Measuring telomerase activity using TRAP assays.

Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.

Gender-specific associations between mixture of polycyclic aromatic hydrocarbons and telomere length.

Evidence shows the relationships of individual environmental PAHs by their urinary metabolites with relative telomere length (RTL), which may be affected by biological gender differences. Since plasma parent PAHs are not metabolized, it may reflect human exposure to PAHs more realistically in daily life. Thus, exploring joint associations between plasma parent PAHs and RTL is urgent, which may identify the major contributor to its adverse effect. In this study, 2577 participants were obtained from the Henan Rural Cohort. The level of PAHs in blood samples was detected by gas chromatography coupled with tandem mass spectrometry. RTL in blood samples was detected by quantitative polymerase chain reaction. Generalized linear models or quantile g-computation were performed to evaluate the associations between the individual or a mixture of PAHs and RTL. Results from generalized linear models showed that each unit increment in BghiP value corresponded to a 0.098 (95%CI: 0.067, 0.129) increment in RTL for men; each unit increment in BaP, BghiP and Flu value corresponded to a 0.041 (95%CI: 0.014, 0.068), 0.081 (95%CI: 0.055, 0.107) and 0.016 (95%CI: 0.005, 0.027) increment in RTL for women. Results from quantile-g computation revealed that each one-quantile increment in the mixture of 10 PAHs corresponded to a 0.057 (95%CI: 0.021, 0.094) and 0.047 (95%CI: 0.003, 0.091) increment in RTL values of women and men, but these associations were mainly ascribed to three PAHs for women (BaP, Flu and BghiP) and men (BaP, BghiP and Pyr), respectively. Similar results were found in smoking men and cooking women without smoking. Our study found that exposure to 10 PAHs mixture was positively associated with RTL across gender, mainly attributed to Flu, BaP and BghiP, implicating that gender-specific associations may be ascribed to tobacco and cooking smoke pollution. The findings provided clues for effective measures to control PAHs pollutants-related aging disease.Clinical trial registration The Henan Rural Cohort Study has been registered at the Chinese Clinical Trial Register (Registration number: ChiCTR-OOC-15006699). Date of registration: 06 July 2015. http://www.chictr.org.cn/showproj.aspx?proj=11375 .

Human POT1 protects the telomeric ds-ss DNA junction by capping the 5' end of the chromosome.

Protection of telomeres 1 (POT1) is the 3' single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) at chromosome ends. What precludes the DDR machinery from accessing the telomeric double-stranded-single-stranded junction is unknown. We demonstrate that human POT1 binds this junction by recognizing the phosphorylated 5' end of the chromosome. High-resolution crystallographic structures reveal that the junction is capped by POT1 through a "POT-hole" surface, the mutation of which compromises junction protection in vitro and telomeric 5'-end definition and DDR suppression in human cells. Whereas both mouse POT1 paralogs bind the single-stranded overhang, POT1a, not POT1b, contains a POT-hole and binds the junction, which explains POT1a's sufficiency for end protection. Our study shifts the paradigm for DDR suppression at telomeres by highlighting the importance of protecting the double-stranded-single-stranded junction.

Early Environment and Telomeres: a Long-Term Toxic Relationship.

PURPOSE OF REVIEW: Telomere length (TL) shortening is a hallmark of biological aging. While studies have extensively focused on the impact of environmental exposures on TL in older populations, consistent evidence indicates that prenatal environmental exposures to air pollutants, polycyclic aromatic hydrocarbons, metals, and endocrine-disrupting chemicals influence TL shortening. Here, we summarize evidence linking prenatal environmental exposures with children's TL and discuss potential long-term effects. RECENT FINDINGS: Current evidence shows that prenatal environmental exposures alter TL and identify pregnancy as a critical window of susceptibility for telomere damage in children. However, results vary across studies, possibly depending on the source, exposure time window, and stage evaluated. Additional research is needed to investigate whether early TL alterations mediate long-term health effects of offspring. Prenatal environmental exposures induce early childhood changes in TL. Based on known links between TL and biological aging, these alterations may have long-term impact on individuals' health throughout life.

[Interaction of polycyclic aromatic hydrocarbon DNA adducts and telomere length on missed abortion].

Objective: To analyze the contribution and interaction of polycyclic aromatic hydrocarbons (PAH)-DNA adducts and changes of telomere length (TL) on missed abortion. Methods: From March to December 2019, patients with missed abortion in the First Hospital of Shanxi Medical University and pregnant women with normal pregnancy but voluntary abortion in the same department during the same period were selected and divided into a case group and a control group. Questionnaire was used to investigate the general situation and the pregnancy situation of the subjects. The abortion villi were collected and the content of PAH-DNA adducts and TL was detected. Logistic regression model was used to analyze the associated factors of missed abortion. R epiR package and Mediation package were used to analyze the effect and relationship between PAH-DNA adducts and TL on missed abortion. Results: The age of the subjects was(29.92±5.69)years old. The M(Q1,Q3)of PAH-DNA adducts was 453.75(404.61, 504.72) pg/ml. The M(Q1,Q3)of TL was 1.21(0.77, 1.72). The content of PAH-DNA adducts in the case group was higher than that in the control group (Z=-2.10, P=0.036), while the TL was lower than that in the control group (Z=-4.05, P<0.001). Multivariate logistic regression showed that low, medium and high levels of PAH-DNA adducts (OR=3.17,95%CI:1.41-7.14;OR=2.85,95%CI:1.25-6.52;OR=2.46,95%CI:1.07-5.64), and long, medium and short levels of TL (OR=2.50,95%CI:1.11-5.63;OR=3.32,95%CI:1.45-7.56;OR=3.22,95%CI:1.42-7.26) were all risk factors for missed abortion. The medium level of PAH-DNA adducts had a 2.76-fold higher risk of shortened TL than those with the lowest level, and no mediating role of TL was found. The stratified analysis showed that when the TL level was longer (>1.21), the low and high levels of PAH-DNA adducts were associated with missed abortion (all P<0.05); when the TL level was shorter (<1.21), the medium level of PAH-DNA adducts was associated with abortion (P=0.025). At lower levels of PAH-DNA adducts, no effect of TL on missed abortion was observed, while, at higher levels, TL was strongly associated with missed abortion (OR=7.50,95%CI:1.95-28.82;OR=6.04,95%CI:1.54-23.65;OR=9.05,95%CI:2.34-35.04). The interaction analysis found that the AP was 0.72 (95%CI: 0.46-0.99), and the SI was 5.21 (95%CI: 2.30-11.77). Conclusion: The high level of PAH-DNA adducts and shortened TL may increase the risk of missed abortion, and there may be a positive additive interaction between the two factors on missed abortion.

Complex interaction network revealed by mutation of human telomerase 'insertion in fingers' and essential N-terminal domains and the telomere protein TPP1.

In the majority of human cancer cells, cellular immortalization depends on the maintenance of telomere length by telomerase. An essential step required for telomerase function is its recruitment to telomeres, which is regulated by the interaction of the telomere protein, TPP1, with the telomerase essential N-terminal (TEN) domain of the human telomerase reverse transcriptase, hTERT. We previously reported that the hTERT 'insertion in fingers domain' (IFD) recruits telomerase to telomeres in a TPP1-dependent manner. Here, we use hTERT truncations and the IFD domain containing mutations in conserved residues or premature aging disease-associated mutations to map the interactions between the IFD and TPP1. We find that the hTERT-IFD domain can interact with TPP1. However, deletion of the IFD motif in hTERT lacking the N-terminus and the C-terminal extension does not abolish interaction with TPP1, suggesting the IFD is not essential for hTERT interaction with TPP1. Several conserved residues in the central IFD-TRAP region that we reported regulate telomerase recruitment to telomeres, and cell immortalization compromise interaction of the hTERT-IFD domain with TPP1 when mutated. Using a similar approach, we find that the IFD domain interacts with the TEN domain but is not essential for intramolecular hTERT interactions with the TEN domain. IFD-TEN interactions are not disrupted by multiple amino acid changes in the IFD or TEN, thus highlighting a complex regulation of IFD-TEN interactions as suggested by recent cryo-EM structures of human telomerase.

Ambient air pollution exposure and telomere length: a systematic review and meta-analysis.

OBJECTIVE: This study aimed to provide evidence of the associations between pre- and post-birth and adulthood air pollution exposure with telomere length. STUDY DESIGN: The databases of PubMed, Embase, and Web of Science were searched up to June 1st, 2022 in order to include relevant observational studies and perform a systematic review and meta-analysis. METHODS: The random-effects meta-analysis was grouped by air pollutant and exposure window (pre- and post-birth and adulthood) to evaluate the summary effect estimate. Cochran's Q and I2 statistics were used to evaluate the heterogeneity among the included studies. The quality of individual studies was evaluated using the national toxicology program/office of health assessment and translation risk of bias rating tool. RESULTS: We identified 18 studies, covering 8506 children and 2263 adults from multiple countries. We found moderate evidence that particulate matter less than 2.5 µm (PM2.5) exposure during the entire pregnancy (-0.043, 95% CI: -0.067, -0.018), nitrogen dioxide (NO2) exposure during the first trimester (-0.016, 95% confidence interval [CI]: -0.027, -0.005), long-term adulthood PM2.5 exposure were associated with shortening telomere length. Mild to high between-study heterogeneity was observed for the most tested air pollutant-telomere length combinations in different exposure windows. CONCLUSIONS: This systematic review and meta-analysis provides the evidence which strongly supports that prenatal PM2.5 and NO2 exposures were related to reduced telomere length, while prenatal sulfur dioxide (SO2) and carbon monoxide (CO) exposures, childhood PM2.5, particulate matter less than 10 µm (PM10), NO2 exposures and short-term adulthood PM2.5 and PM10 exposures were not associated with telomere length. Further high-quality studies are needed to elaborate our suggestive associations.

Association of Genetic Polymorphisms of TERT with Telomere Length in Coke Oven Emissions-Exposed Workers.

We explored the association between variations in the telomere maintenance genes and change in telomere length (TL) in workers. The TL of peripheral blood leukocytes from 544 coke oven workers and 238 controls were detected using the Real-time PCR method. Variations in four genes were then detected using the PCR based restriction fragment length polymorphism. The effects of environmental and genetic factors on TL were subsequently analyzed through covariance analysis and a generalized linear model .The TL of subjects with GG genotypes were longer than those with AG genotype in the TERT rs2736098 locus amongst the controls (P = .032). The combined effect of COEs exposure and AG+AA genotypes had a significant effect on TL (P < .001). The interaction between the COEs exposure factor and the rs2736098AG+AA genotypes had a significant effect on the TL (P < .05). The TL in coke oven workers is associated with the interactions between TERT rs2736098 AG+AA and COEs exposure.

Interaction of polycyclic aromatic hydrocarbon DNA adducts and telomere length on missed abortion / 中华预防医学杂志

Objective: To analyze the contribution and interaction of polycyclic aromatic hydrocarbons (PAH)-DNA adducts and changes of telomere length (TL) on missed abortion. Methods: From March to December 2019, patients with missed abortion in the First Hospital of Shanxi Medical University and pregnant women with normal pregnancy but voluntary abortion in the same department during the same period were selected and divided into a case group and a control group. Questionnaire was used to investigate the general situation and the pregnancy situation of the subjects. The abortion villi were collected and the content of PAH-DNA adducts and TL was detected. Logistic regression model was used to analyze the associated factors of missed abortion. R epiR package and Mediation package were used to analyze the effect and relationship between PAH-DNA adducts and TL on missed abortion. Results: The age of the subjects was(29.92±5.69)years old. The M(Q1,Q3)of PAH-DNA adducts was 453.75(404.61, 504.72) pg/ml. The M(Q1,Q3)of TL was 1.21(0.77, 1.72). The content of PAH-DNA adducts in the case group was higher than that in the control group (Z=-2.10, P=0.036), while the TL was lower than that in the control group (Z=-4.05, P<0.001). Multivariate logistic regression showed that low, medium and high levels of PAH-DNA adducts (OR=3.17,95%CI:1.41-7.14;OR=2.85,95%CI:1.25-6.52;OR=2.46,95%CI:1.07-5.64), and long, medium and short levels of TL (OR=2.50,95%CI:1.11-5.63;OR=3.32,95%CI:1.45-7.56;OR=3.22,95%CI:1.42-7.26) were all risk factors for missed abortion. The medium level of PAH-DNA adducts had a 2.76-fold higher risk of shortened TL than those with the lowest level, and no mediating role of TL was found. The stratified analysis showed that when the TL level was longer (>1.21), the low and high levels of PAH-DNA adducts were associated with missed abortion (all P<0.05); when the TL level was shorter (<1.21), the medium level of PAH-DNA adducts was associated with abortion (P=0.025). At lower levels of PAH-DNA adducts, no effect of TL on missed abortion was observed, while, at higher levels, TL was strongly associated with missed abortion (OR=7.50,95%CI:1.95-28.82;OR=6.04,95%CI:1.54-23.65;OR=9.05,95%CI:2.34-35.04). The interaction analysis found that the AP was 0.72 (95%CI: 0.46-0.99), and the SI was 5.21 (95%CI: 2.30-11.77). Conclusion: The high level of PAH-DNA adducts and shortened TL may increase the risk of missed abortion, and there may be a positive additive interaction between the two factors on missed abortion.

Columnar structure of human telomeric chromatin.

Telomeres, the ends of eukaryotic chromosomes, play pivotal parts in ageing and cancer and are targets of DNA damage and the DNA damage response1-5. Little is known about the structure of telomeric chromatin at the molecular level. Here we used negative stain electron microscopy and single-molecule magnetic tweezers to characterize 3-kbp-long telomeric chromatin fibres. We also obtained the cryogenic electron microscopy structure of the condensed telomeric tetranucleosome and its dinucleosome unit. The structure displayed close stacking of nucleosomes with a columnar arrangement, and an unusually short nucleosome repeat  length that comprised about 132 bp DNA wound in a continuous superhelix around histone octamers. This columnar structure is primarily stabilized by the H2A carboxy-terminal and histone amino-terminal tails in a synergistic manner. The columnar conformation results in exposure of the DNA helix, which may make it susceptible to both DNA damage and the DNA damage response. The conformation also exists in an alternative open state, in which one nucleosome is unstacked and flipped out, which exposes the acidic patch of the histone surface. The structural features revealed in this work suggest mechanisms by which protein factors involved in telomere maintenance can access telomeric chromatin in its compact form.

Structures of the human CST-Polα-primase complex bound to telomere templates.

The mammalian DNA polymerase-α-primase (Polα-primase) complex is essential for DNA metabolism, providing the de novo RNA-DNA primer for several DNA replication pathways1-4 such as lagging-strand synthesis and telomere C-strand fill-in. The physical mechanism underlying how Polα-primase, alone or in partnership with accessory proteins, performs its complicated multistep primer synthesis function is unknown. Here we show that CST, a single-stranded DNA-binding accessory protein complex for Polα-primase, physically organizes the enzyme for efficient primer synthesis. Cryogenic electron microscopy structures of the CST-Polα-primase preinitiation complex (PIC) bound to various types of telomere overhang reveal that template-bound CST partitions the DNA and RNA catalytic centres of Polα-primase into two separate domains and effectively arranges them in RNA-DNA synthesis order. The architecture of the PIC provides a single solution for the multiple structural requirements for the synthesis of RNA-DNA primers by Polα-primase. Several insights into the template-binding specificity of CST, template requirement for assembly of the CST-Polα-primase PIC and activation are also revealed in this study.

G-quadruplex structure of the C. elegans telomeric repeat: a two tetrads basket type conformation stabilized by a non-canonical C-T base-pair.

The Caenorhabditis elegans model has greatly contributed to the understanding of the role of G-quadruplexes in genomic instability. The GGCTTA repeats of the C. elegans telomeres resemble the GGGTTA repeats of the human telomeres. However, the comparison of telomeric sequences (Homo sapiens, Tetrahymena, Oxytricha, Bombyx mori and Giardia) revealed that small changes in these repeats can drastically change the topology of the folded G-quadruplex. In the present work we determined the structure adopted by the C. elegans telomeric sequence d[GG(CTTAGG)3]. The investigated C. elegans telomeric sequence is shown to fold into an intramolecular two G-tetrads basket type G-quadruplex structure that includes a C-T base pair in the diagonal loop. This work sheds light on the telomeric structure of the widely used C. elegans animal model.

Vectorial folding of telomere overhang promotes higher accessibility.

Human telomere overhang composed of tandem repeats of TTAGGG folds into G-quadruplex (G4). Unlike in an experimental setting in the test tube in which the entire length is allowed to fold at once, inside the cell, the overhang is expected to fold as it is synthesized directionally (5' to 3') and released segmentally by a specialized enzyme, the telomerase. To mimic such vectorial G4 folding process, we employed a superhelicase, Rep-X which can unwind DNA to release the TTAGGG repeats in 5' to 3' direction. We demonstrate that the folded conformation achieved by the refolding of full sequence is significantly different from that of the vectorial folding for two to eight TTAGGG repeats. Strikingly, the vectorially folded state leads to a remarkably higher accessibility to complementary C-rich strand and the telomere binding protein POT1, reflecting a less stably folded state resulting from the vectorial folding. Importantly, our study points to an inherent difference between the co-polymerizing and post-polymerized folding of telomere overhang that can impact telomere architecture and downstream processes.

Triglyceride-Glucose Index Correlate With Telomere Length in Healthy Adults From the National Health and Nutrition Examination Survey.

Aim: The present investigation was designed to test the association between leukocyte telomere length (LTL) and two simple markers of insulin resistance, that is, homeostatic model assessment of insulin resistance (HOMA-IR) and triglyceride-glucose (TyG) index in U.S. adults without metabolic diseases. Methods: A total of 6489 U.S. adults without diabetes from NHANES 1999-2002 were analyzed. TyG index was calculated as ln [fasting triglycerides (mg/dL) × fasting glucose (mg/dL)/2]. HOMA-Index was calculated as fasting plasma glucose (mmol/L) × fasting serum insulin (mU/mL)/22.5. LTL was obtained using the quantitative polymerase chain reaction method. Multivariate linear regression analysis was assessed to evaluate the association of TyG index HOMA-IR with LTL. We further conducted a generalized additive model (GAM) and a fitted smoothing curve with penalized spline method. Results: It was found that the mean LTL was 5796.1 bp in the measured healthy adults. Overall, TyG index was significantly associated with LTL, while HOMA-IR was not. Compared with participants in tertile 1 of the TyG index, the ß (95% CI) for those in the second (8.27 to 8.77) and third (≥ 8.77) were -4.31 (95% CI: -48.12~39.49) and -95.98 (95% CI: -145.08~-46.89), respectively. Subjects with TyG index ≥ 8.77 had statistically significant shorter LTL (ß = -93.33, 95%CI: -134.33~-52.32), compared with TyG index < 8.77. We further explored a dose-response relation between TyG index by a decile approach [≤ 7.81 (reference), 7.81-8.04, 8.04-8.21, 8.21-8.37, 8.37-8.52, 8.52-8.68, 8.68-8.83, 8.83-9.03, 9.03-9.33, and >9.33] and LTL. Five subgroups (TyG index 7.81-8.04, 8.04-8.21, 8.21-8.37, 8.37-8.52, and 8.52-8.68) did not show significant effect on LTL; while there was a significantly shorter LTL for participants with the TyG index > 8.68, supporting a threshold effect of TyG index on LTL. Conclusions: The results suggested that higher TyG index (> 8.68) was closely related to shorter LTL and the TyG index was better associated with LTL than HOMA-IR.

Riboflavin Stabilizes Abasic, Oxidized G-Quadruplex Structures.

The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.

The telomeric 5' end nucleotide is regulated in the budding yeast Naumovozyma castellii.

The junction between the double-stranded and single-stranded telomeric DNA (ds-ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5' end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5' end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5' end, indicating that not all ds-ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds-ss junction ensures the protection of both 5' ends and 3' overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.

Early steps of oxidative damage in DNA quadruplexes are position-dependent: Quantum mechanical and molecular dynamics analysis of human telomeric sequence containing ionized guanine.

Guanine radical cation (G•+) is a key intermediate in many oxidative processes occurring in nucleic acids. Here, by combining mixed Quantum Mechanical/Molecular Mechanics calculations and Molecular Dynamics (MD) simulations, we study how the structural behaviour of a tract GGG(TTAGGG)3 (hereafter Tel21) of the human telomeric sequence, folded in an antiparallel quadruple helix, changes when one of the G bases is ionized to G•+ (Tel21+). Once assessed that the electron-hole is localized on a single G, we perform MD simulations of twelve Tel21+ systems, differing in the position of G•+ in the sequence. When G•+ is located in the tetrad adjacent to the diagonal loop, we observe substantial structural rearrangements, which can decrease the electrostatic repulsion with the inner Na+ ions and increase the solvent exposed surface of G•+. Analysis of solvation patterns of G•+ provides new insights on the main reactions of G•+, i.e. the deprotonation at two different sites and hydration at the C8 atom, the first steps of the processes producing 8oxo-Guanine. We suggest the main structural determinants of the relative reactivity of each position and our conclusions, consistent with the available experimental trends, can help rationalizing the reactivity of other G-quadruplex topologies.

Investigation of the binding site of ruthenium complexes to short single-stranded oligodeoxynucleotides using electrospray ionization tandem mass spectrometry.

RATIONALE: In order to elucidate the nature of the interaction between metal complexes and DNA, use was made of short telomere single-stranded oligodeoxynucleotide (ODN) strand 5'-T1 T2 A3 G4 G5 G6 -3' (1) and strands 5'-T1 C2 A3 G4 G5 G6 -3' (2), 5'-T1 T2 A3 C4 G5 G6 -3' (3) and 5'-T1 C2 C3 C4 C5 G6 -3' (4) for the verification of the binding site with four different ruthenium complexes as possible anticancer drug candidates. METHODS: The ability to form adducts between ruthenium complexes with short single-stranded 6-mers was investigated through the use of electrospray ionization mass spectrometry (ESI-MS). Full scan ESI mass spectra and collision-induced dissociation (CID) mass spectra were recorded on a high-resolution quadrupole time-of-flight mass spectrometer. The elemental compositions of the adducts and the most important product ions were calculated by exact mass measurements. RESULTS: ESI-MS measurements showed that the mono-ruthenated ODNs were the main products produced under the conditions for the four ruthenium complexes and each of the ODNs. The CID results revealed that thymine and guanine are the preferred binding sites depending on the different compositions in the ODNs. However, for the ODN of the type: 5'-T1 C2 C3 C4 C5 G6 -3' the coordination site on cytosine was observed as well. The different ruthenium complexes interacted in the same way. CONCLUSIONS: This study showed that the characterization of new ruthenium complexes with short single-stranded telomeric DNA (TTAGGG) and further different ODNs is possible with positive ESI-MS/MS measurement. The identification of thymine and cytosine besides guanine as possible binding sites suggests that the interaction site is highly affected by the ODN's structure.

Regulation of Gene Expression by Telomere Position Effect.

Many diseases that involve malignant tumors in the elderly affect the quality of human life; therefore, the relationship between aging and pathogenesis in geriatric diseases must be under-stood to develop appropriate treatments for these diseases. Recent reports have shown that epigenetic regulation caused by changes in the local chromatin structure plays an essential role in aging. This review provides an overview of the roles of telomere shortening on genomic structural changes during an age-dependent shift in gene expression. Telomere shortening is one of the most prominent events that is involved in cellular aging and it affects global gene expression through genome rearrangement. This review provides novel insights into the roles of telomere shortening in disease-affected cells during pathogenesis and suggests novel therapeutic approaches.