De novo telomere addition by Tetrahymena telomerase in vitro.

Previous molecular genetic studies have shown that during programmed chromosomal healing, telomerase adds telomeric repeats directly to non-telomeric sequences in Tetrahymena, forming de novo telomeres. However, the biochemical mechanism underlying this process is not well understood. Here, we show for the first time that telomerase activity is capable in vitro of efficiently elongating completely non-telomeric DNA oligonucleotide primers, consisting of natural telomere-adjacent or random sequences, at low primer concentrations. Telomerase activity isolated from mated or vegetative cells had indistinguishable specificities for nontelomeric and telomeric primers. Consistent with in vivo results, the sequence GGGGT... was the predominant initial DNA sequence added by telomerase in vitro onto the 3' end of the non-telomeric primers. The 3' and 5' sequences of the primer both influenced the efficiency and pattern of de novo telomeric DNA addition. Priming of telomerase by double-stranded primers with overhangs of various lengths showed a requirement for a minimal 3' overhang of 20 nucleotides. With fully single-stranded non-telomeric primers, primer length up to approximately 30 nucleotides strongly affected the efficiency of telomeric DNA addition. We propose a model for the primer binding site of telomerase for non-telomeric primers to account for these length and structural requirements. We also propose that programmed de novo telomere addition in vivo is achieved through a hitherto undetected intrinsic ability of telomerase to elongate completely non-telomeric sequences.     

Human POT1 facilitates telomere elongation by telomerase

Mammalian telomeric DNA is mostly composed of double-stranded 5'-TTAGGG-3' repeats and ends with a single-stranded 3' overhang. Telomeric proteins stabilize the telomere by protecting the overhang from degradation or by remodeling the telomere into a T loop structure. Telomerase is a ribonucleoprotein that synthesizes new telomeric DNA. In budding yeast, other proteins, such as Cdc13p, that may help maintain the telomere end by regulating the recruitment or local activity of telomerase have been identified. Pot1 is a single-stranded telomeric DNA binding protein first identified in fission yeast, where it was shown to protect telomeres from degradation [10]. Human POT1 (hPOT1) protein is known to bind specifically to the G-rich telomere strand. We now show that hPOT1 can act as a telomerase-dependent, positive regulator of telomere length. Three splice variants of hPOT1 were overexpressed in a telomerase-positive human cell line. All three variants lengthened telomeres, and splice variant 1 was the most effective. hPOT1 was unable to lengthen the telomeres of telomerase-negative cells unless telomerase activity was induced. These data suggest that a normal function of hPOT1 is to facilitate telomere elongation by telomerase.     

Recognition and elongation of telomeres by telomerase

Telomeres stabilize chromosomal ends and allow their complete replication in vivo. In diverse eukaryotes, the essential telomeric DNA sequence consists of variable numbers of tandem repeats of simple, G + C rich sequences, with a strong strand bias of G residues on the strand oriented 5' to 3' toward the chromosomal terminus. This strand forms a protruding 3' over-hang at the chromosomal terminus in three different eukaryotes analyzed. Analysis of yeast and protozoan telomeres showed that telomeres are dynamic structures in vivo, being acted on by shortening and lengthening activities. We previously identified and partially purified an enzymatic activity, telomere terminal transferase, or telomerase, from the ciliate Tetrahymena. Telomerase is a ribonucleoprotein enzyme with essential RNA and protein components. This activity adds repeats of the Tetrahymena telomeric sequence, TTGGGG, onto the 3' end of a single-stranded DNA primer consisting of a few repeats of the G-rich strand of known telomeric, and telomere-like, sequences. The shortest oligonucleotide active as a primer was the decamer G4T2G4. Structural analysis of synthetic DNA oligonucleotides that are active as primers showed that they all formed discrete intramolecular foldback structures at temperatures below 40 degrees C. Addition of TTGGGG repeats occurs one nucleotide at a time by de novo synthesis, which is not templated by the DNA primer. Up to 8000 nucleotides of G4T2 repeats were added to the primer in vitro. We discuss the implications of this finding for regulation of telomerase in vivo and a model for telomere elongation by telomerase.     

Sequence-specific DNA primer effects on telomerase polymerization activity.

The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo.     

The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers.

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.     

Ontogeny of telomerase in chicken: impact of downregulation on pre- and postnatal telomere length in vivo

Telomeres are the termini of linear chromosomes composed of tandem repeats of a conserved DNA sequence. Telomerase provides a mechanism for proliferating cells to offset telomeric sequence erosion by synthesizing new repeats onto the end of each parental DNA strand. Reduced or absent telomerase activity can lead to telomere shortening and genome instability. Telomeres and telomerase have not previously been characterized during ontogeny of any avian species. In the present study, telomerase activity in the chicken model was examined from early differentiation embryos through to adulthood. Telomerase activity was detected in all early embryos (preblastula through neurula) and in tissues throughout organogenesis. Subsequently, telomerase was downregulated in the majority of somatic tissues, either pre- or postnatally. A subset of tissues, such as intestine, immune and reproductive organs, exhibited constitutive activity. The impact of telomerase downregulation on telomere length was investigated and a telomere reduction of 3.2 kb in somatic tissues compared with germ line was observed in 5-year-old adults. The present results suggest that the telomere clock function is a conserved feature of avians as well as mammals. Knowledge regarding the relationships among telomerase regulation, proliferation/senescence profiles and differentiation status will be useful for numerous applications of chicken cells.     

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.     

Developmentally regulated initiation of DNA synthesis by telomerase: evidence for factor-assisted de novo telomere formation.

Telomerase serves a dual role at telomeres, maintaining tracts of telomere repeats and forming telomeres de novo on broken chromosomes in a process called chromosome healing. In ciliates, both mechanisms are readily observed. Vegetatively growing cells maintain pre-existing telomeres, while cells undergoing macronuclear development fragment their chromosomes and form telomeres de novo. Here we provide the first evidence for developmentally regulated initiation of DNA synthesis by telomerase. In vitro assays were conducted with telomerase from vegetative and developing Euplotes macronuclei using chimeric primers that contained non-telomeric 3' ends and an upstream stretch of telomeric DNA. In developing macronuclei, chimeric primers had two fates: nucleotides were either polymerized directly onto the 3' terminus or residues were removed from the 3' end by endonucleolytic cleavage before polymerization began. In contrast, telomerase from vegetative macronuclei used only the cleavage pathway. Telomere repeat addition onto non-telomeric 3' ends was lost when developing macronuclei were lysed and the contents purified on glycerol gradients. However, when fractions from the glycerol gradient were added back to partially purified telomerase, telomere synthesis was restored. The data indicate that a dissociable chromosome healing factor (CHF) collaborates with telomerase to initiate developmentally programmed de novo telomere formation.     

端粒与端粒酶研究进展

细胞分裂中染色体因其末端（端粒）的DNA不能完全复制而短缩,使细胞逐渐失去增殖能力而衰老．端粒酶可延长染色体末端DNA,端粒酶的活化使细胞无限增殖．85％左右的恶性肿瘤端粒酶表达阳性,生殖细胞和无限繁殖的细胞系中端粒酶表达也呈阳性．文章综述了端粒的构成和功能、端粒酶在端粒合成中的作用,介绍了端粒酶活性的测定方法、细胞恶变与端粒酶激活的关系,并论及通过抑制端粒酶活性来治疗癌症的可能性．     

端粒酶的研究进展

端粒酶是由RNA模板和蛋白质催化亚基组成的核糖蛋白颗粒,它解决染色体的未端复制问题,归属于逆转录酶家族又和逆转录酶有一定的差别。端粒酶的缺失能在细胞和机体水平引起各种疾病的发生,且端粒的过度表达和细胞的永生化和癌变直接相关,其中端粒蛋白在此过程中可能起调控作用,端粒酶的结构和功能决定了它有广泛的应用前景。     

Complexation of single strand telomere and telomerase RNA template polyanions by deoxyribonucleic guanidine (DNG) polycations: plausible anticancer agents

Cancerous cell immortality is due to relatively high concentrations of telomerase enzyme which maintains telomere sequence during cell division. Deoxyribonucleic guanidine (DNG) is a positively charged DNA analog in which guanidine replaces the phosphordiester linkage of DNA. Mixed sequences of DNG and DNA oligonucleotides are referred to as chimera. Complexation of DNG and chimeric polycations with the complementary negatively charged non-coding telomere single strand d(5'-TTAGGG-3')(n) and the 11-base telomeric RNA template (5'-CUAACCCUAAC-3') in the active site of telomerase has been studied. Calculated by ensemble sampling simulations in GBMV solvent model, we found that binding of complementary DNG hexamer with telomere is favored over that of DNA-telomere by approximately 10(6)-fold and binding of chimera hexamer is favored by approximately 10(4)-fold. Binding of complementary DNG with telomeric RNA is favored by 43 kcal/mol over telomere substrate binding with telomeric RNA.     

Generation of telomeric G strand overhangs involves both G and C strand cleavage

Processing of telomeric DNA is required to generate the 3' G strand overhangs necessary for capping chromosome ends. We have investigated the steps involved in telomere processing by examining G overhang structure in Tetrahymena cells that lack telomerase or have altered telomeric sequences. We show that overhangs are generated by two precise cleavage steps involving nucleases that are robust but lack sequence specificity. Our data suggest that a G overhang binding protein delineates the boundaries for G and C strand cleavage. We also show that telomerase is not the nuclease responsible for G strand cleavage, although telomerase depletion alters the precision of processing. This change in processing indicates that telomerase affects multiple transactions at the telomere and provides a physical footprint for the continued association of telomerase with the telomere after repeat addition is complete.     

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G‐rich sequence, and with noncoding RNA, Telomeric repeat‐containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2′‐OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G‐quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate – the 3′‐end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3′‐end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Telomerase activity in early bovine embryos derived from parthenogenetic activation and nuclear transfer

This study examined the telomerase activity in preimplantation bovine embryos derived from either parthenogenetic activation or nuclear transfer. Telomeres are the DNA-protein structures located at the ends of eukaryotic chromosomes. Telomerase is the ribonuclear enzyme that helps to restore telomere length by synthesizing telomeric DNA repeat (5'-TTAGGG-3') from its own RNA template. Without telomerase activity, telomeres shorten with each cell division through conventional DNA replication. In most mammalian species, telomerase activity is present in germ cells but not in somatic cells. Previously, we reported the dynamics of telomerase activity in bovine in vitro fertilized (IVF) embryos. In the present study, we examined the telomerase activity in bovine embryos derived either from parthenogenetic activation or somatic cell nuclear transfer (i.e., cloning). Embryos from both sources were harvested at different stages, from zygote to blastocyst. Telomerase activity in embryos derived from parthenogenetic activation and nuclear transfer showed a dynamic profile similar to that of those derived from IVF. Telomerase activity was detected in embryos at all stages examined, with the highest level in the blastocyst stage, regardless of the method of embryo production.     

Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro

The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3' overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.