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1.
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.  相似文献   

2.
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.  相似文献   

3.
Appropriate control of the chromosome end-replicating enzyme telomerase is crucial for maintaining telomere length and genomic stability. The essential telomeric DNA-binding protein Cdc13p both positively and negatively regulates telomere length in budding yeast. Here we test the effect of purified Cdc13p on telomerase action in vitro. We show that the full-length protein and its DNA-binding domain (DBD) inhibit primer extension by telomerase. This inhibition occurs by competitive blocking of telomerase access to DNA. To further understand the requirements for productive telomerase 3′-end access when Cdc13p or the DBD is bound to a telomerase substrate, we constrained protein binding at various distances from the 3′-end on two sets of increasingly longer oligonucleotides. We find that Cdc13p inhibits the action of telomerase through three distinct biochemical modes, including inhibiting telomerase even when a significant tail is available, representing a novel ‘action at a distance’ inhibitory activity. Thus, while yeast Cdc13p exhibits the same general activity as human POT1, providing an off switch for telomerase when bound near the 3′-end, there are significant mechanistic differences in the ways telomere end-binding proteins inhibit telomerase action.  相似文献   

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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.  相似文献   

6.
Recognition and elongation of telomeres by telomerase   总被引:9,自引:0,他引:9  
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.  相似文献   

7.
端粒是真核细胞染色体末端的重复DNA序列 ,其生物学功能是防止染色体DNA降解、末端融合、非正常重组和染色体的缺失[1] .由于存在“末端复制问题” ,随着老化人体细胞端粒重复序列长度不断缩短 ,但在生殖细胞中由于端粒酶的存在 ,端粒序列并不缩短 .端粒酶是由蛋白质和RNA构成的核蛋白 ,是依赖RNA的DNA聚合酶 ,在DNA3’端合成端粒重复序列[2 ] .研究表明 ,在 85 %~ 95 %的人肿瘤细胞中可以检测到端粒酶的活性[3 ,4 ] ,而在正常体细胞中除生殖细胞和造血干细胞等极少数细胞中存在端粒酶活性外 ,均检测不到端粒酶活性 ,这…  相似文献   

8.
王娟 《现代生物医学进展》2007,7(6):923-925,937
端粒酶几乎在所有的人类癌细胞中均异常表达,它的持久活性对肿瘤的增殖是必需的。因此,抑制端粒酶活性代表了一种新的癌症治疗机制。端粒酶全酶复合物有多处可以做为抑制剂的靶点,包括hTR、hTERT、引物锚定位点等。本文对以端粒酶RNA模板区为靶点的抗肿瘤药物设计策略进行了综述,包括对该区域进行点突变、使用反义寡核苷酸封闭模板区、改变端粒酶RNA空间构象等,并探讨了目前抑制端粒酶活性研究中存在的一些问题。  相似文献   

9.
Evans SK  Lundblad V 《Genetics》2002,162(3):1101-1115
The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.  相似文献   

10.
Telomerase activity in bovine embryos during early development   总被引:11,自引:0,他引:11  
Xu J  Yang X 《Biology of reproduction》2000,63(4):1124-1128
The telomere is the end structure of the DNA molecule. Telomerase is the ribonuclear enzyme that helps the cell's telomere to elongate; otherwise, the telomere will 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. Recent research shows that telomerase activity is also present in early embryos, but to our knowledge, the dynamics of this enzyme during early embryo development have not been studied. In the present work, we conducted telomerase activity assays on bovine embryos fertilized in vitro and harvested at different stages from zygote to blastocyst. A polymerase chain reaction-based assay (Telomeric Repeat Amplification Protocol) was used to detect the telomerase activity in these embryos. We demonstrated that the telomerase activity is present in the early embryos, but that its level varies with the different developmental stages. The activity was relatively low in mature oocytes. It increased after in vitro fertilization and then decreased gradually until the embryo reached the eight-cell stage. After the eight-cell stage, the telomerase activity increased again and reached its highest level in the blastocyst stage. This study provides insight regarding how telomerase activity and, possibly, the length of the telomere are reprogrammed during early embryo development.  相似文献   

11.
12.
Telomere lengths are maintained in many cancer cells by the ribonucleoprotein enzyme telomerase but can be further elongated by increasing telomerase activity through the overexpression of telomerase components. We report here that increased telomerase activity results in increased telomere length that eventually reaches a plateau, accompanied by the generation of telomere length heterogeneity and the accumulation of extrachromosomal telomeric repeat DNA, principally in the form of telomeric circles (t-circles). Telomeric DNA was observed in promyelocytic leukemia bodies, but no intertelomeric copying or telomere exchange events were identified, and there was no increase in telomere dysfunction-induced foci. These data indicate that human cells possess a mechanism to negatively regulate telomere length by trimming telomeric DNA from the chromosome ends, most likely by t-loop resolution to form t-circles. Additionally, these results indicate that some phenotypic characteristics attributed to alternative lengthening of telomeres (ALT) result from increased mean telomere length, rather than from the ALT mechanism itself.  相似文献   

13.
Bianchi A  Shore D 《Cell》2007,128(6):1051-1062
The maintenance of an appropriate number of telomere repeats by telomerase is essential for proper chromosome protection. The action of telomerase at the telomere terminus is regulated by opposing activities that either recruit/activate the enzyme at shorter telomeres or inhibit it at longer ones, thus achieving a stable average telomere length. To elucidate the mechanistic details of telomerase regulation we engineered specific chromosome ends in yeast so that a single telomere could be suddenly shortened and, as a consequence of its reduced length, elongated by telomerase. We show that shortened telomeres replicate early in S phase, unlike normal-length telomeres, due to the early firing of origins of DNA replication in subtelomeric regions. Early telomere replication correlates with increased telomere length and telomerase activity. These data reveal an epigenetic effect of telomere length on the activity of nearby replication origins and an unanticipated link between telomere replication timing and telomerase action.  相似文献   

14.
Li Y  Li X  Ji X  Li X 《Biosensors & bioelectronics》2011,26(10):4095-4098
In the present study, a chemiluminescence method for sensitive detection of human telomerase activity was developed based on the formation of G-quadruplex-hemin DNAzyme. In the presence of telomerase, the telomerase substrate (TS) primer elongated and a long single-strand DNA containing the telomere repeat units (TTAGGG)n was formed. When K(+) was introduced, the telomere repeat units could form G-quadruplex and then combined with hemin to form DNAzymes which could stimulate the generation of chemiluminescence (CL) in the presence of luminol and H(2)O(2). The amount of telomerase elongation product was controlled by the content of telomerase extracted from HeLa cells, so the amount of DNAzymes and the intensity of chemiluminescence signal were all related to the number of HeLa cells. Using this simple method, the telomerase activity extracted from 100 cultured cancer cells could be detected without the polymerase chain reaction (PCR) amplification of telomerase elongated product.  相似文献   

15.
哺乳动物早期胚胎端粒和端粒酶重编程   总被引:1,自引:0,他引:1  
端粒位于真核染色体末端,是稳定染色体末端的重要元件。端粒酶(TER)是一种特殊的细胞核糖核蛋白(RNP)反转录酶(RT),其核心酶包括蛋白亚基和RNA元件。在DNA复制过程中的端粒丢失可以被有活性的端粒酶修复回来。哺乳动物端粒酶在发育中受调控,端粒的重编程可能是由于早期胚胎不同时期的端粒酶活性而造成的。因此,研究端粒和端粒酶重编程在早期胚胎发育中是非常重要的。该文综述了端粒和端粒酶的结构和功能,及其与哺乳动物早期胚胎发育的关系,并在此基础上展望了端粒和端粒酶在克隆动物胚胎发育的基础研究。  相似文献   

16.
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18.
Tárkányi I  Aradi J 《Biochimie》2008,90(1):156-172
Telomerase enzyme is a ribonucleoprotein maintaining the length of the telomeres by adding G-rich repeats to the end of the eukaryotic chromosomes. Normal human somatic cells, cultured in vitro, have a strictly limited proliferative potential undergoing senescence after about 50-70 population doublings. In contrast, most of the tumor cells have unlimited replicative potential. Although the mechanisms of immortalization are not understood completely at a genetic level, the key role of the telomere/telomerase system in the process is clear. The DNA replication machinery is not able to replicate fully the DNA at the very end of the chromosomes; therefore, about 50-200 nucleotides are lost during each of the replication cycles resulting in a gradual decrease of telomere length. Critically short telomere induces senescence, subsequent crisis and cell death. In tumor cells, however, the telomerase enzyme prevents the formation of critically short telomeres, adding GGTTAG repeats to the 3' end of the chromosomes immortalizing the cells. Immortality is one of the hallmarks of cancer. Besides the catalytic activity dependent telomere maintenance, catalytic activity-independent effects of telomerase may also be involved in the regulation of cell cycle. The telomere/telomerase system offers two possibilities to intervene the proliferative activity of the cell: (1) inhibition the telomere maintenance by inhibiting the telomerase activity; (2) activating the residual telomerase enzyme or inducing telomerase expression. Whilst the former approach could abolish the limitless replicative potential of malignant cells, the activation of telomerase might be utilized for treating degenerative diseases. Here, we review the current status of telomerase therapeutics, summarizing the activities of those pharmacological agents which either inhibit or activate the enzyme. We also discuss the future opportunities and challenges of research on pharmacological intervention of telomerase activity.  相似文献   

19.
Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.  相似文献   

20.
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