dGTP-dependent processivity and possible template switching of euplotes telomerase.

We have measured the processivity of telomeric DNA extension by Euplotes aediculatus telomerase at various concentrations of the nucleotide substrates dGTP and dTTP. The maximum processivity (approximately 3 repeats) was observed at approximately 100 microM of each dNTP. Processivity decreased as the dNTP concentrations were reduced and, surprisingly, as the concentration of dGTP was increased. Also, the characteristic banding pattern generated by telomerase extension of DNA primers shifted in response to changes in dGTP concentration. One pattern with 8 nt periodicity was predominant at dGTP concentrations </=16 microM, while at >/= 250 microM an 8 nt repeat pattern out-of-phase with the first was observed; at intermediate concentrations the two patterns coexisted. We propose that two different segments of the RNA subunit can serve as the template for repeat synthesis; nt 42-49 at low dGTP concentrations and nt 36-43 at high dGTP concentrations. An alternative model for the low dGTP pattern involves an internal pause site but no pause at the end of the template and is, therefore, considered less likely. Because the effects of dGTP on processivity and banding pattern appear to be distinct from nucleotide binding in the polymerase active site, we propose a second dGTP binding site involved in template selection and processivity.     

A single nucleotide incorporation step limits human telomerase repeat addition activity

Human telomerase synthesizes telomeric DNA repeats (GGTTAG)_n onto chromosome ends using a short template from its integral telomerase RNA (hTR). However, telomerase is markedly slow for processive DNA synthesis among DNA polymerases. We report here that the unique template‐embedded pause signal restricts the first nucleotide incorporation for each repeat synthesized, imparting a significantly greater K_M. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate under physiological conditions, which is alleviated with augmented concentrations of dGTP or dGDP, and not with dGMP nor other nucleotides. The activity stimulation by dGDP is due to nucleoside diphosphates functioning as substrates for telomerase. Converting the first nucleotide of the repeat synthesized from dG to dA through the telomerase template mutation, hTR‐51U, correspondingly shifts telomerase repeat addition activity stimulation to dATP‐dependent. In accordance, telomerase without the pause signal synthesizes DNA repeats with extremely high efficiency under low dGTP concentrations and lacks dGTP stimulation. Thus, the first nucleotide incorporation step of the telomerase catalytic cycle is a potential target for therapeutic enhancement of telomerase activity.     

Regulation of catalytic activity and processivity of human telomerase

The ends of eukaryotic chromosomes are specialized sequences, called telomeres comprising tandem repeats of simple DNA sequences. Those sequences are essential for preventing aberrant recombination and protecting genomic DNA against exonucleolytic DNA degradation. Telomeres are maintained at a stable length by telomerase, an RNA-dependent DNA polymerase. Recently, human telomerase has been recognized as a unique diagnostic marker for human tumors and is potentially a highly selective target for antitumor drugs. In this study, we have examined the major factors affecting the catalytic activity and processivity of human telomerase. Specifically, both the catalytic activity and processivity of human telomerase were modulated by temperature, substrate (dNTP and primer) concentration, and the concentration of K+. The catalytic activity of telomerase increased as temperature (up to 37 degrees C), concentrations of dGTP, primer, and K+ were increased. However, the processivity of human telomerase decreased as temperature, primer concentration, and K+ were increased. Our results support the current model for human telomerase reaction and strengthen the hypothesis that a G-quadruplex structure of telomere DNA plays an important role in the regulation of the telomerase reaction.     

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.     

Mechanism of primer synthesis by the herpes simplex virus 1 helicase-primase

We utilized templates of defined sequence to investigate the mechanism of primer synthesis by herpes simplex virus 1 helicase-primase. Under steady-state conditions, the rate of primer synthesis and the size distribution of products remained constant with time, suggesting that the rate-limiting step(s) of primer synthesis occur(s) during primer initiation (at or before the formation of the pppNpN dinucleotide). Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis. Due to these effects on primer initiation, varying the NTP concentration could affect start site selection on templates containing multiple G-pyr-pyr initiation sites. Increasing the NTP concentration also increased the processivity of primase. However, even at very high concentrations of NTPs, elongation of the dinucleotide into longer products remained relatively inefficient. Primase did not readily elongate preexisting primers under conditions where free template was present in large excess of enzyme. However, if template concentrations were lowered such that primase synthesized primers on all or most of the template present in the reaction, then primase would elongate previously synthesized primers.     

A possible mechanism of processive nucleotide and repeat additions by the telomerase

Telomerase is a specialized cellular ribonucleoprotein complex that can synthesize long stretches of a DNA primer by using an intrinsic RNA template sequence. This requires that the telomerase must be able to carry out both nucleotide and repeat additions. Here, based on available structures and experimental data, a model is presented to describe these two addition activities. In the model, the forward movement of the polymerase active site along the template during the processive nucleotide addition is rectified through the incorporation of a matched base, via the Brownian ratchet mechanism. The unpairing of the DNA:RNA hybrid and then repositioning of product 3′-end after each round of repeat synthesis, which are prerequisites for the processive repeat addition, are caused by a force acting on the primer. The force results from the conformational transition of the stem III pseudoknot, which is mechanically induced by the rotation of TERT fingers together with stem IV loop towards the polymerase active site upon a nucleotide binding. Based on the model, the dynamics of processive nucleotide and repeat additions by recombinant Tetrahymena telomerase is studied analytically, which gives good quantitative explanations to the previous experimental results. Moreover, some predicted results are presented. In particular, it is shown that the repeat addition processivity is mainly determined by the difference between the free-energy change required to disrupt the DNA:RNA hybrid and that required to unfold the stem III pseudoknot. A large difference in free energy corresponds to a low repeat addition processivity while a small difference in free energy corresponds to a high repeat addition processivity.     

POT1–TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation

Telomerase contributes to chromosome end replication by synthesizing repeats of telomeric DNA, and the telomeric DNA‐binding proteins protection of telomeres (POT1) and TPP1 synergistically increase its repeat addition processivity. To understand the mechanism of increased processivity, we measured the effect of POT1–TPP1 on individual steps in the telomerase reaction cycle. Under conditions where telomerase was actively synthesizing DNA, POT1–TPP1 bound to the primer decreased primer dissociation rate. In addition, POT1–TPP1 increased the translocation efficiency. A template‐mutant telomerase that synthesizes DNA that cannot be bound by POT1–TPP1 exhibited increased processivity only when the primer contained at least one POT1–TPP1‐binding site, so a single POT1–TPP1–DNA interaction is necessary and sufficient for stimulating processivity. The POT1–TPP1 effect is specific, as another single‐stranded DNA‐binding protein, gp32, cannot substitute. POT1–TPP1 increased processivity even when substoichiometric relative to the DNA, providing evidence for a recruitment function. These results support a model in which POT1–TPP1 enhances telomerase processivity in a manner markedly different from the sliding clamps used by DNA polymerases.     

Effects of nucleotide analogues on Euplotes aediculatus telomerase processivity: evidence for product-assisted translocation.

Telomerase is a unique ribonucleoprotein that reverse transcribes a defined region of its RNA subunit onto the ends of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is typically a G-rich repeated sequence, (TTTTGGGG)(n) in the ciliate Euplotes aediculatus and (TTAGGG)(n) in humans. Telomerase can extend oligonucleotide primers in vitro in a processive fashion. We used dNTP analogues to study the structure-activity relationship between substrate nucleotides and processivity of telomerase from E. aediculatus. Several analogues, including 2'-deoxyuridine triphosphate (dUTP), 2'-deoxyinosine triphosphate (dITP), and 7-deaza-2'-deoxyguanosine triphosphate (7-deaza-dGTP), were good substrates for telomerase with K(m) and V(max) values near those of the natural substrates, dTTP and dGTP. However, telomerase processivity was affected with these substrates, decreasing in the order dUTP > 7-deaza-dGTP > dITP. Telomerase did not completely reverse transcribe the template when dITP was the substrate, and it efficiently extended a primer by the addition of two repeats when 7-deaza-dGTP and dUTP were utilized. When the same nucleotide analogues were incorporated into the primers, no effects were observed except in the case of a 3'-terminal deoxyinosine. The data support a model that includes the formation of an intramolecular secondary structure within the product DNA to facilitate translocation. The most likely structure is a G-G hairpin.     

Mutual dependence of Candida albicans Est1p and Est3p in telomerase assembly and activation

Telomerase is an RNA-protein complex responsible for extending one strand of the telomere terminal repeats. Analysis of the telomerase complex in budding yeasts has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The TERT subunit is essential for telomerase catalysis, while the functions of Est1p and Est3p have not been precisely elucidated. In an earlier study, we showed that telomerase derived from a Candida est1-null mutant is defective in primer utilization in vitro; it exhibits reduced initiation and processivity on primers that terminate in two regions of the telomere repeat. Here we show that telomerase derived from a Candida est3-null mutant has nearly identical defects in primer utilization and processivity. Further analysis revealed an unexpected mutual dependence of Est1p and Est3p in their assembly into the full telomerase complex, which accounts for the similarity between the mutant enzymes. We also developed an affinity isolation and an in vitro reconstitution protocol for the telomerase complex that will facilitate future mechanistic studies.     

有限延伸法检测端粒酶活性

人端粒酶是一种核蛋白体,通过其内含的RNA模板与端粒末端配对把重复端粒片段添加在端粒3＇末端|因此,端粒酶活性与细胞凋亡、衰老、永生化有密切关系,是癌症临床预测诊断的一个生物标签．现有的端粒酶活性检测方法,存在灵敏度低和不易定量等问题．本研究采用错配有限延伸法检测端粒酶活性：在人端粒酶延伸人工合成的游离端粒酶底物时,只加入dATP和dGTP,端粒酶只能把底物延伸4个脱氧核糖核苷酸AGGG．然后加入dNTP,让端粒酶延伸的产物和一条长的引物配对从而延伸出PCR模板|再加入引物进行热启动PCR．PCR后进行非变性PAGE (polyacrylamide gel electrophoresis),得到希望的唯一1条目标带．同时,用不同的端粒酶浓度梯度进行优化,发现有限延伸法检测端粒酶活性的下限达到250个HeLa细胞．     

Inhibition of human telomerase by 7-deaza-2'-deoxyguanosine nucleoside triphosphate analogs: potent inhibition by 6-thio-7-deaza-2'-deoxyguanosine 5'-triphosphate

We have examined analogs of the previously reported 7-deaza-2'-deoxypurine nucleoside triphosphate series of human telomerase inhibitors. Two new telomerase-inhibiting nucleotides are reported: 6-methoxy-7-deaza-2'-deoxyguanosine 5'-triphosphate (OMDG-TP) and 6-thio-7-deaza-2'-deoxyguanosine 5'-triphosphate (TDG-TP). In particular, TDG-TP is a very potent inhibitor of human telomerase with an IC(50) of 60 nM. TDG-TP can substitute for dGTP as a substrate for telomerase, but only at relatively high concentrations. Under conditions in which TDG-TP is the only available guanosine substrate, telomerase becomes nonprocessive, synthesizing short products that appear to contain only one to three TDG residues. Similarly, the less potent telomerase inhibitor OMDG-TP gives rise to short telomerase products, but less efficiently than TDG-TP. We show here that TDG-TP, and to a lesser extent OMDG-TP, can serve as substrates for both templated (Klenow exo) and nontemplated (terminal transferase) DNA polymerases. For either polymerase, the products arising from TDG-TP are relatively short, and give rise to bands of unusual mobility under PAGE conditions. These anomalous bands revert, under treatment with DTT, to normal mobility bands, indicating that these products may contain thiol-labile disulfide linkages involving the incorporated TDG residues. This observation of potential TDG-crosslinks may have bearing on the mechanism of telomerase inhibition by this nucleotide analog.     

Determinants in mammalian telomerase RNA that mediate enzyme processivity and cross-species incompatibility

Telomerase contains two essential components: an RNA molecule that templates telomeric repeat synthesis and a catalytic protein component. Human telomerase is processive, while the mouse enzyme has much lower processivity. We have identified nucleotide determinants in the telomerase RNA that are responsible for this difference in processivity. Mutations adjacent to the template region of human and mouse telomerase RNA significantly altered telomerase processivity both in vitro and in vivo. We also identified functionally important nucleotides in the pseudoknot domain of telomerase RNA that potentially mediate the incompatibility between human TERT and mouse telomerase RNA. These experiments identify essential residues of the telomerase RNA that regulate telomerase activity and processivity.