What is the role of telomerase in end replication of chromosomes in eukaryotic cells?

Introduction

Telomeres are essential chromosomal structures that cap the ends of linear eukaryotic chromosomes. Incomplete DNA replication every time cells divide results in a gradual loss of telomeric sequences in human somatic tissues. Telomere erosion ultimately results in the loss of telomeric function, which in turn triggers the cells to enter a non-dividing but biochemically active state termed replicative senescence. Telomere-dependent replicative senescence is considered to have evolved as a tumor suppressive mechanism; indeed, 85% of human malignancies express the enzyme telomerase that synthesizes telomere repeat sequences, preventing telomere loss and facilitating unlimited cell division. Senescent cells have the potential to interfere with the tissue microenvironment, and the accumulation of senescent cells is considered to contribute to age-related tissue deterioration and disease. Consistent with this hypothesis, telomere loss is observed as a function of age in human tissue, and this loss has been linked to specific disease phenotypes.

Budding Yeast, Saccharomyces cerevisiae

Telomeres in budding yeast are bound by two distinct protein complexes: the CST complex that binds the G-overhang and the Rap1 complex that binds the DNA duplex. The CST complex forms as Cdc13 binds to G-overhangs and recruits two binding partners (Stn1 and Ten1). CST deficiency results in C-strand degradation and accumulation of G-overhangs. The CST complex also plays a role in telomere replication. In addition, Rap1, together with Rif1 and Rif2, binds the double-stranded telomeres and functions to regulate telomere length through a negative feedback loop. Increased binding of Rif1 and Rif2 at telomeres inhibits telomerase recruitment and prevents further elongation. Disruption of Rap1 binding results in the lengthening of telomeric sequence and a faster turnover rate of the terminal repeats. Moreover, Rap1 can also recruit a second group of proteins – Sir2, Sir3, and Sir4, which enhances the formation of heterochromatin, telomere silencing, and influence positioning of the telomeres at the nuclear periphery.

The Need for Telomeres

Telomeres are found at the ends of chromosomes; they provide the answer to two problems of chromosome management. First, there has to be something to distinguish true chromosome ends from the accidental ends resulting from chromosome breakage. There is much evidence from studies of the effect of radiation on chromosomes that broken ends are prone to indiscriminate rejoining, with the possibility of segmental rearrangement. Presumably they provide substrates for double-stranded DNA ligase, and they may also be subject to erosion by exonuclease. True chromosome termini must be sealed in some way to protect them against these hazards.

Second, there has to be a way of completing DNA replication. DNA polymerase extends DNA strands from their 3′ ends, and so the two strands of double-stranded DNA are replicated in opposite directions. The synthesis of one follows the replication fork and can be continuous, but the synthesis of the other runs ‘backwards’ in piecemeal fashion, each piece having to be initiated afresh by an RNA primer that is subsequently removed. The consequence is that the DNA strand with a 5′ terminus cannot be fully replicated by the regular mechanism, since there is no 3′ end to prime the filling of the gap left by removal of the last RNA primer (Figure 1). Indeed, there is evidence that the 5′-terminated strand, already shorter, may be further shortened, leaving an even longer single-stranded 3′ ‘tail.’ So, without some additional end-replication mechanism, one would expect the chromosome to get a little shorter with each cycle of replication. Cell viability depends on the constant replenishment of terminal sequences.

11 Telomeres as Epigenetic Agents

Telomeres are not only targets for epigenetic processes but also are chromosome domains acting themselves as epigenetic regulatory elements on other chromatin domains both in cis and trans positions. Telomere shortening affects the epigenetic status of telomeres and subtelomeres which in turn influence the telomere position effect (TPE). TPE is a phenomenon that refers to the ability of mammalian telomeres to silence subtelomeric genes. Telomere shortening in humans is associated with a decrease in TPE while TPE increases with telomere elongation [99]. Progressive loss of telomeres as a result of telomerase deficiency in mice showed a decrease in the density of heterochromatin marks in both telomeric and subtelomeric regions. Additionally, it showed an associated raise in acetylation of histone H3 and H4 suggesting that distal changes at telomere ends may influence the epigenetic state of subtelomeric chromatin [100]. Furthermore, the loss of subtelomeric DNA methylation elucidated by telomere shortening results in telomere instability, and since telomere shortening attract telomerase, this may indicate that telomeres have specific marks recognizable by the telomerase complex.

Abbreviations

ALT

Alternative lengthening of telomeres

ALT-associated promyelocitic leukemia (PML) nuclear body

Ataxia telangiectasia mutated kinase

ATM and Rad3-related kinase

α-Thalassemia/mental retardation syndrome X-linked

Chromatin immunoprecipitation

CTC1-STN1-TEN1

CCCTC-binding factor

Death domain–associated protein 6

DNA damage response

Displacement loop

Double-strand DNA break

Gly/Arg-rich domain

Phosphorylated H2AX on Ser-139

Histone deacetylase

Heterochromatin protein 1

Homologous recombination

Interferon-stimulated gene 15

Micrococcal nuclease

MRE11/RAD50/NBS1 complex

Nonhomologous end joining

Promyelocytic leukemia

Protection of telomeres 1

Repressor-activator protein 1

Silent information regulator complex

SWItch/sucrose nonfermentable complex

Topologically associated domain

Telomere-associated sequences

Telomerase RNA component

Telomeric repeat–containing RNA

Telomerase reverse transcriptase

Telomere dysfunction–induced focus

TRF1-interacting protein 2

Telomere loop

Telomere position effect

TPE over long distances

TINT1/PIP1/PTOP1 complex

Telomeric repeat–binding factor 1

Telomeric repeat–binding factor 2

TRF homology domain

Summary

Telomeres are nucleoprotein structures at the end of each chromosome. The nucleic acid sequence of telomeres is a TTAGGG hexamer repeat. The number of hexamer repeats can vary greatly from very few to thousands of repeats with most reported telomere lengths varying between 4-11 kilobases in humans. Several proteins (TRF1, TRF2, POT1, TIN2, TPP1, RAP1 and Tankyrase) are associated with telomere DNA to form the shelterin complex which interacts with enzymes such as telomerase and other proteins required for proper telomere maintenance and function. Shortening of telomeres may increase excessively with age and lead to telomere-end-fusions, chromosomal instability and accelerated senescence. Telomeres may become dysfunctional if base damage and/or DNA strand breaks accumulate in the telomere sequence. Several lines of evidence indicate that telomere shortening, telomere base damage and telomere strand breaks may increase due to poor nutrition and that dietary intervention has the potential to improve telomere integrity.

Telomeric Repeat-Containing RNA

Telomeres were initially viewed as transcriptionally silent, packed into the higher-order heterochromatin structure. It was later discovered that the C-strand of the telomere is used as template for transcription of the long non-coding RNA, termed telomeric repeat-containing RNA (TERRA), which as the name suggests contains telomeric DNA repeat sequences. TERRA has been identified within distantly related species, ranging from vertebrates to yeasts to plants, and is actively transcribed from approximately 25% of the telomeres within a cell (Azzalin et al., 2007). Human TERRA is highly heterogeneous, varying in length from 100 nucleotides to more than 9000 nucleotides, while yeast TERRA has a more consistent length of approximately 400 nucleotides. The levels of TERRA transcribed are seemingly dependent on telomere length, with shorter telomeres promoting TERRA transcription and longer telomeres repressing expression (Iglesias et al., 2011; Arnoult et al., 2012). Shorter telomeres have fewer telomere-binding proteins that are responsible for transcriptional silencing of the telomere. TERRA appears to aid in telomerase recruitment and the increased levels of TERRA at short telomeres increase the rate of telomere extension at these eroded telomere ends (Cusanelli et al., 2013). Additional functions for TERRA have been proposed, which include preventing separate telomeres from interacting and supporting chromosome end stability.

9.2.3.2 Telomere Stability

Telomeres shorten after each mitotic cell division in adult somatic cells leading to cell senescence and apoptosis (see also Chapter 13). However, telomeres can be replenished by the telomerase reverse transcriptase enzyme, an enzyme that is very active in stem cells and also in tumor cells. The shelterin complex of proteins is known to regulate telomerase activity by binding to telomeres and inducing the formation of a t-loop, a cap structure of 300 bp single-stranded DNA that stabilizes the telomere, preventing the telomere ends from being recognized as potential break points by the DNA repair machinery [70]. Six proteins form the shelterin complex in humans: TRF1, TRF2, POT1, TIN2, TPP1, and RAP1 [71]. Telomere repeat-containing RNAs (TERRAs) are a group of lncRNAs transcribed at the telomeres that actively participate in the regulation of telomere homeostasis and telomere function [58] through different mechanisms, including the stabilization of chromosome ends or the prevention of senescence induced by DNA damage response. TERRA transcripts are actively recruited to chromosome ends through interactions with stable components of the telomeric structure [59]. First, TERRA associates with the shelterin components TRF1 and TRF2 [59], but also with members of the epigenetic machinery including the heterochromatin protein 1 (HP1), the lysine 9-histone 3 methyltransferase SUV39H1 or the component of the NuA2 histone acetyltransferase complex, MORF4L2 [72,73]. Recent findings thus suggest that TERRA could act as a scaffold molecule promoting the binding of proteins associated with heterochromatin formation to the chromosome ends. Second, TERRA transcripts can base-pair with their template DNA strand, forming RNA:DNA hybrid structures known as R-loops at telomere regions [60]. Interestingly, accumulation of telomeric R-loops promotes also the alternative lengthening of telomeres (ALT) pathway by facilitating homologous recombination between telomeric repeats of different chromosomes, and delays senescence in cells lacking telomerase [60]. Furthermore, tight regulation of the levels of TERRA during the cell cycle is involved in telomere protection and in the suppression of the DNA damage response at telomeres [74]. During telomeric DNA replication, exposed ssDNA is thus bound by the ssDNA-binding protein RPA, which is essential for DNA replication and activation of the ATR checkpoint. RPA can be displaced by another ssDNA binding protein, hnRNAP1, which can be sequestered by TERRA [75]. TERRA expression changes during the cell cycle: it is expressed at low levels in the late S phase while its expression increases in early G1 phase. As TERRA binds hnRNPA1, decreased TERRA during S phase facilitates hnRNPA1 binding to telomeric DNA and RPA displacement [75]. Release of RPA from telomeres prevents erroneous activation of ATR during cell cycle progression, enhancing telomere protection. Finally, it has been postulated that TERRA is also involved in the direct regulation of telomerase activity. In vitro assays showed that TERRA inhibits telomerase activity [76]; however, its regulatory role in vivo is still unclear.

Abstract

Telomeres stabilize open chromosome ends and protect them against chromosomal end-to-end fusions, breakage, instability, and nonreciprocal translocations. Telomere dysfunction is known to lead to an impaired regenerative capacity of hepatocytes and an increased cirrhosis formation in the context of acute and chronic liver injury. In addition, telomere dysfunction and telomerase mutations have been associated with the induction of chromosomal instability and consequently with cirrhosis development and hepatocarcinogenesis. The identification of molecular mechanisms related to telomere dysfunction and telomerase activation might lead to new therapeutic strategies. In this chapter, we are reviewing the current knowledge about the importance of telomere dysfunction in liver diseases.

Role of telomere length in oxidative stress and antioxidant defense in elderly women

Telomere maintenance might be a key factor in relation to the cumulative effects of genetic, environmental, and lifestyle factors involved in aging and aging-related diseases.16 Various nutrients influence telomere length via mechanisms that reflect their roles in cellular functions including DNA repair and chromosome maintenance, DNA methylation, inflammation, oxidative stress, and the activity of the enzyme telomerase (Fig. 3). Damage to telomeric DNA due to either oxidative stress or nucleotide precursors results in shorter telomeres.17 In this context, antioxidant nutrients can reduce the erosion of telomeres through the potential to influence the regulation of telomere length.17

Although oxidative stress gene expression and telomere shortening may be epiphenomena of an underlying aging process, allelic variation in oxidative stress genotypes is fixed and may contribute to individual differences in both telomere length and biological aging.18 An investigation on elderly persons demonstrated that oxidative genes that are associated with shorter telomeres are also associated with the impairment of physical biomarkers of aging.18 The authors hypothesized that pathways involving altered Cu/Fe metabolism, glutathione transferase, and mitochondrial function are likely to be centered on sirtuins, providing a possible functional link between redox biology and telomere biology. Hence, associations between telomere length and physical biomarkers of aging may, in part, reflect cellular redox status as underlying common causes.18

The relationship between estimated endogenous estrogen exposure and telomere length was evaluated in a sample of postmenopausal women at risk of cognitive decline.8 The authors found that greater endogenous estrogen exposure, as measured by a longer duration of reproductive years, was related to longer telomere length.

The telomerase molecule, which synthesizes telomeres, contains an estrogen-responsive element in its promoter region, which determines that telomerase activity is higher in females than in males.19 Longer telomeres in women have been ascribed to the ability of estrogen to upregulate telomerase and at the same time reduce oxidative stress.8 In addition, it has been observed that telomerase is also regulated by glutathione.19