RESUMO
Telomerase is the essential reverse transcriptase required for linear chromosome maintenance in most eukaryotes. Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication. The template for telomerase reverse transcriptase is within the RNA subunit of the ribonucleoprotein complex, which in cells contains additional telomerase holoenzyme proteins that assemble the active ribonucleoprotein and promote its function at telomeres. Telomerase is distinct among polymerases in its reiterative reuse of an internal template. The template is precisely defined, processively copied, and regenerated by release of single-stranded product DNA. New specificities of nucleic acid handling that underlie the catalytic cycle of repeat synthesis derive from both active site specialization and new motif elaborations in protein and RNA subunits. Studies of telomerase provide unique insights into cellular requirements for genome stability, tissue renewal, and tumorigenesis as well as new perspectives on dynamic ribonucleoprotein machines.
Assuntos
Replicação do DNA , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Telomerase/metabolismo , Telômero/enzimologia , Animais , Domínio Catalítico , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Humanos , Repetições de Microssatélites , Conformação de Ácido Nucleico , Oxytricha/genética , Oxytricha/metabolismo , RNA/genética , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telômero/química , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismoRESUMO
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 , Complexo Shelterina , Telomerase/química , Telômero/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Telomeric repeat binding factor 1 (TRF1) is essential to the maintenance of telomere chromatin structure and integrity. However, how telomere integrity is maintained, especially in response to damage, remains poorly understood. Here, we identify Nek7, a member of the Never in Mitosis Gene A (NIMA) kinase family, as a regulator of telomere integrity. Nek7 is recruited to telomeres and stabilizes TRF1 at telomeres after damage in an ATM activation-dependent manner. Nek7 deficiency leads to telomere aberrations, long-lasting γH2AX and 53BP1 foci, and augmented cell death upon oxidative telomeric DNA damage. Mechanistically, Nek7 interacts with and phosphorylates TRF1 on Ser114, which prevents TRF1 from binding to Fbx4, an Skp1-Cul1-F box E3 ligase subunit, thereby alleviating proteasomal degradation of TRF1, leading to a stable association of TRF1 with Tin2 to form a shelterin complex. Our data reveal a mechanism of efficient protection of telomeres from damage through Nek7-dependent stabilization of TRF1.
Assuntos
Dano ao DNA , Quinases Relacionadas a NIMA/metabolismo , Estresse Oxidativo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/enzimologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Quinases Relacionadas a NIMA/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Interferência de RNA , Complexo Shelterina , Telômero/genética , Telômero/efeitos da radiação , Proteínas de Ligação a Telômeros/genética , Fatores de Tempo , Transfecção , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , UbiquitinaçãoRESUMO
The role of telomerase reverse transcriptase (TERT) induction and telomere maintenance in carcinogenesis including cervical cancer (CC) pathogenesis has been well established. However, it remains unclear whether they affect infection of high-risk human papillomavirus (hrHPV), an initiating event for CC development. Similarly, genetic variants at the TERT locus are shown to be associated with susceptibility to CC, but it is unclear whether these SNPs modify the risk for cervical HPV infection. Here we show that in CC-derived HeLa cells, TERT overexpression inhibits, while its depletion upregulates expression of Syndecan-1 (SDC-1), a key component for HPV entry receptors. The TCGA cohort of CC analyses reveals an inverse correlation between TERT and SDC-1 expression (R = -0.23, P = 0.001). We further recruited 1330 females (520 non-HPV and 810 hrHPV-infected) without CC or high-grade cervical intraepithelial neoplasia to analyze telomeres in cervical epithelial cells and SNPs at rs2736098, rs2736100 and rs2736108, previously identified TERT SNPs for CC risk. Non-infected females exhibited age-related telomere shortening in cervical epithelial cells and their telomeres were significantly longer than those in hrHPV-infected group (1.31 ± 0.62 vs 1.19 ± 0.48, P < 0.001). There were no differences in rs2736098 and rs2736100 genotypes, but non-infected individuals had significantly a higher C-allele frequency (associated with higher TERT expression) while lower T-allele levels at rs2736108 compared with those in the hrHPV group (P = 0.020). Collectively, appropriate telomere maintenance and TERT expression in normal cervical cells may prevent CC by modulating hrHPV infection predisposition, although they are required for CC development and progression.
Assuntos
Predisposição Genética para Doença/genética , Infecções por Papillomavirus/genética , Telomerase/genética , Telômero/genética , Neoplasias do Colo do Útero/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Epitélio/metabolismo , Epitélio/virologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Polimorfismo de Nucleotídeo Único , Telomerase/metabolismo , Telômero/enzimologia , Neoplasias do Colo do Útero/complicações , Neoplasias do Colo do Útero/metabolismo , Adulto JovemRESUMO
Telomere maintenance by telomerase is essential for continuous proliferation of human cells and is vital for the survival of stem cells and 90% of cancer cells. To compensate for telomeric DNA lost during DNA replication, telomerase processively adds GGTTAG repeats to chromosome ends by copying the template region within its RNA subunit. Between repeat additions, the RNA template must be recycled. How telomerase remains associated with substrate DNA during this critical translocation step remains unknown. Using a single-molecule telomerase activity assay utilizing high-resolution optical tweezers, we demonstrate that stable substrate DNA binding at an anchor site within telomerase facilitates the processive synthesis of telomeric repeats. The product DNA synthesized by telomerase can be recaptured by the anchor site or fold into G-quadruplex structures. Our results provide detailed mechanistic insights into telomerase catalysis, a process of critical importance in aging and cancer.
Assuntos
DNA/metabolismo , Quadruplex G , RNA/metabolismo , Telomerase/metabolismo , Telômero/enzimologia , Biocatálise , DNA/genética , Replicação do DNA , Expressão Gênica , Células HEK293 , Humanos , Pinças Ópticas , RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Telomerase/genética , Telômero/ultraestruturaRESUMO
The telomeric complex has been analyzed in detail for its role in regulating telomere protection and telomere length. Now, modern genome-editing techniques in human embryonic stem cells reveal TPP1 as the essential recruitment factor for telomerase, with additional functions in telomerase activation and definition of telomere length homeostasis.
Assuntos
Telomerase/metabolismo , Homeostase do Telômero/genética , Telômero/enzimologia , Humanos , Complexo Shelterina , Proteínas de Ligação a TelômerosRESUMO
Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.
Assuntos
Telomerase/metabolismo , Homeostase do Telômero/genética , Telômero/enzimologia , Células-Tronco Embrionárias , Ativação Enzimática/genética , Técnicas de Inativação de Genes , Teste de Complementação Genética , Humanos , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Complexo Shelterina , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP-ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage-responsive E3 ligase RNF8 conjugates K63-linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36-containing deubiquitinase complex. We show that K63-linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM-mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle-regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.
Assuntos
Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Tanquirases/metabolismo , Telômero/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Enzimas Desubiquitinantes , Células HeLa , Humanos , Telômero/enzimologia , Ubiquitina-Proteína LigasesRESUMO
The maintenance of telomere length is critical to longevity and survival. Specifically, the failure to properly replicate, resect, and/or form appropriate telomeric structures drives telomere shortening and, in turn, genomic instability. The endonuclease CtIP is a DNA repair protein that is well-known to promote genome stability through the resection of endogenous DNA double-stranded breaks. Here, we describe a novel role for CtIP. We show that in the absence of CtIP, human telomeres shorten rapidly to non-viable lengths. This telomere dysfunction results in an accumulation of fusions, breaks, and frank telomere loss. Additionally, CtIP suppresses the generation of circular, extrachromosomal telomeric DNA. These latter structures appear to arise from arrested DNA replication forks that accumulate in the absence of CtIP. Hence, CtIP is required for faithful replication through telomeres via its roles at stalled replication tracts. Our findings demonstrate a new role for CtIP as a protector of human telomere integrity.
Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Proteínas Nucleares/metabolismo , Encurtamento do Telômero/genética , Telômero/metabolismo , Reparo do DNA por Junção de Extremidades , Replicação do DNA , DNA Circular/metabolismo , Endodesoxirribonucleases , Humanos , Telômero/enzimologia , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFß-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição da Família Snail/genética , Homeostase do Telômero/genética , Telômero/genética , Animais , Linhagem Celular , Células Cultivadas , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica/métodos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos Knockout , Células-Tronco Neoplásicas/metabolismo , RNA Longo não Codificante/genética , Fatores de Transcrição da Família Snail/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/enzimologiaRESUMO
BACKGROUND: Human telomerase reverse transcriptase (hTERT) and its components play a significant role in cancer progression, but recent data demonstrated that telomeres and telomerase alterations could be found in other diseases; increasing evidence suggests a key role of this enzyme in the fields of hepatobiliary and pancreatic diseases. DATA SOURCES: We performed a PubMed search with the following keywords: telomerase, hepatocellular carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma by December 2019. We reviewed the relevant publications that analyzed the correlation between telomerase activity and hepatobiliary and pancreatic diseases. RESULTS: Telomerase reactivation plays a significant role in the development and progression of hepatobiliary and pancreatic tumors and could be used as a diagnostic biomarker for hepatobiliary and pancreatic cancers, as a predictor for prognosis and a promising therapeutic target. CONCLUSIONS: Our review summarized the evidence about the critical role of hTERT in cancerous and precancerous lesions of the alteration and its activity in hepatobiliary and pancreatic diseases.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Sistema Digestório/enzimologia , Telomerase/metabolismo , Homeostase do Telômero , Telômero/enzimologia , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Animais , Neoplasias dos Ductos Biliares/enzimologia , Neoplasias dos Ductos Biliares/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Colangiocarcinoma/enzimologia , Colangiocarcinoma/genética , Neoplasias do Sistema Digestório/genética , Ativação Enzimática , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Prognóstico , Telomerase/genética , Telômero/metabolismoRESUMO
In fission yeast, the DNA damage sensor kinases Tel1(ATM) and Rad3(ATR) exist at telomeres and are required for telomere maintenance, but the biological role they play at telomeres is not known. Here we show that the telomere protein Ccq1 is phosphorylated at Thr 93 (threonine residue at amino acid 93) by Tel1(ATM) and Rad3(ATR) both in vitro and in vivo. A ccq1 mutant in which alanine was substituted for Thr 93 failed to recruit telomerase to telomeres and showed gradual shortening of telomeres. These results indicate that the direct phosphorylation of Ccq1 Thr 93 by Tel1 and Rad3 is involved in the recruitment of telomerase to elongate telomeres.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Telomerase/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Mutação , Fosforilação , Proteínas Quinases/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Telômero/enzimologiaRESUMO
Chromosome stability relies on an adequate length and complete replication of telomeres, the physical ends of chromosomes. Telomeres are composed of short direct repeat DNA and the associated nucleoprotein complex is essential for providing end-stability. In addition, the so-called end-replication problem of the conventional replication requires that telomeres be elongated by a special mechanism which, in virtually all organisms, is based by a reverse transcriptase, called telomerase. Although, at the conceptual level, telomere functions are highly similar in most organisms, the telomeric nucleoprotein composition appears to diverge significantly, in particular if it is compared between mammalian and budding yeast cells. However, over the last years, the CST complex has emerged as a central hub for telomere replication in most systems. Composed of three proteins, it is related to the highly conserved replication protein A complex, and in all systems studied, it coordinates telomerase-based telomere elongation with lagging-strand DNA synthesis. In budding yeast, the Cdc13 protein of this complex also is essential for telomerase recruitment and this specialisation is accompanied by additional regulatory adaptations. Based on recent results obtained in yeast, here, we review these issues and present an updated telomere replication hypothesis. We speculate that the similarities between systems far outweigh the differences, once we detach ourselves from the historic descriptions of the mechanisms in the various organisms.
Assuntos
Instabilidade Cromossômica/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Replicação do DNA/genética , Humanos , Modelos Genéticos , Mutação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telomerase/metabolismo , Telômero/enzimologia , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Oxidative DNA damage triggers telomere erosion and cellular senescence. However, how repair is initiated at telomeres is largely unknown. Here, we found unlike PARP1-mediated Poly-ADP-Ribosylation (PARylation) at genomic damage sites, PARylation at telomeres is mainly dependent on tankyrase1 (TNKS1). TNKS1 is recruited to damaged telomeres via its interaction with TRF1, which subsequently facilitates the PARylation of TRF1 after damage. TNKS inhibition abolishes the recruitment of the repair proteins XRCC1 and polymerase ß at damaged telomeres, while the PARP1/2 inhibitor only has such an effect at non-telomeric damage sites. The ANK domain of TNKS1 is essential for the telomeric damage response and TRF1 interaction. Mutation of the tankyrase-binding motif (TBM) on TRF1 (13R/18G to AA) disrupts its interaction with TNKS1 concomitant recruitment of TNKS1 and repair proteins after damage. Either TNKS1 inhibition or TBM mutated TRF1 expression markedly sensitizes cells to telomere oxidative damage as well as XRCC1 inhibition. Together, our data reveal a novel role of TNKS1 in facilitating SSBR at damaged telomeres through PARylation of TRF1, thereby protecting genome stability and cell viability.
Assuntos
Reparo do DNA , Tanquirases/metabolismo , Telômero/enzimologia , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular , Sobrevivência Celular , Dano ao DNA , Instabilidade Genômica , HumanosRESUMO
AURKB (Aurora Kinase B) is a serine/threonine kinase better known for its role at the mitotic kinetochore during chromosome segregation. Here, we demonstrate that AURKB localizes to the telomeres in mouse embryonic stem cells, where it interacts with the essential telomere protein TERF1. Loss of AURKB function affects TERF1 telomere binding and results in aberrant telomere structure. In vitro kinase experiments successfully identified Serine 404 on TERF1 as a putative AURKB target site. Importantly, in vivo overexpression of S404-TERF1 mutants results in fragile telomere formation. These findings demonstrate that AURKB is an important regulator of telomere structural integrity.
Assuntos
Aurora Quinase B/metabolismo , Telômero/enzimologia , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Animais , Aurora Quinase B/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/enzimologia , Humanos , Interfase/genética , Camundongos , Mitose/genética , Mutação , Ligação Proteica , Telômero/ultraestrutura , Proteína 1 de Ligação a Repetições Teloméricas/química , Proteína 1 de Ligação a Repetições Teloméricas/genéticaRESUMO
Werner syndrome (WS) is a progeroid-like syndrome caused by WRN gene mutations. WS cells exhibit shorter telomere length compared to normal cells, but it is not fully understood how WRN deficiency leads directly to telomere dysfunction. By generating localized telomere-specific DNA damage in a real-time fashion and a dose-dependent manner, we found that the damage response of WRN at telomeres relies on its RQC domain, which is different from the canonical damage response at genomic sites via its HRDC domain. We showed that in addition to steady state telomere erosion, WRN depleted cells are also sensitive to telomeric damage. WRN responds to site-specific telomeric damage via its RQC domain, interacting at Lysine 1016 and Phenylalanine1037 with the N-terminal acidic domain of the telomere shelterin protein TRF1 and demonstrating a novel mechanism for WRN's role in telomere protection. We also found that tankyrase1-mediated poly-ADP-ribosylation of TRF1 is important for both the interaction between WRN and TRF1 and the damage recruitment of WRN to telomeres. Mutations of potential tankyrase1 ADP-ribosylation sites within the RGCADG motif of TRF1 strongly diminish the interaction with WRN and the damage response of WRN only at telomeres. Taken together, our results reveal a novel mechanism as to how WRN protects telomere integrity from damage and telomere erosion.
Assuntos
Reparo do DNA , Tanquirases/metabolismo , Telômero/enzimologia , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Helicase da Síndrome de Werner/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Dano ao DNA , Humanos , Oxirredução , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/metabolismo , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/química , Helicase da Síndrome de Werner/químicaRESUMO
Telomeres are highly susceptible to oxidative DNA damage, which if left unrepaired can lead to dysregulation of telomere length homeostasis. Here we employed single molecule FRET, single molecule pull-down and biochemical analysis to investigate how the most common oxidative DNA lesions, 8-oxoguanine (8oxoG) and thymine glycol (Tg), regulate the structural properties of telomeric DNA and telomerase extension activity. In contrast to 8oxoG which disrupts the telomeric DNA structure, Tg exhibits substantially reduced perturbation of G-quadruplex folding. As a result, 8oxoG induces high accessibility, whereas Tg retains limited accessibility, of telomeric G-quadruplex DNA to complementary single stranded DNA and to telomere binding protein POT1. Surprisingly, the Tg lesion stimulates telomerase loading and activity to a similar degree as an 8oxoG lesion. We demonstrate that this unexpected stimulation arises from Tg-induced conformational alterations and dynamics in telomeric DNA. Despite impacting structure by different mechanisms, both 8oxoG and Tg enhance telomerase binding and extension activity to the same degree, potentially contributing to oncogenesis.
Assuntos
Dano ao DNA , Estresse Oxidativo , Telomerase/metabolismo , Telômero/enzimologia , Sequência de Bases , DNA/química , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Quadruplex G , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Complexo Shelterina , Telômero/genética , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Timina/análogos & derivados , Timina/química , Timina/metabolismoRESUMO
Cancer cells maintain telomere length equilibrium to avoid senescence and apoptosis induced by short telomeres, which trigger the DNA damage response. Limiting the potential for telomere maintenance in cancer cells has been long been proposed as a therapeutic target. Using an unbiased shRNA screen targeting known kinases, we identified bromodomain-containing protein 4 (BRD4) as a telomere length regulator. Four independent BRD4 inhibitors blocked telomere elongation, in a dose-dependent manner, in mouse cells overexpressing telomerase. Long-term treatment with BRD4 inhibitors caused telomere shortening in both mouse and human cells, suggesting BRD4 plays a role in telomere maintenance in vivo. Telomerase enzymatic activity was not directly affected by BRD4 inhibition. BRD4 is in clinical trials for a number of cancers, but its effects on telomere maintenance have not been previously investigated.
Assuntos
Proteínas Nucleares/genética , Homeostase do Telômero/genética , Encurtamento do Telômero/genética , Fatores de Transcrição/genética , Acetanilidas/farmacologia , Animais , Azepinas/farmacologia , Southern Blotting , Proteínas de Ciclo Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HeLa , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Hibridização in Situ Fluorescente , Camundongos , Morfolinas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Pironas/farmacologia , Interferência de RNA , Telomerase/genética , Telomerase/metabolismo , Telômero/efeitos dos fármacos , Telômero/enzimologia , Telômero/genética , Homeostase do Telômero/efeitos dos fármacos , Encurtamento do Telômero/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Triazóis/farmacologiaRESUMO
The Fanconi anemia protein SLX4 assembles a genome and telomere maintenance toolkit, consisting of the nucleases SLX1, MUS81 and XPF. Although it is known that SLX4 acts as a scaffold for building this complex, the molecular basis underlying this function of SLX4 remains unclear. Here, we report that functioning of SLX4 is dependent on its dimerization via an oligomerization motif called the BTB domain. We solved the crystal structure of the SLX4BTB dimer, identifying key contacts (F681 and F708) that mediate dimerization. Disruption of BTB dimerization abrogates nuclear foci formation and telomeric localization of not only SLX4 but also of its associated nucleases. Furthermore, dimerization-deficient SLX4 mutants cause defective cellular response to DNA interstrand crosslinking agent and telomere maintenance, underscoring the contribution of BTB domain-mediated dimerization of SLX4 in genome and telomere maintenance.
Assuntos
Endonucleases/metabolismo , Recombinases/química , Linhagem Celular , Interações Hidrofóbicas e Hidrofílicas , Mitomicina/toxicidade , Domínios Proteicos , Multimerização Proteica , Recombinases/metabolismo , Telômero/enzimologia , Telômero/ultraestruturaRESUMO
Damaged DNA can be repaired by removal and re-synthesis of up to 30 nucleotides during base or nucleotide excision repair. An important question is what happens when many more nucleotides are removed, resulting in long single-stranded DNA (ssDNA) lesions. Such lesions appear on chromosomes during telomere damage, double strand break repair or after the UV damage of stationary phase cells. Here, we show that long single-stranded lesions, formed at dysfunctional telomeres in budding yeast, are re-synthesized when cells are removed from the telomere-damaging environment. This process requires Pol32, an accessory factor of Polymerase δ. However, re-synthesis takes place even when the telomere-damaging conditions persist, in which case the accessory factors of both polymerases δ and ε are required, and surprisingly, salt. Salt added to the medium facilitates the DNA synthesis, independently of the osmotic stress responses. These results provide unexpected insights into the DNA metabolism and challenge the current view on cellular responses to telomere dysfunction.