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1.
Nucleic Acids Res ; 49(20): 11653-11665, 2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34718732

RESUMO

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

2.
Science ; 368(6495): 1081-1085, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499435

RESUMO

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.


Assuntos
Complexos Multiproteicos/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Telômero/química , Microscopia Crioeletrônica , DNA de Cadeia Simples/química , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Proteína de Replicação A/química
3.
Mol Biol Cell ; 29(7): 869-880, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29386295

RESUMO

The reverse transcriptase telomerase adds telomeric repeats to chromosome ends to counteract telomere shortening and thereby assures genomic stability in dividing human cells. Key parameters in telomere homeostasis are the frequency with which telomerase engages the chromosome end and the number of telomeric repeats it adds during each association event. To study telomere elongation in vivo, we have established a live-cell imaging assay to track individual telomerase ribonucleoproteins in CRISPR-edited HeLa cells. Using this assay and the drug imetelstat, which is a competitive inhibitor of telomeric DNA binding, we demonstrate that stable association of telomerase with the single-stranded overhang of the chromosome end requires telomerase-DNA base pairing. Furthermore, we show that telomerase processivity contributes to telomere elongation in vivo. Together, these findings provide new insight into the dynamics of telomerase recruitment and the importance of processivity in maintaining telomere length in human cancer cells.

4.
Nat Commun ; 8(1): 1075, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-29057866

RESUMO

The human shelterin proteins associate with telomeric DNA to confer telomere protection and length regulation. They are thought to form higher-order protein complexes for their functions, but studies of shelterin proteins have been mostly limited to pairs of proteins. Here we co-express various human shelterin proteins and find that they form defined multi-subunit complexes. A complex harboring both TRF2 and POT1 has the strongest binding affinity to telomeric DNA substrates comprised of double-stranded DNA with a 3' single-stranded extension. TRF2 interacts with TIN2 with an unexpected 2:1 stoichiometry in the context of shelterin (RAP12:TRF22:TIN21:TPP11:POT11). Tethering of TPP1 to the telomere either via TRF2-TIN2 or via POT1 gives equivalent enhancement of telomerase processivity. We also identify a peptide region from TPP1 that is both critical and sufficient for TIN2 interaction. Our findings reveal new information about the architecture of human shelterin and how it performs its functions at telomeres.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Ligação Proteica , Telomerase/genética , Proteínas de Ligação a Telômeros/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo
5.
Cell ; 166(5): 1188-1197.e9, 2016 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-27523609

RESUMO

Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end.


Assuntos
Telomerase/metabolismo , Telômero/enzimologia , Transporte Ativo do Núcleo Celular , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Linhagem Celular , Núcleo Celular/enzimologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Corpos Enovelados/enzimologia , Endonucleases , Edição de Genes , Genoma Humano , Células HeLa , Humanos , Imageamento Tridimensional , Domínios Proteicos , Fase S , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Telomerase/química , Telômero/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo
6.
Genome Biol ; 16: 231, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26553065

RESUMO

BACKGROUND: To facilitate indefinite proliferation, stem cells and most cancer cells require the activity of telomerase, which counteracts the successive shortening of telomeres caused by incomplete DNA replication at the very end of each chromosome. Human telomerase activity is often determined by the expression level of telomerase reverse transcriptase (TERT), the catalytic subunit of the ribonucleoprotein complex. The low expression level of TERT and the lack of adequate antibodies have made it difficult to study telomerase-related processes in human cells. RESULTS: To overcome the low CRISPR-Cas9 editing efficiency at the TERT locus, we develop a two-step "pop-in/pop-out" strategy to enrich cells that underwent homologous recombination (HR). Using this technique, we fuse an N-terminal FLAG-SNAP-tag to TERT, which allows us to reliably detect TERT in western blots, immunopurify it for biochemical analysis, and determine its subcellular localization by fluorescence microscopy. TERT co-localizes detectably with only 5-7 % of the telomeres at a time in S-phase HeLa cells; no nucleolar localization is detected. Furthermore, we extend this approach to perform single base-pair modifications in the TERT promoter; reverting a recurrent cancer-associated TERT promoter mutation in a urothelial cancer cell line results in decreased telomerase activity, indicating the mutation is causal for telomerase reactivation. CONCLUSIONS: We develop a two-step CRISPR-Cas9 genome editing strategy to introduce precise modifications at the endogenous TERT locus in human cell lines. This method provides a useful tool for studying telomerase biology, and suggests a general approach to edit loci with low targeting efficiency and to purify and visualize low abundance proteins.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de RNA/genética , Telomerase/genética , Telômero/genética , Cromossomos/genética , Replicação do DNA/genética , Regulação da Expressão Gênica , Genoma Humano , Células HeLa , Humanos , Mutação , Regiões Promotoras Genéticas , Telomerase/biossíntese
7.
Science ; 347(6225): 1006-10, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25722414

RESUMO

Reactivation of telomerase, the chromosome end-replicating enzyme, drives human cell immortality and cancer. Point mutations in the telomerase reverse transcriptase (TERT) gene promoter occur at high frequency in multiple cancers, including urothelial cancer (UC), but their effect on telomerase function has been unclear. In a study of 23 human UC cell lines, we show that these promoter mutations correlate with higher levels of TERT messenger RNA (mRNA), TERT protein, telomerase enzymatic activity, and telomere length. Although previous studies found no relation between TERT promoter mutations and UC patient outcome, we find that elevated TERT mRNA expression strongly correlates with reduced disease-specific survival in two independent UC patient cohorts (n = 35; n = 87). These results suggest that high telomerase activity may be a better marker of aggressive UC tumors than TERT promoter mutations alone.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Telomerase/genética , Telomerase/metabolismo , Homeostase do Telômero , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Mutação Puntual , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Neoplasias da Bexiga Urinária/patologia , Urotélio/enzimologia , Urotélio/patologia
8.
J Cell Sci ; 128(2): 331-41, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25416818

RESUMO

Most human cancers depend on the telomerase to maintain telomeres; however, about 10% of cancers are telomerase negative and utilize the alternative lengthening of telomeres (ALT) mechanism. Mutations in the DAXX gene have been found frequently in both telomerase-positive and ALT cells, and how DAXX mutations contribute to cancers remains unclear. We report here that endogenous DAXX can localize to Cajal bodies, associate with the telomerase and regulate telomerase targeting to telomeres. Furthermore, disease mutations that are located in different regions of DAXX differentially impact on its ability to interact with its binding partners and its targeting to Cajal bodies and telomeres. In addition, DAXX knockdown by RNA interference led to reduced telomerase targeting to telomeres and telomere shortening. These findings collectively support a DAXX-centric pathway for telomere maintenance, where DAXX interaction with the telomerase regulates telomerase assembly in Cajal bodies and telomerase targeting to telomeres.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Nucleares/genética , Telomerase/genética , Homeostase do Telômero/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Correpressoras , Corpos Enovelados/genética , Corpos Enovelados/metabolismo , DNA Helicases/genética , Humanos , Hibridização in Situ Fluorescente , Chaperonas Moleculares , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Interferência de RNA , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo
9.
Nucleic Acids Res ; 41(19): 8969-78, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23901009

RESUMO

Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays.


Assuntos
Telomerase/genética , Telomerase/metabolismo , Linhagem Celular , Humanos , Mutação , Proteínas de Ligação a Telômeros/metabolismo
10.
Nature ; 492(7428): 285-9, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-23103865

RESUMO

Human chromosome ends are capped by shelterin, a protein complex that protects the natural ends from being recognized as sites of DNA damage and also regulates the telomere-replicating enzyme, telomerase. Shelterin includes the heterodimeric POT1-TPP1 protein, which binds the telomeric single-stranded DNA tail. TPP1 has been implicated both in recruiting telomerase to telomeres and in stimulating telomerase processivity (the addition of multiple DNA repeats after a single primer-binding event). Determining the mechanisms of these activities has been difficult, especially because genetic perturbations also tend to affect the essential chromosome end-protection function of TPP1 (refs 15-17). Here we identify separation-of-function mutants of human TPP1 that retain full telomere-capping function in vitro and in vivo, yet are defective in binding human telomerase. The seven separation-of-function mutations map to a patch of amino acids on the surface of TPP1, the TEL patch, that both recruits telomerase to telomeres and promotes high-processivity DNA synthesis, indicating that these two activities are manifestations of the same molecular interaction. Given that the interaction between telomerase and TPP1 is required for telomerase function in vivo, the TEL patch of TPP1 provides a new target for anticancer drug development.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética
11.
Nature ; 474(7351): 399-402, 2011 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-21602826

RESUMO

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.


Assuntos
Disceratose Congênita/genética , Disceratose Congênita/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Telômero/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Reprogramação Celular , Fibroblastos , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/enzimologia , Telômero/genética , Telômero/metabolismo
12.
Genes Dev ; 24(6): 613-22, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20231318

RESUMO

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3' ssDNA extension of human telomeres. POT1-TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1-TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1-TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1-TPP1, in contrast to other single amino acid mutations. Chimeric human-fish telomerases that contained the human TEN domain were active but not stimulated by POT1-TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A-mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1-POT1-telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/genética , Humanos , Camundongos , Modelos Moleculares , Mutação/genética , Oryzias , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação a Telômeros/genética
13.
Nat Struct Mol Biol ; 15(8): 870-2, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18641663

RESUMO

Telomerase shows repeat-addition processivity (RAP): synthesis of multiple telomeric DNA repeats without primer dissociation. Leu14 mutants in the telomerase essential N-terminal domain of Tetrahymena thermophila telomerase reverse transcriptase retain full activity and anchor-site function but lose RAP, suggesting models for how this domain facilitates DNA translocation.


Assuntos
Mutação , Telomerase/química , Tetrahymena thermophila/enzimologia , Animais , Sítios de Ligação , Domínio Catalítico , DNA/química , Primers do DNA/química , Humanos , Cinética , Leucina/química , Estrutura Terciária de Proteína , Telomerase/metabolismo , Temperatura
14.
Nature ; 445(7127): 506-10, 2007 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17237768

RESUMO

Telomeres were originally defined as chromosome caps that prevent the natural ends of linear chromosomes from undergoing deleterious degradation and fusion events. POT1 (protection of telomeres) protein binds the single-stranded G-rich DNA overhangs at human chromosome ends and suppresses unwanted DNA repair activities. TPP1 is a previously identified binding partner of POT1 that has been proposed to form part of a six-protein shelterin complex at telomeres. Here, the crystal structure of a domain of human TPP1 reveals an oligonucleotide/oligosaccharide-binding fold that is structurally similar to the beta-subunit of the telomere end-binding protein of a ciliated protozoan, suggesting that TPP1 is the missing beta-subunit of human POT1 protein. Telomeric DNA end-binding proteins have generally been found to inhibit rather than stimulate the action of the chromosome end-replicating enzyme, telomerase. In contrast, we find that TPP1 and POT1 form a complex with telomeric DNA that increases the activity and processivity of the human telomerase core enzyme. We propose that POT1-TPP1 switches from inhibiting telomerase access to the telomere, as a component of shelterin, to serving as a processivity factor for telomerase during telomere extension.


Assuntos
Telomerase/química , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , Cristalografia por Raios X , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Homologia Estrutural de Proteína , Telomerase/antagonistas & inibidores
15.
Proc Natl Acad Sci U S A ; 102(31): 10864-9, 2005 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-16043710

RESUMO

The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation. We now show that telomeric oligonucleotides, such as d[GGG(TTAGGG)(3)], which form intramolecular G-quadruplexes through Hoogsteen base-pairing, serve as only marginal primers for extension by recombinant human telomerase; telomerase stalls after every nucleotide addition. Addition of hPOT1 to the reaction restores the normal processive elongation pattern seen with primers that cannot form G-quadruplexes. hPOT1 does not act catalytically but, instead, forms a stoichiometric complex with the DNA, freeing its 3' tail. An antisense oligonucleotide, which base-pairs near the 5' end of the telomeric sequence, leaving a telomerase-extendable 3' tail, duplicates the effect of hPOT1 on activation of G-quadruplex primers. Thus, hPOT1 may function simply by trapping the unfolded forms of these telomeric primers in an equilibrium population. We propose an additional role for hPOT1 in telomere maintenance: disrupting G-quadruplex structures in telomeric DNA, thereby allowing proper elongation by telomerase.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Sequência de Bases , DNA/química , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Técnicas In Vitro , Substâncias Macromoleculares , Modelos Biológicos , Conformação de Ácido Nucleico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Telomerase/genética , Proteínas de Ligação a Telômeros/genética
16.
J Biol Chem ; 280(21): 20449-56, 2005 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-15792951

RESUMO

POT1 (protection of telomeres 1) protein binds the G-rich single-stranded telomeric DNA at the ends of chromosomes. In human cells hPOT1 is involved in telomere length regulation, but the mechanism of this regulation remains unknown. Examination of the high-resolution crystal structure of the hPOT1-TTAGGGTTAG complex suggested that it would not be extended by telomerase, a hypothesis that we confirm by in vitro assays with recombinant telomerase. On the other hand, when hPOT1 is bound at a position one telomeric repeat before the 3'-end, leaving an 8-nucleotide 3'-tail, the complex is extended with improved activity and processivity. Thus, depending on its location relative to the DNA 3'-end, hPOT1 can either inhibit telomerase action or form a preferred substrate for telomerase. We propose that another factor catalyzes the interconversion of these states in vivo.


Assuntos
Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Processamento Alternativo , Sítios de Ligação , Cristalização , DNA/metabolismo , Primers do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Inibidores Enzimáticos , Hemaglutininas , Humanos , Modelos Moleculares , Estrutura Molecular , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico , Relação Estrutura-Atividade , Especificidade por Substrato , Telomerase/antagonistas & inibidores , Telômero/química , Telômero/genética , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/genética
17.
RNA ; 9(11): 1323-32, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14561882

RESUMO

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.


Assuntos
RNA Fúngico/metabolismo , Saccharomyces cerevisiae/enzimologia , Telomerase/metabolismo , Sequência de Bases , Clonagem Molecular , Primers do DNA , Mutação , Telomerase/genética , Moldes Genéticos
18.
Nucleic Acids Res ; 31(6): 1646-55, 2003 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-12626706

RESUMO

Telomeres, the protective caps of eukaryotic chromosomes, are maintained by the enzyme telomerase. This telomere-specific reverse transcriptase (RT) uses a small region of its RNA subunit as template to synthesize telomeric DNA, which is generally G/T rich in the strand that contains the 3' end. To further our understanding of why telomeres are usually G/T rich, we screened Saccharomyces cerevisiae telomerase RNA (TLC1) libraries with randomized template sequences for complementation of a tlc1 deletion and decapping of existing telomeres. Surprisingly, the vast majority of the 60 000 different mutant telomerase templates tested showed no activity in vivo. This deficiency was not due to impaired assembly with the catalytic subunit (Est2p) nor could it be alleviated by enforced telomerase recruitment to the telomeres. Rather, the mutant templates reduced the nucleotide addition processivity of telomerase. The functional RNA template sequences recovered in our screens preferentially contained two or more consecutive rC nucleotides, reminiscent of the wild-type template. Thus, in contrast to retroviral RTs that can reverse transcribe any RNA sequence into DNA, the budding yeast telomerase RT is specialized for its C-rich RNA template.


Assuntos
RNA Fúngico/metabolismo , Saccharomyces cerevisiae/enzimologia , Telomerase/metabolismo , Adenina/metabolismo , Sequência de Bases , Citosina/metabolismo , Teste de Complementação Genética , Mutação , Ligação Proteica , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade por Substrato , Telomerase/genética , Telômero/genética , Telômero/metabolismo
19.
Mol Cell Biol ; 22(7): 2366-74, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11884619

RESUMO

The Saccharomyces cerevisiae telomerase RNA subunit is encoded by the TLC1 gene. A selection for viable alleles of TLC1 RNA from a large library of random deletion alleles revealed that less than half (approximately 0.5 kb of the approximately 1.3-kb RNA) is required for telomerase function in vivo. The main essential region (430 nucleotides), which contains the template for telomeric DNA synthesis, was required for coimmunoprecipitation with Est1p and Est2p. Furthermore, the subregion required for interaction with Est1p, the telomerase recruitment subunit, differed from those required for interaction with Est2p, the reverse transcriptase subunit. Two regions of the RNA distant from the template in the nucleotide sequence were required for Est2p binding, but the template itself was not. Having the RNA secured to the protein away from the template is proposed to facilitate the translocation of the RNA template through the active site. More generally, our results support a role for the telomerase RNA serving as a scaffold for binding key protein subunits.


Assuntos
Proteínas Fúngicas/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Telomerase/genética , Telomerase/metabolismo , Alelos , Sítios de Ligação , Proteínas de Ligação a DNA , Dimerização , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade , Telomerase/química , Moldes Genéticos
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