Factors that influence telomeric oxidative base damage and repair by DNA glycosylase OGG1

Telomeres are nucleoprotein complexes at the ends of linear chromosomes in eukaryotes, and are essential in preventing chromosome termini from being recognized as broken DNA ends. Telomere shortening has been linked to cellular senescence and human aging, with oxidative stress as a major contributing factor. 7,8-Dihydro-8-oxogaunine (8-oxodG) is one of the most abundant oxidative guanine lesions, and 8-oxoguanine DNA glycosylase (OGG1) is involved in its removal. In this study, we examined if telomeric DNA is particularly susceptible to oxidative base damage and if telomere-specific factors affect the incision of oxidized guanines by OGG1. We demonstrated that telomeric TTAGGG repeats were more prone to oxidative base damage and repaired less efficiently than non-telomeric TG repeats in vivo. We also showed that the 8-oxodG-incision activity of OGG1 is similar in telomeric and non-telomeric double-stranded substrates. In addition, telomere repeat binding factors TRF1 and TRF2 do not impair OGG1 incision activity. Yet, 8-oxodG in some telomere structures (e.g., fork-opening, 3'-overhang, and D-loop) were less effectively excised by OGG1, depending upon its position in these substrates. Collectively, our data indicate that the sequence context of telomere repeats and certain telomere configurations may contribute to telomere vulnerability to oxidative DNA damage processing.     

Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells

One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.     

Deletion of Ogg1 DNA glycosylase results in telomere base damage and length alteration in yeast

Telomeres consist of short guanine‐rich repeats. Guanine can be oxidized to 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG). 8‐oxoguanine DNA glycosylase (Ogg1) repairs these oxidative guanine lesions through the base excision repair (BER) pathway. Here we show that in Saccharomyces cerevisiae ablation of Ogg1p leads to an increase in oxidized guanine level in telomeric DNA. The ogg1 deletion (ogg1Δ) strain shows telomere lengthening that is dependent on telomerase and/or Rad52p‐mediated homologous recombination. 8‐oxoG in telomeric repeats attenuates the binding of the telomere binding protein, Rap1p, to telomeric DNA in vitro. Moreover, the amount of telomere‐bound Rap1p and Rif2p is reduced in ogg1Δ strain. These results suggest that oxidized guanines may perturb telomere length equilibrium by attenuating telomere protein complex to function in telomeres, which in turn impedes their regulation of pathways engaged in telomere length maintenance. We propose that Ogg1p is critical in maintaining telomere length homoeostasis through telomere guanine damage repair, and that interfering with telomere length homoeostasis may be one of the mechanism(s) by which oxidative DNA damage inflicts the genome.     

Oxidative stress shortens telomeres

Telomeres in most human cells shorten with each round of DNA replication, because they lack the enzyme telomerase. This is not, however, the only determinant of the rate of loss of telomeric DNA. Oxidative damage is repaired less well in telomeric DNA than elsewhere in the chromosome, and oxidative stress accelerates telomere loss, whereas antioxidants decelerate it. I suggest here that oxidative stress is an important modulator of telomere loss and that telomere-driven replicative senescence is primarily a stress response. This might have evolved to block the growth of cells that have been exposed to a high risk of mutation.     

Telomere length assessment: biomarker of chronic oxidative stress?

Telomeres are nucleoprotein structures, located at the ends of chromosomes and are subject to shortening at each cycle of cell division. They prevent chromosomal ends from being recognized as double strand breaks and protect them from end to end fusion and degradation. Telomeres consist of stretches of repetitive DNA with a high G-C content and are reported to be highly sensitive to damage induced by oxidative stress. The resulting DNA strand breaks can be formed either directly or as an intermediate step during the repair of oxidative bases. In contrast to the majority of genomic DNA, there is evidence that telomeric DNA is deficient in the repair of single strand breaks. Since chronic oxidative stress plays a major role in the pathophysiology of several chronic inflammatory diseases, it is hypothesized that telomere length is reducing at a faster rate during oxidative stress. Therefore, assessment of telomere length might be a useful biomarker of disease progression. In this review several features of telomere length regulation, their relation with oxidative stress, and the potential application of measurement of telomere length as biomarker of chronic oxidative stress, will be discussed.     

Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2

The ends of linear chromosomes are capped by protein–DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2.     

DNA damage in telomeres and mitochondria during cellular senescence: is there a connection?

Cellular senescence is the ultimate and irreversible loss of replicative capacity occurring in primary somatic cell culture. It is triggered as a stereotypic response to unrepaired nuclear DNA damage or to uncapped telomeres. In addition to a direct role of nuclear DNA double-strand breaks as inducer of a DNA damage response, two more subtle types of DNA damage induced by physiological levels of reactive oxygen species (ROS) can have a significant impact on cellular senescence: Firstly, it has been established that telomere shortening, which is the major contributor to telomere uncapping, is stress dependent and largely caused by a telomere-specific DNA single-strand break repair inefficiency. Secondly, mitochondrial DNA (mtDNA) damage is closely interrelated with mitochondrial ROS production, and this might also play a causal role for cellular senescence. Improvement of mitochondrial function results in less telomeric damage and slower telomere shortening, while telomere-dependent growth arrest is associated with increased mitochondrial dysfunction. Moreover, telomerase, the enzyme complex that is known to re-elongate shortened telomeres, also appears to have functions independent of telomeres that protect against oxidative stress. Together, these data suggest a self-amplifying cycle between mitochondrial and telomeric DNA damage during cellular senescence.     

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.     

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.     

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.     

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.     

Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death

Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.     

Mammalian DNA2 helicase/nuclease cleaves G‐quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G‐quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2‐deficient mice develop aneuploidy‐associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.     

P66SHC deletion improves fertility and progeric phenotype of late‐generation TERC‐deficient mice but not their short lifespan

Oxidative stress and telomere attrition are considered the driving factors of aging. As oxidative damage to telomeric DNA favors the erosion of chromosome ends and, in turn, telomere shortening increases the sensitivity to pro‐oxidants, these two factors may trigger a detrimental vicious cycle. To check whether limiting oxidative stress slows down telomere shortening and related progeria, we have investigated the effect of p66SHC deletion, which has been shown to reduce oxidative stress and mitochondrial apoptosis, on late‐generation TERC (telomerase RNA component)‐deficient mice having short telomeres and reduced lifespan. Double mutant (TERC^?/? p66SHC^?/?) mice were generated, and their telomere length, fertility, and lifespan investigated in different generations. Results revealed that p66SHC deletion partially rescues sterility and weight loss, as well as organ atrophy, of TERC‐deficient mice, but not their short lifespan and telomere erosion. Therefore, our data suggest that p66SHC‐mediated oxidative stress and telomere shortening synergize in some tissues (including testes) to accelerate aging; however, early mortality of late‐generation mice seems to be independent of any link between p66SHC‐mediated oxidative stress and telomere attrition.     

Telomeric D-loops Containing 8-Oxo-2��-deoxyguanosine Are Preferred Substrates for Werner and Bloom Syndrome Helicases and Are Bound by POT1

8-Oxo-2′-deoxyguanosine (8-oxodG) is one of the most important oxidative DNA lesions, and G-rich telomeric DNA is especially susceptible to oxidative DNA damage. RecQ helicases WRN and BLM and telomere-binding protein POT1 are thought to play roles in telomere maintenance. This study examines the ability of WRN, BLM, and RecQ5 to unwind and POT1 to bind telomeric D-loops containing 8-oxodG. The results demonstrate that WRN and BLM preferentially unwind telomeric D-loops containing 8-oxodG and that POT1 binds with higher affinity to telomeric D-loops with 8-oxodG but shows no preference for telomeric single-stranded DNA with 8-oxodG. We speculate that telomeric D-loops with 8-oxodG may have a greater tendency to form G-quadruplex DNA structures than telomeric DNA lacking 8-oxodG.     

Telomeric DNA damage by topoisomerase I. A possible mechanism for cell killing by camptothecin

Topoisomerase I adjusts torsional stress in the genome by breaking and resealing one strand of the helix through a transient covalent coupling between enzyme and DNA. Camptothecin, a specific topoisomerase I poison, traps this covalent intermediate, thereby damaging the genome. Here we examined the activity of topoisomerase I at telomeric repeats to determine whether telomere structures are targets for DNA damage. We show that topoisomerase I is catalytically active in cleaving the G-rich telomeric strand in vitro in the presence of camptothecin but not in cleaving the C-rich strand. The topoisomerase I cleavage site is 5'-TT (downward arrow) AGGG-3' (cleavage site marked by the downward arrow). We also show that endogenous topoisomerase I can access telomeric DNA in vivo and form camptothecin-dependent covalent complexes. Therefore, each telomeric repeat represents a potential topoisomerase I cleavage site in vivo. Because telomere structures are comprised of a large number of repeats, telomeres in fact represent a high concentration of nested topoisomerase I sites. Therefore, more telomeric DNA damage by camptothecin could occur in cells with longer telomeres when cells possess equivalent levels of topoisomerase I. The evidence presented here suggests that DNA damage at telomeric repeats by topoisomerase I is a prominent feature of cell killing by camptothecin and triggers camptothecin-induced apoptosis.     

端粒结合蛋白在端粒复制与损伤修复中的作用

端粒位于真核细胞线性染色体末端,正常的端粒长度与结构对于细胞基因组稳定的维持有重要作用.端粒DNA序列的高度重复性使其容易形成一些特殊的二级结构,相比染色体其他位置更难复制.结合在端粒上的Shelterin蛋白复合体由六个端粒结合蛋白组成,该复合体可以通过抑制端粒处异常DNA损伤修复途径的激活维持端粒的稳定.此外,近几...     

The RECQL4 protein,deficient in Rothmund–Thomson syndrome is active on telomeric D-loops containing DNA metabolism blocking lesions

Telomeres are critical for cell survival and functional integrity. Oxidative DNA damage induces telomeric instability and cellular senescence that are associated with normal aging and segmental premature aging disorders such as Werner Syndrome and Rothmund–Thomson Syndrome, caused by mutations in WRN and RECQL4 helicases respectively. Characterizing the metabolic roles of RECQL4 and WRN in telomere maintenance is crucial in understanding the pathogenesis of their associated disorders. We have previously shown that WRN and RECQL4 display a preference in vitro to unwind telomeric DNA substrates containing the oxidative lesion 8-oxoguanine. Here, we show that RECQL4 helicase has a preferential activity in vitro on telomeric substrates containing thymine glycol, a critical lesion that blocks DNA metabolism, and can be modestly stimulated further on a D-loop structure by TRF2, a telomeric shelterin protein. Unlike that reported for telomeric D-loops containing 8-oxoguanine, RECQL4 does not cooperate with WRN to unwind telomeric D-loops with thymine glycol, suggesting RECQL4 helicase is selective for the type of oxidative lesion. RECQL4's function at the telomere is not yet understood, and our findings suggest a novel role for RECQL4 in the repair of thymine glycol lesions to promote efficient telomeric maintenance.     

A stochastic model of cell replicative senescence based on telomere shortening, oxidative stress, and somatic mutations in nuclear and mitochondrial DNA.

Human diploid fibroblast cells can divide for only a limited number of times in vitro, a phenomenon known as replicative senescence or the Hayflick limit. Variability in doubling potential is observed within a clone of cells, and between two sister cells arising from a single mitotic division. This strongly suggests that the process by which cells become senescent is intrinsically stochastic. Among the various biochemical mechanisms that have been proposed to explain replicative senescence, particular interest has been focussed on the role of telomere reduction. In the absence of telomerase--an enzyme switched off in normal diploid fibro-blasts-cells lose telomeric DNA at each cell division. According to the telomere hypothesis of cell senescence, cells eventually reach a critically short telomere length and cell cycle arrest follows. In support of this concept, forced expression of telomerase in normal fibroblasts appears to prevent cell senescence. Nevertheless, the telomere hypothesis in its basic form has some difficulty in explaining the marked stochastic variations seen in the replicative lifespans of individual cells within a culture, and there is strong empirical and theoretical support for the concept that other kinds of damage may contribute to cellular ageing. We describe a stochastic network model of cell senescence in which a primary role is played by telomere reduction but in which other mechanisms (oxidative stress linked particularly to mitochondrial damage, and nuclear somatic mutations) also contribute. The model gives simulation results that are in good agreement with published data on intra-clonal variability in cell doubling potential and permits an analysis of how the various elements of the stochastic network interact. Such integrative models may aid in developing new experimental approaches aimed at unravelling the intrinsic complexity of the mechanisms contributing to human cell ageing.     

Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects

Telomeres are chromosome end structures and are essential for maintenance of genome stability. Highly repetitive telomere sequences appear to be susceptible to oxidative stress-induced damage. Oxidation may therefore have a severe impact on telomere integrity and function. A wide spectrum of oxidative pyrimidine-derivatives has been reported, including thymine glycol (Tg), that are primarily removed by a DNA glycosylase, Endonuclease III-like protein 1 (Nth1). Here, we investigate the effect of Nth1 deficiency on telomere integrity in mice. Nth1 null (Nth1^−/−) mouse tissues and primary MEFs harbor higher levels of Endonuclease III-sensitive DNA lesions at telomeric repeats, in comparison to a non-telomeric locus. Furthermore, oxidative DNA damage induced by acute exposure to an oxidant is repaired slowly at telomeres in Nth1^−/− MEFs. Although telomere length is not affected in the hematopoietic tissues of Nth1^−/− adult mice, telomeres suffer from attrition and increased recombination and DNA damage foci formation in Nth1^−/− bone marrow cells that are stimulated ex vivo in the presence of 20% oxygen. Nth1 deficiency also enhances telomere fragility in mice. Lastly, in a telomerase null background, Nth1^−/− bone marrow cells undergo severe telomere loss at some chromosome ends and cell apoptosis upon replicative stress. These results suggest that Nth1 plays an important role in telomere maintenance and base repair against oxidative stress-induced base modifications. The fact that telomerase deficiency can exacerbate telomere shortening in Nth1 deficient mouse cells supports that base excision repair cooperates with telomerase to maintain telomere integrity.