently, telomeres shorten with age in many endotherms, and this shortening is accelerated under stressful environmental conditions. Data in ectotherm vertebrates remain scarce so far, so our goal was to review existing data for fish and to test the influence of age and stress on telomere length in a very long-lived fish, the Siberian sturgeon (Acipenser baerii). Our review of the literature revealed age-related telomere shortening in approximately half of the published studies. In the Siberian sturgeon, we found a significant telomere shortening with age, both at the intraindividual level using red blood cells (-12.5% in 16 mo) and at the interindividual level using cross-sectional samples of fin over an age range of 8 yr. We also found that heat stress (30°C) significantly reduced telomere length by 15.0% after only 1 mo of exposure. Our results highlight that both age and stressful environmental conditions might be important determinants of telomere length in fish.

Regulation of the Telomerase Reverse Transcriptase Subunit through Epigenetic Mechanisms.

Chromosome-shortening is characteristic of normal cells, and is known as the end replication problem. Telomerase is the enzyme responsible for extending the ends of the chromosomes in de novo synthesis, and occurs in germ cells as well as most malignant cancers. There are three subunits of telomerase: human telomerase RNA (hTERC), human telomerase associated protein (hTEP1), or dyskerin, and human telomerase reverse transcriptase (hTERT). hTERC and hTEP1 are constitutively expressed, so the enzymatic activity of telomerase is dependent on the transcription of hTERT. DNA methylation, histone methylation, and histone acetylation are basic epigenetic regulations involved in the expression of hTERT. Non-coding RNA can also serve as a form of epigenetic control of hTERT. This epigenetic-based regulation of hTERT is important in providing a mechanism for reversibility of hTERT control in various biological states. These include embryonic down-regulation of hTERT contributing to aging and the upregulation of hTERT playing a critical role in over 90% of cancers. Normal human somatic cells have a non-methylated/hypomethylated CpG island within the hTERT promoter region, while telomerase-positive cells paradoxically have at least a partially methylated promoter region that is opposite to the normal roles of DNA methylation. Histone acetylation of H3K9 within the promoter region is associated with an open chromatin state such that transcription machinery has the space to form. Histone methylation of hTERT has varied control of the gene, however. Mono- and dimethylation of H3K9 within the promoter region indicate silent euchromatin, while a trimethylated H3K9 enhances gene transcription. Non-coding RNAs can target epigenetic-modifying enzymes, as well as transcription factors involved in the control of hTERT. An epigenetics diet that can affect the epigenome of cancer cells is a recent fascination that has received much attention. By combining portions of this diet with epigenome-altering treatments, it is possible to selectively regulate the epigenetic control of hTERT and its expression.

G-Quadruplex ligands: Potent inhibitors of telomerase activity and cell proliferation in Plasmodium falciparum

Telomeres are DNA and protein structures located at the ends of eukaryotic chromosomes. These structures maintain chromosomal stability by impeding chromosomal ends from being recognized and processed as fragmented DNA. They also support the complete replication of the genome by providing mechanisms that solve the end-replication problem. Telomeric DNA is formed of short, repeating, guanine-rich sequences. The G-rich sequence extends towards the 3′ end, forming a protruding single-stranded end that can acquire a conformation known as G-quadruplex. The ligands stabilizing this structure are potent inhibitors of telomerase activity, a catalytic activity necessary to compensate for the loss of telomeric DNA that occurs in each round of replication. In the absence of telomerase, telomeres shorten after a given number of cell divisions, after which the cell enters a senescence state and finally dies. In the presence of telomerase activity, telomeres are preserved, and cells reach a state of indefinite replication or immortalization.This study analyzed the effect of two ligands of the G-quadruplex (TMPyP4 and Telomestatin) on the telomerase activity and cell proliferation of Plasmodium falciparum, given that this parasite has a high proliferation rate and an almost indefinite replication capacity. The effect of the ligands on telomerase activity was evaluated using the TRAP (Telomere Repeat Amplification Protocol) activity assay, which was performed in the presence of increasing concentrations of each ligand. In this study, TMPyP4 showed the highest inhibitory effect, reaching 50% inhibition at a 5μM concentration. Regarding proliferation, both ligands drastically affected parasite growth, but Telomestatin had a stronger effect. After three days of treatment, parasite growth decreased by 90%. Thus, it is possible that this compound interferes with other vital pathways for the parasite beyond the elongation of telomeric DNA by telomerase.

The Borrelia burgdorferi telomere resolvase, ResT, anneals ssDNA complexed with its cognate ssDNA-binding protein.

Spirochetes of the genus Borrelia possess unusual genomes that consist in a linear chromosome and multiple linear and circular plasmids. The linear replicons are terminated by covalently closed hairpin ends, referred to as hairpin telomeres. The hairpin telomeres represent a simple solution to the end-replication problem. Deoxyribonucleic acid replication initiates internally and proceeds bidirectionally toward the hairpin telomeres. The telomere resolvase, ResT, forms the hairpin telomeres from replicated telomere intermediates in a reaction with similarities to those promoted by type IB topoisomerases and tyrosine recombinases. ResT has also been shown to possess DNA single-strand annealing activity. We report here that ResT promotes single-strand annealing of both free DNA strands and ssDNA complexed with single-stranded DNA binding protein (SSB). The annealing of complementary strands bound by SSB requires a ResT–SSB interaction that is mediated by the conserved amphipathic C-terminal tail of SSB. These properties of ResT are similar to those demonstrated for the recombination mediator protein, RecO, of the RecF pathway. Borrelia burgdorferi is unusual in lacking identifiable homologs of the RecFOR proteins. We propose that ResT may provide missing RecFOR functions.

Editorial: The Evolving Telomeres.

Editorial: The Evolving Telomeres.

Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system

Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.

Protein-Primed Replication of Bacteriophage Φ29 DNA.

The requirement of DNA polymerases for a 3'-hydroxyl (3'-OH) group to prime DNA synthesis raised the question about how the ends of linear chromosomes could be replicated. Among the strategies that have evolved to handle the end replication problem, a group of linear phages and eukaryotic and archaeal viruses, among others, make use of a protein (terminal protein, TP) that primes DNA synthesis from the end of their genomes. The replicative DNA polymerase recognizes the OH group of a specific residue in the TP to initiate replication that is guided by an internal 3' nucleotide of the template strand. By a sliding-back mechanism or variants of it the terminal nucleotide(s) is(are) recovered and the TP becomes covalently attached to the genome ends. Bacillus subtilis phage ϕ29 is the organism in which such a mechanism has been studied more extensively, having allowed to lay the foundations of the so-called protein-primed replication mechanism. Here we focus on the main biochemical and structural features of the two main proteins responsible for the protein-primed initiation step: the DNA polymerase and the TP. Thus, we will discuss the structural determinants of the DNA polymerase responsible for its ability to use sequentially a TP and a DNA as primers, as well as for its inherent capacity to couple high processive synthesis to strand displacement. On the other hand, we will review how TP primes initiation followed by a transition step for further DNA-primed replication by the same polymerase molecule. Finally, we will review how replication is compartmentalized in vivo.

Age-Adjusted Telomere Length May Predict Treatment-Related Mortality in Children Undergoing Hematopoietic Stem Cell Transplantation

IntroductionTelomeres are specialized DNA structures found at the end of linear chromosomes. In humans, they contain the repetitive sequence (TTAGGG)nalong with its complementary strand and associated proteins. With each cell division, the telomere shortens due to incomplete replication of the lagging strand, a phenomenon known as the end replication problem. Oxidative damage, as well as the action of nucleases also leads to telomere attrition. Once the telomere reaches a critically short length, cells enter senescence or apoptosis. In the general population, telomere length (TL) varies greatly, and is seen to decline with age. Patients with Dyskeratosis Congenita (DC) have inherently short telomeres, which leads to multiple organ dysfunction. These patients suffer increased organ toxicity when given standard myeloablative conditioning regimens during hematopoietic stem cell transplantation (HSCT). This is most likely due to the reduced ability of their cells to replicate and recover following cytotoxic treatment. It is not known whether patients without DC, who have telomere lengths at the lower end of the normal spectrum also suffer increased rates of toxicity. Thus we undertook a retrospective analysis to determine if there was an association between short TL and increased transplant-related mortality (TRM) following HSCT in children.MethodMedical records of patients who underwent allogeneic HSCT at the Children's Hospital at Westmead (Sydney, Australia) between 2002 to 2013 were examined and demographic, prior treatment, transplant, toxicity and transplant information was extracted. The primary outcome measured was TRM, while overall survival, relapse and graft versus host disease (GVHD) being secondary outcomes of interest. Stored DNA, extracted from bone marrow, taken within 3 months prior to HSCT was used for telomere length testing, as a surrogate marker for TL in other tissues in the body. Quantitative PCR was carried out to determine relative telomere length, which was converted to an age adjusted TL (AATL) by subtracting the expected relative TL (i.e. 50th percentile for age of the patient) from the measured relative TL, so that patients of all ages could be analyzed together.ResultsBetween 2002 and 2013, there were 121 patients who underwent allogeneic HSCT at our institution. Of these, 78 had DNA stored, with appropriate consent and ethics approval to be used in our study. Most children were undergoing HSCT for hematological malignancies (acute lymphoblastic leukemia 52%, acute myeloid leukemia 39%), with 55% having relapse or refractory disease. Matched sibling donors made up 46% of cases. At a median follow up of 3.2 years, 64% of patients remained alive. The single leading cause of death was relapse (48%) with 52% of deaths attributable to transplant-related causes.The median AATL for the entire cohort was -0.21 (range -0.61-0.28). Telomere length was not associated with relapse or refractory disease. There was no correlation between AATL and specific organ toxicity, relapse or overall survival. When the cohort was divided into 2 groups based on AATL, there was a trend (p=0.055) towards increased TRM in patients who had AATL below the median for the cohort (Figure 1). Multivariate analysis adjusting for age at transplant, CMV status, disease status and HLA matching showed a reduced risk (HR 0.33; p value 0.072) of TRM in patients with AATL above the median for the cohort.ConclusionAATL was shorter in patients pre-HSCT compared with age matched controls. There was a trend towards increased TRM in patients with shorter telomere length, however most likely due to small sample size, this did not achieve significance. If extended to a larger cohort of patients, TL may prove to be a significant marker for children at increased risk of TRM prior to HSCT. This could influence the choice to use less myeloablative conditioning in these patients.Support: NHMRC GNT1056667 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Eµ-TCL1mTerc-/- Mouse Model for Telomere Dysfunction in Chronic Lymphocytic Leukemia

Telomeres are nucleo-protein complexes at the ends of the chromosomes that play a key role in protection of the ends from being recognized as DNA damage and to prevent fusion of the chromosomes. The telomeric DNA shortens with each cell division in the absence of telomerase, due to end replication problem. In chronic lymphocytic leukemia (CLL), short telomeres were found to be associated with poor prognostic factors and poor survival in various univariable and multivariable analyses. Short telomeres in CLL are known to be frequently associated with increased DNA damage response and to undergo fusion events, conferring genomic instability. But the contribution of telomere dysfunction to CLL pathogenesis and disease progression has never been studied in vivo using mouse models.Here, we hypothesized that genomic instability resulting from telomere dysfunction could drive acquisition of genetic lesions, contributing to CLL pathogenesis, progression and disease evolution. Thus, the CLL mouse model with telomere dysfunction was generated by crossing the Eµ-TCL1 (TCL1+) mouse with mTerc-/- mouse. The first generation TCL1+ mTerc-/- (G1) mice were inter-crossed to obtain generations G2 and G3, as telomeres are known to shorten with subsequent generations. The TCL1+ mTerc-/- mice from the generations G1 (N=14), G2 (N=33) and G3 (N=26), including TCL1+ (N=34), wildtype (WT, N=18) and mTerc-/- G1 (N=4), G2 (N=5) and G3 (N=13) as controls were initially analyzed for disease burden in peripheral blood (PB) by bleeding at an interval of 4 weeks, starting from 12 weeks and the percentage of CD19+ CD5+ cells was estimated by FACS. No difference in disease onset or progression was observed between the TCL1+ mTerc-/- G1, G2 and G3 in comparison toTCL1+ mice (Fig. 1a). Similarly, analysis of survival showed no significant difference between the TCL1+ mTerc-/- G1 (N=14), G2 (N=33) and G3 (N=26) mice, compared to TCL1+ (N=34) (median: 53, 55, 52 weeks vs. 50.5 weeks, Fig. 1b).Spleen and liver weights in the TCL1+ mTerc-/- G1 (N=12), G2 (N=33) and G3 (N=26) mice were highly variable (spleen: 0.1g to 3.5g, liver: 0.1g to 8.0g) as in the TCL1+ (N=27, spleen: 0.3g to 5.0g, liver: 1.7g to 7.4g) mice but no significant difference in spleen (Fig. 1d) and liver weights was observed between the subgroups. Interestingly, spleen weights were associated with survival only in the TCL1+ mice, with larger spleens associated with worse survival (48.5 vs. 57.5 weeks, P=0.091). Since no difference in disease characteristics was observed, it was verified using Q-PCR, if telomere lengths vary in the tumors from the different subgroups. Telomere lengths of CLL cells from the spleen were significantly shorter (Fig. 1c) in the G1 (median: 20.5kb, P=0.0002), G2 (median: 18.5kb, P=0.0016) and G3 (median: 13.2kb, P<0.0001) compared to TCL1+ (median: 28.7kb). The absence of correlation of telomere length with survival in the murine CLL models with telomere dysfunction may indicate that a critical telomere length in the tumor is yet to be reached to elicit genetic alterations and clonal selection.Additionally, the G3 mTerc-/- microenvironment is known to restrict B and T lymphopoiesis and thus might influence CLL cell proliferation, masking disease aggressiveness in the TCL1+ mTerc-/- G3 mice. To overcome the influence of mTerc-/- microenvironment, CLL cells obtained from spleens of TCL1+ and TCL1+ mTerc-/- G3 mice were transferred into syngeneic C57Bl6 mice. Briefly, 20 million cells were intravenously injected into the tail vein and disease was monitored by analysis of CD19+ CD5+ cells in PB, once every 4 weeks. Early follow up of 8 weeks clearly show a trend towards increase in CLL cells in PB of mice transferred with TCL1+ mTerc-/- G3 tumors compared to those with TCL1+ tumors (median tumor load: 15.75% vs. 6.1%, P=0.0553). Longer follow up of the experiment is ongoing.In summary, the TCL1+ mTerc-/- mice across the generations G1, G2 and G3 showed no difference in disease onset, progression, disease burden and survival in comparison to TCL1+ mice. The absence of increased disease manifestation in the TCL1+ mTerc-/- may be attributed to the microenvironmental influence on lymphopoiesis, as syngeneic transfer of CLL from TCL1+ mTerc-/- G3 mice showed an increase in tumor load compared to that of TCL1+ tumors, indicating a contribution of telomere shortening to disease aggressiveness in CLL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3870: Telomeric non-coding RNA causes genome-wide alteration of gene expression in cancer

Abstract The end replication problem of linear chromosome DNA causes telomere shortening and eventual senescence of normal cells. Cancer cells reactivate telomerase to maintain their telomere length and exhibit an infinite replicative potential. However, telomerase activation does not necessarily increase the average telomere length in cancer cells. Actually, cancer cells often maintain shorter telomeres than the cells in the surrounding normal tissues. We previously demonstrated that forced telomere elongation in cancer cells promotes their differentiation in a xenograft tumor and represses innate immune genes that are upregulated in various human cancers. This implies that short telomeres of cancer cells contribute to their malignancy. However, it is completely unknown how such genetic repression is caused by elongated telomeres. Here, we report that telomeric repeat-containing RNA (TERRA) induces a genome-wide alteration of gene expression in telomere-elongated human cancer cells. Using three different cancer cell lines, including those from prostate, breast and stomach, we found that telomere elongation by hTERT overexpression up-regulates TERRA signals, which were detected by northern blot analysis, and down-regulates innate immune genes such as STAT1, ISG15, and OAS3 in vivo. These changes were caused by telomere elongation and not by enhanced telomerase activity or hTERT overexpression per se. Down-regulation of the innate immune genes was also observed in the telomere-elongated cells under three-dimensional (3D) culture conditions, which mimicked the tumor microenvironment in vivo. Introduction of TERRA oligonucleotides repressed the innate immune genes even in cells with short telomeres under 3D culture conditions. Intriguingly, this gene repression appeared to occur from the action of the non-canonical nucleic acid structure called G-quadruplex (G4) in TERRA, because non-telomeric (i.e., non-G4-forming) control oligonucleotides had no effect whereas another non-telomeric but G4-forming oligonucleotide phenocopied the TERRA oligonucleotides. Transcriptome analysis revealed that telomere elongation and G4-forming oligonucleotides showed similar gene expression signatures. Most of the commonly suppressed genes were involved in the innate immune system and were up-regulated in various human cancers. Taken together, these observations suggest that the telomeric non-coding RNA G4 counteracts cancer malignancy through suppression of innate immune genes, and give a new insight into our understanding of telomere shortening in human cancer. Citation Format: Kyotaro Hirashima, Hiroyuki Seimiya. Telomeric non-coding RNA causes genome-wide alteration of gene expression in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3870. doi:10.1158/1538-7445.AM2015-3870

Telomere G-Quadruplex as a Potential Target to Accelerate Telomere Shortening by Expanding the Incomplete End-Replication of Telomere DNA.

Chromosomes in human cells are protected by telomeres. Telomere shortens during each round of cell division because of the DNA end-replication problem. Cancer cells maintain telomere length homeostasis by either telomerase or/and the alternative lengthening of telomere (ALT) mechanism to sustain their division potential. Telomeric DNA tends to form G-quadruplex preferentially at the extreme 3' end. This unique feature prevents the 3' end from being used as a substrate of telomerase and as a primer in the ALT. Therefore, stabilizing telomere G-quadruplex is expected to inhibit both pathways and limit the proliferation of cancer cells. Based on a mathematical modeling and experimental results, this mini-review proposes a hypothesis that the formation of G-quadruplex in telomere may constitute a significant contribution to the incomplete end-replication of telomere DNA by preventing the priming of DNA synthesis near the 3' end during telomere replication. According to this, stabilization of telomere G-quadruplex by chemical ligand may promise to accelerate telomere shortening in proliferating cells.

Emerging therapeutic approaches based on nanotechnology for the treatment of diseases associated with telomere dysfunction.

Telomeric diseases are a group of rare progeroid genetic syndromes, presenting premature aging phenotypes, characterized for defects on telomere maintenance. In humans, telomeres are heterochromatic structures consisting of long TTAGGG repeats located at the chromosomal ends, which shorten progressively after each DNA replication because of the 'end replication problem'. Critically short telomeres activate a DNA damage response that leads to the arrest of the cell cycle and resulting in cellular senescence or apoptosis. Furthermore, excessively short telomeres are prone to create telomeric fusions, causing genomic instability and malignant transformation. In order to counteract this process, there are two enzymatic complexes, the telomerase complex, with the capacity to elongate telomeres; and the shelterin complex, which protects them from being recognized as DNA breaks. Over the last few decades, several studies have confirmed that critically short telomeres and defects in telomere-associated enzymatic complexes are involved in the development of a group of rare human genetic diseases, with the accumulation of excessive telomere attrition as the underlying cause of these pathologies. Despite the severity of these disorders, there is no curative treatment for any of them. In light of this, this review summarizes the most important defective telomere diseases, their current management, and it presents possible therapeutic strategies based on nanotechnology which may open up new possibilities for their treatment.

Telomerase exerts physiological and tumor promoting functions by stimulating ribosomal biogenesis

The enzymatic activity of telomerase counters erosion of telomeric DNA that occurs as a biochemical consequence during replication. This primary function of telomerase resolves the ‘end-replication problem’ and prevents telomere dysfunction and replicative senescence ensuring cellular immortality. The vast majority of human cells are devoid of telomerase activity but reactivation of telomerase occurs in more than 80% of human cancers. Moreover, telomerase activity is a pre-requisite of sustained organ maintenance under physiological hyper-proliferative conditions such as liver regeneration and for the proliferation of activated B- and T- cells. Increasing evidence indicates at telomere length independent functions of telomerase, including DNA-damage repair, mitochondrial function and stem cell activity. Mechanisms of how telomerase can fulfill such diverse functions remain yet to be elucidated. Our recent findings showing that telomerase activates ribosomal biogenesis under oncogenic and regenerative conditions can shed a light to some of these questions.

Telomeric aging: mitotic clock or stress indicator?

Telomeres are regions of tandem arrays of TTAGGG repeats and associated proteins located at chromosomal ends that allow cells to distinguish chromosome ends from double-strand breaks and protect chromosomes from end-to-end fusion, recombination, and degradation (Houben et al., 2008). Telomeres are not linear structures, telomeric DNA is maintained in a loop structure due to many key proteins. This structure serves to protect the ends of chromosomes (Neidle and Parkinson, 2003). Telomeres are subjected to shortening at each cycle of cell division due to incomplete synthesis of the lagging strand during DNA replication owing to the inability of DNA polymerase to completely replicate the ends of chromosome DNA (“end-replication problem”) (Muraki et al., 2012). Therefore, they assume to limit the number of cell cycles and act as a “mitotic clock” (Olovnikov, 1996). Shortened telomeres cause decreased proliferative potential, thus triggering senescence (Blackburn et al., 2006).

Telomere Restriction Fragment (TRF) Analysis.

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al., 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells. Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al., 1990; Ouellette et al., 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Telomerase Repeated Amplification Protocol (TRAP).

Telomeres are found at the end of eukaryotic linear chromosomes, and proteins that bind to telomeres protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions (Griffith et al., 1999). However, due to the end replication problem and other factors such as oxidative damage, the limited life span of cultured cells (Hayflick limit) results in progressive shortening of these protective structures (Hayflick and Moorhead, 1961; Olovnikov, 1973). The ribonucleoprotein enzyme complex telomerase-consisting of a protein catalytic component hTERT and a functional RNA component hTR or hTERC- counteracts telomere shortening by adding telomeric repeats to the end of chromosomes in ~90% of primary human tumors and in some transiently proliferating stem-like cells (Shay and Wright, 1996; Shay and Wright, 2001). This results in continuous proliferation of cells which is a hallmark of cancer. Therefore, telomere biology has a central role in aging, cancer progression/metastasis as well as targeted cancer therapies. There are commonly used methods in telomere biology such as Telomere Restriction Fragment (TRF) (Mender and Shay, 2015b), Telomere Repeat Amplification Protocol (TRAP) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this detailed protocol we describe Telomere Repeat Amplification Protocol (TRAP). The TRAP assay is a popular method to determine telomerase activity in mammalian cells and tissue samples (Kim et al., 1994). The TRAP assay includes three steps: extension, amplification, and detection of telomerase products. In the extension step, telomeric repeats are added to the telomerase substrate (which is actually a non telomeric oligonucleotide, TS) by telomerase. In the amplification step, the extension products are amplified by the polymerase chain reaction (PCR) using specific primers (TS upstream primer and ACX downstream primer) and in the detection step, the presence or absence of telomerase is analyzed by electrophoresis. TSNT is, an internal standard control, amplified by TS primer. NT is its own reverse primer, which is not a substrate for telomerase. These primers are used to identify false-negative results by if the gel lacks internal control bands.

Hairpin Telomere Resolvases

Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.

Abstract 2250: Role of single strand binding protein 1 in TERT recruitment to telomeres and in maintaining telomere G-overhangs

Abstract Telomeres cap the ends of all eukaryotic chromosomes from degradation or end-to-end fusions. Mammalian telomeres are maintained by telomerase which is a ribonucleoprotein complex that includes a reverse transcriptase catalytic unit called TERT and a functional telomerase RNA (TR or TERC), which acts as a template to maintain telomere length. Telomerase maintains telomere length, by overcoming incomplete lagging strand synthesis known as the “end replication problem” during DNA replication. Mammalian stem and cancer cells utilize telomerase to extend telomeric G-overhangs to partially (stem cells) or completely (tumor cells) maintain telomere ends. Cells expressing telomerase also have higher levels of single strand binding 1 (SSB1) protein, which has a known critical role in the DNA damage responses and double strand break repair specifically by homologous recombination. We have established that SSB1 binds specifically to G-strand telomeric DNA in vitro, and associates in vivo with telomeres both in human and mouse cell lines. Interestingly, SSB1 interacts with the TERT protein, and determines the amount of TERT interaction with telomeres. Deletion/depletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss, but has no effect on in vitro telomerase activity. We will discuss, how SSB1 is required for telomerase recruitment to telomeres to facilitate G-strand DNA extension and to maintain telomeres. Citation Format: Tej K. Pandita, Raj Pandita. Role of single strand binding protein 1 in TERT recruitment to telomeres and in maintaining telomere G-overhangs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2250. doi:10.1158/1538-7445.AM2014-2250

닭 텔로미어 길이의 유전력 추정과 유전 전이 양상

Department of Animal Science and Biotechnology, Gyeongnam National University of Science and Technology, Jinju 660-758, KoreaABSTRACT Telomeres are the ends of the eukaryotic chromosomes and consist of a tandem repetitive DNA sequence and shelterin protein complex. The function of telomere is to protect chromosome. Telomere length in somatic cells tends to decrease with organismal age due to the end replication problem. However, several factors at the genetic, epigenetic and environmental level affect telomere length. In this study, we estimated heritability of telomere length and investigated inheritance of telomeres in a chicken. Telomere length of lymphocytes was analyzed by semi-quantitative polymerase chain reaction using telomere primer and quantitative fluorescence in situ hybridization using telomeric DNA probe. In results, heritability of telomere length was estimated 0.9 at birth by offspring-parent regression analysis and was estimated 0.03 and 0.04 at 10 and 30 weeks old, respectively, by parental variance analysis. There was a significant positive correlation in telomere length between father and their offspring (r=0.348), and mother and their offspring (r=0.380). In inheritance patterns of telomere length, the influence of paternal and maternal effect on their offspring was similar. The influence of inherited telomeres on male and female progeny was also roughly alike. These results implicated that imprinting of parental telomere length was regulated by autosomal genes, not sex linked genes. In addition, telomere length of offspring at birth did not differ along with their maternal age. Thus, maternal age does not affects telomere length in their offspring at birth owing to cellular reprogramming at early embryonic stage.(Key words : heritability, telomere length, inheritance, chicken)

Single-molecule real-time detection of telomerase extension activity.

The ends of eukaryotic chromosomes are capped by telomeres which consist of tandem G-rich DNA repeats stabilized by the shelterin protein complex. Telomeres shorten progressively in most normal cells due to the end replication problem. In more than 85% of cancers however, the telomere length is maintained by telomerase; a reverse transcriptase that adds telomeric TTAGGG repeats using its integral RNA template. The strong association between telomerase activity and malignancy in many cancers suggests that telomerase activity could serve as a diagnostic marker. We demonstrate single-molecule, real-time telomerase extension activity observed digitally as the telomeric repeats are added to a substrate. The human telomerase complex pulled down from mammalian cells displays extension activity dependent on dNTP concentration. In complex with the processivity factor, POT1-TPP1, telomerase adds repeats at an accelerated rate and yields longer products. Our assay provides a unique detection platform that enables the study of telomerase kinetics with single molecule resolution.