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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.
Nat Genet ; 52(9): 931-938, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32632336

RESUMO

Many chromatin-binding proteins and protein complexes that regulate transcription also bind RNA. One of these, Polycomb repressive complex 2 (PRC2), deposits the H3K27me3 mark of facultative heterochromatin and is required for stem cell differentiation. PRC2 binds RNAs broadly in vivo and in vitro. Yet, the biological importance of this RNA binding remains unsettled. Here, we tackle this question in human induced pluripotent stem cells by using multiple complementary approaches. Perturbation of RNA-PRC2 interaction by RNase A, by a chemical inhibitor of transcription or by an RNA-binding-defective mutant all disrupted PRC2 chromatin occupancy and localization genome wide. The physiological relevance of PRC2-RNA interactions is further underscored by a cardiomyocyte differentiation defect upon genetic disruption. We conclude that PRC2 requires RNA binding for chromatin localization in human pluripotent stem cells and in turn for defining cellular state.


Assuntos
Cromatina/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes/fisiologia , Complexo Repressor Polycomb 2/genética , RNA/genética , Sítios de Ligação/genética , Proteínas de Transporte , Diferenciação Celular/genética , Genoma/genética , Histonas/genética , Humanos , Ligação Proteica/genética
3.
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
4.
RNA ; 25(8): 935-947, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31048495

RESUMO

Some neurological disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), fragile X syndrome, Huntington's disease, myotonic dystrophy, and various ataxias, can be caused by expansions of short nucleic acid sequence repeats in specific genes. A possible disease mechanism involves the transcribed repeat RNA binding an RNA-binding protein (RBP), resulting in its sequestration and thus dysfunction. Polycomb repressive complex 2 (PRC2), the histone methyltransferase that deposits the H3K27me3 mark of epigenetically silenced chromatin, binds G-rich RNAs and has especially high affinity for G-quadruplex (G-Q) structures. Here, we find that PRC2 target genes are derepressed and the RNA binding subunit EZH2 largely insoluble in postmortem brain samples from ALS/FTD patients with C9ORF72 (C9) repeat expansions, leading to the hypothesis that the (G4C2)n repeat RNA might be sequestering PRC2. Contrary to this expectation, we found that C9 repeat RNAs (n = 6 or 10) bind weakly to purified PRC2, and studies with the G-Q specific BG4 antibody and circular dichroism studies both indicated that these C9 RNAs have little propensity to form G-Qs in vitro. Several GC-rich triplet-repeat expansion RNAs also have low affinity for PRC2 and do not appreciably form G-Qs in vitro. The results are consistent with these sequences forming hairpin structures that outcompete G-Q folding when the repeat length is sufficiently large. We suggest that binding of PRC2 to these GC-rich RNAs is fundamentally weak but may be modulated in vivo by protein factors that affect secondary structure, such as helicases and other RBPs.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/química , Proteína C9orf72/genética , Demência Frontotemporal/genética , Complexo Repressor Polycomb 2/metabolismo , Repetições de Trinucleotídeos , Esclerose Amiotrófica Lateral/metabolismo , Autopsia , Dicroísmo Circular , Proteína Potenciadora do Homólogo 2 de Zeste/química , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Demência Frontotemporal/metabolismo , Quadruplex G , Humanos , Complexo Repressor Polycomb 2/química , Solubilidade
5.
Elife ; 62017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-29185984

RESUMO

Polycomb repressive complex 2 (PRC2) is a key chromatin modifier responsible for methylation of lysine 27 in histone H3. PRC2 has been shown to interact with thousands of RNA species in vivo, but understanding the physiological function of RNA binding has been hampered by the lack of separation-of-function mutants. Here, we use comprehensive mutagenesis and hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify critical residues for RNA interaction in PRC2 core complexes from Homo sapiens and Chaetomium thermophilum, for which crystal structures are known. Preferential binding of G-quadruplex RNA is conserved, surprisingly using different protein elements. Key RNA-binding residues are spread out along the surface of EZH2, with other subunits including EED also contributing, and missense mutations of some of these residues have been found in cancer patients. The unusual nature of this protein-RNA interaction provides a paradigm for other epigenetic modifiers that bind RNA without canonical RNA-binding motifs.


Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Quadruplex G , RNA/metabolismo , Chaetomium/enzimologia , Análise Mutacional de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/química , Humanos , Espectrometria de Massas , Ligação Proteica
6.
Mol Cell ; 65(6): 1056-1067.e5, 2017 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-28306504

RESUMO

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that trimethylates H3K27, a mark of repressed chromatin. Mammalian PRC2 binds RNA promiscuously, with thousands of target transcripts in vivo. But what does PRC2 recognize in these RNAs? Here we show that purified human PRC2 recognizes G > C,U ≫ A in single-stranded RNA and has a high affinity for folded guanine quadruplex (G4) structures but little binding to duplex RNAs. Importantly, G-tract motifs are significantly enriched among PRC2-binding transcripts in vivo. DNA sequences coding for PRC2-binding RNA motifs are enriched at PRC2-binding sites on chromatin and H3K27me3-modified nucleosomes. Collectively, the abundance of PRC2-binding RNA motifs rationalizes the promiscuous RNA binding of PRC2, and their enrichment at Polycomb target genes provides a means for RNA-mediated regulation.


Assuntos
Cromatina/enzimologia , Guanina/metabolismo , Nucleossomos/enzimologia , Complexo Repressor Polycomb 2/metabolismo , RNA/metabolismo , Sítios de Ligação , Cromatina/química , Cromatina/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Motivos de Nucleotídeos , Complexo Repressor Polycomb 2/genética , Ligação Proteica , RNA/química , RNA/genética , Relação Estrutura-Atividade , Transfecção
7.
Cell ; 165(5): 1267-1279, 2016 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-27180905

RESUMO

RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.


Assuntos
Ficusina/química , RNA de Cadeia Dupla/química , Animais , Pareamento de Bases , Células HEK293 , Células HeLa , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , RNA Longo não Codificante/química
8.
Mol Cell ; 57(3): 552-8, 2015 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-25601759

RESUMO

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Early works suggested binding specificity of PRC2 to certain long non-coding RNAs for recruitment to chromatin. More recent studies provided evidence both in favor and against this idea. Here, we bridge the two existing models of PRC2-RNA interaction. RepA RNA is a good binding partner for PRC2, while multiple non-relevant RNAs, including bacterial mRNAs, also bind PRC2; Kds depend to some extent on the experimental conditions. Human and mouse PRC2 have broadly similar RNA-binding properties in vitro. Examination of evidence supporting an existing model for site-specific recruitment of PRC2 by a well-defined RNA motif in cells reveals that results are PRC2 independent. We conclude that promiscuous and specific RNA-binding activities of PRC2 in vitro are not mutually exclusive, and that binding specificity in vivo remains to be demonstrated.


Assuntos
Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica , RNA/metabolismo , Animais , Células HEK293 , Humanos , Técnicas In Vitro , Sequências Repetidas Invertidas , Camundongos , RNA/química , RNA Longo não Codificante/metabolismo
9.
Nucleic Acids Res ; 42(14): 9236-48, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24992961

RESUMO

Polycomb repressive complex-2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long non-coding RNAs (lncRNAs) can recruit PRC2 to chromatin. Previous studies identified PRC2 subunits in a complex with the apparent molecular weight of a dimer, which might be accounted for by the incorporation of additional protein subunits or RNA rather than PRC2 dimerization. Here we show that reconstituted human PRC2 is in fact a dimer, using multiple independent approaches including analytical size exclusion chromatography (SEC), SEC combined with multi-angle light scattering and co-immunoprecipitation of differentially tagged subunits. Even though it contains at least two RNA-binding subunits, each PRC2 dimer binds only one RNA molecule. Yet, multiple PRC2 dimers bind a single RNA molecule cooperatively. These observations suggest a model in which the first RNA binding event promotes the recruitment of multiple PRC2 complexes to chromatin, thereby nucleating repression.


Assuntos
Complexo Repressor Polycomb 2/metabolismo , Animais , Humanos , Proteínas Ligantes de Maltose/genética , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/genética , Multimerização Proteica , RNA/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera
10.
Nat Struct Mol Biol ; 20(11): 1250-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24077223

RESUMO

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase required for epigenetic silencing during development and cancer. Long noncoding RNAs (lncRNAs) recruit PRC2 to chromatin, but the general role of RNA in maintaining repressed chromatin is unknown. Here we measure the binding constants of human PRC2 to various RNAs and find comparable affinity for human lncRNAs targeted by PRC2 as for irrelevant transcripts from ciliates and bacteria. PRC2 binding is size dependent, with lower affinity for shorter RNAs. In vivo, PRC2 predominantly occupies repressed genes; PRC2 is also associated with active genes, but most of those are not regulated by PRC2. These findings support a model in which PRC2's promiscuous binding to RNA transcripts allows it to scan for target genes that have escaped repression, thus leading to maintenance of the repressed state. Such RNAs may also provide a decoy for PRC2.


Assuntos
Complexo Repressor Polycomb 2/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Bactérias , Cilióforos , Perfilação da Expressão Gênica , Humanos , Ligação Proteica
11.
RNA ; 19(6): 841-51, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23610127

RESUMO

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.


Assuntos
Proteínas de Ligação a DNA/química , RNA Fúngico/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Bases , Sítios de Ligação , CME-Carbodi-Imida/análogos & derivados , CME-Carbodi-Imida/química , Núcleo Celular/química , Núcleo Celular/genética , Pegada de DNA/métodos , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Sequências Repetidas Invertidas , Mutação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Oligonucleotídeos Fosforotioatos/química , Mapeamento de Interação de Proteínas , RNA/genética , RNA/metabolismo , Clivagem do RNA , RNA Fúngico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ésteres do Ácido Sulfúrico/química , Telomerase/química , Telomerase/genética , Telomerase/metabolismo
12.
Cell ; 148(5): 922-32, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22365814

RESUMO

In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes Ku's telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt nonhomologous end-joining (NHEJ), telomeric gene silencing, and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in vitro and in vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PAPERCLIP:


Assuntos
Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Saccharomyces cerevisiae/química , Deleção de Sequência , Telomerase/química , Telômero/genética
13.
RNA ; 17(2): 298-311, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21177376

RESUMO

Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.


Assuntos
Proteínas de Ligação a DNA/química , RNA/química , Ribonucleoproteínas/química , Proteínas de Saccharomyces cerevisiae/química , Telomerase/química , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Cinética , Modelos Biológicos , RNA/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo , Telômero/química , Telômero/metabolismo
14.
Proc Natl Acad Sci U S A ; 107(11): 4914-8, 2010 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-20194781

RESUMO

Telomerase is a ribonucleoprotein complex consisting of a protein reverse transcriptase (TERT) and an RNA subunit (TR). Telomerase normally adds telomeric DNA repeats to chromosome ends. Here, we engineer human and Tetrahymena cis-telomerase RNAs, each having a DNA primer covalently linked to its 3' end. We find that cis-telomerase synthesizes DNA with increased repeat addition processivity (RAP) but does not completely rescue the RAP defect of the L14A mutant of Tetrahymena TERT. This supports the conclusion that L14 has a function beyond binding the DNA primer and preventing dissociation during multiple rounds of repeat addition. By comparing cis-telomerases with various linker lengths, we find that a 5 nt linker gives near-optimal activity, indicating that the distance between the 3' end of the telomerase RNA pseudoknot region and the 5' end of the DNA primer is approximately 33 A. Even a 2 nt linker (approximately 14 A) gives some activity, indicating a high degree of conformational flexibility in this ribonucleoprotein complex. More generally, the cis system will allow structure-function relationships of each RNA molecule to be read directly through the reaction that it performs on itself.


Assuntos
Engenharia Genética , RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Telomerase/metabolismo , Telômero/metabolismo , Animais , Humanos , Mutação/genética , Tetrahymena/enzimologia
15.
Nucleic Acids Res ; 37(2): 354-67, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19043074

RESUMO

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.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Telomerase/antagonistas & inibidores , Proteínas de Ligação a Telômeros/metabolismo , Ensaios de Proteção de Nucleases , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Telômero/metabolismo , Proteínas de Ligação a Telômeros/química
16.
J Biol Chem ; 280(7): 5249-57, 2005 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-15591066

RESUMO

RecQ helicases play roles in telomere maintenance in cancerous human cells using the alternative lengthening of telomeres mechanism and in budding yeast lacking telomerase. Fission yeast lacking the catalytic subunit of telomerase (trt1(+)) up-regulate the expression of a previously uncharacterized sub-telomeric open reading frame as survivors emerge from crisis. Here we show that this open reading frame encodes a protein with homology to RecQ helicases such as the human Bloom's and Werner's syndrome proteins and that copies of the helicase gene are present on multiple chromosome ends. Characterization of the helicase transcript revealed a 7.6-kilobase RNA that was associated with polyribosomes, suggesting it is translated. A 3.6-kilobase domain of the helicase gene predicted to encode the region with catalytic activity was cloned, and both native and mutant forms of this domain were overexpressed in trt1(-) cells as they progressed through crisis. Overexpression of the native form caused cells to recover from crisis earlier than cells with a vector-only control, whereas overexpression of the mutant form caused delayed recovery from crisis. Taken together, the sequence homology, functional analysis, and site-directed mutagenesis indicate that the protein is likely a second fission yeast RecQ helicase (in addition to Rqh1) that participates in telomere metabolism during crisis. These results strengthen the notion that in multiple organisms RecQ helicases contribute to survival after telomere damage.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Homologia de Sequência de Aminoácidos , Telomerase/deficiência , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Cromossomos Fúngicos/enzimologia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Clonagem Molecular , DNA Helicases/genética , Regulação Fúngica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Polirribossomos/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RecQ Helicases , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Telomerase/genética , Telômero/enzimologia , Telômero/genética , Telômero/metabolismo
17.
Mol Biol Cell ; 14(12): 4794-804, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-13679509

RESUMO

Telomerase is an enzyme that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. The catalytic core of telomerase consists of the RNA subunit and a protein reverse transcriptase subunit, known as telomerase reverse transcriptase (TERT). It has previously been shown that both yeast and human telomerase can form dimers or multimers in which one RNA in the complex can influence the activity of another. To test the proposal that dimerization might be essential for telomerase activity, we sought to determine whether Tetrahymena thermophila telomerase is active as a dimer or a monomer. Recombinant Tetrahymena telomerase eluted from a gel filtration column at the size of a monomeric complex (one RNA plus one TERT), and those fractions showed processive telomerase activity. We were unable to detect dimerization of Tetrahymena telomerase by coprecipitation experiments, by using tags on either the TERT protein or telomerase RNA. Therefore, a majority, if not all, of the recombinant Tetrahymena telomerase in our reconstitution system is present as a monomeric complex. We were also unable to detect dimerization of native telomerase from mating and vegetative Tetrahymena cell extracts. These results demonstrate that Tetrahymena telomerase does not need to dimerize to be active and processive.


Assuntos
RNA de Protozoário/metabolismo , Telomerase/metabolismo , Tetrahymena/enzimologia , Animais , Northern Blotting , Clonagem Molecular , Proteínas de Ligação a DNA , Dimerização , Ativação Enzimática/fisiologia , Ligação Proteica , RNA de Protozoário/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Telomerase/genética , Tetrahymena/genética
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