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1.
Genes Dev ; 34(23-24): 1619-1636, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33122293

RESUMO

Mutations in the telomere-binding protein POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we define the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR interference and biotin-based proximity labeling, respectively. These screens reveal that replication stress is a major vulnerability in cells expressing mutant POT1, which manifests as increased telomere mitotic DNA synthesis at telomeres. Our study also unveils a role for the nuclear pore complex in resolving replication defects at telomeres. Depletion of nuclear pore complex subunits in the context of POT1 dysfunction increases DNA damage signaling, telomere fragility and sister chromatid exchanges. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.


Assuntos
Replicação do DNA/genética , Poro Nuclear/patologia , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Humanos , Mitose/genética , Mutação , Neoplasias/genética , Neoplasias/fisiopatologia , Complexo Shelterina , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismo
2.
Nucleic Acids Res ; 48(13): 7239-7251, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542379

RESUMO

Telomeres cap the ends of eukaryotic chromosomes and distinguish them from broken DNA ends to suppress DNA damage response, cell cycle arrest and genomic instability. Telomeres are elongated by telomerase to compensate for incomplete replication and nuclease degradation and to extend the proliferation potential of germ and stem cells and most cancers. However, telomeres in somatic cells gradually shorten with age, ultimately leading to cellular senescence. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and diverse symptoms including bone marrow failure, immunodeficiency, and neurodevelopmental defects. HHS is caused by germline mutations in telomerase subunits, factors essential for its biogenesis and recruitment to telomeres, and in the helicase RTEL1. While diverse phenotypes were associated with RTEL1 deficiency, the telomeric role of RTEL1 affected in HHS is yet unknown. Inducible ectopic expression of wild-type RTEL1 in patient fibroblasts rescued the cells, enabled telomerase-dependent telomere elongation and suppressed the abnormal cellular phenotypes, while silencing its expression resulted in gradual telomere shortening. Our observations reveal an essential role of the RTEL1 C-terminus in facilitating telomerase action at the telomeric 3' overhang. Thus, the common etiology for HHS is the compromised telomerase action, resulting in telomere shortening and reduced lifespan of telomerase positive cells.


Assuntos
DNA Helicases/metabolismo , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Deficiência Intelectual/genética , Microcefalia/genética , Homeostase do Telômero , Células Cultivadas , DNA Helicases/química , DNA Helicases/genética , Fibroblastos/metabolismo , Humanos , Domínios Proteicos , Telomerase/genética , Telomerase/metabolismo , Encurtamento do Telômero
3.
Proc Natl Acad Sci U S A ; 110(36): E3408-16, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23959892

RESUMO

Telomeres repress the DNA damage response at the natural chromosome ends to prevent cell-cycle arrest and maintain genome stability. Telomeres are elongated by telomerase in a tightly regulated manner to ensure a sufficient number of cell divisions throughout life, yet prevent unlimited cell division and cancer development. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and a broad range of pathologies, including bone marrow failure, immunodeficiency, and developmental defects. HHS-causing mutations have previously been found in telomerase and the shelterin component telomeric repeat binding factor 1 (TRF1)-interacting nuclear factor 2 (TIN2). We identified by whole-genome exome sequencing compound heterozygous mutations in four siblings affected with HHS, in the gene encoding the regulator of telomere elongation helicase 1 (RTEL1). Rtel1 was identified in mouse by its genetic association with telomere length. However, its mechanism of action and whether it regulates telomere length in human remained unknown. Lymphoblastoid cell lines obtained from a patient and from the healthy parents carrying heterozygous RTEL1 mutations displayed telomere shortening, fragility and fusion, and growth defects in culture. Ectopic expression of WT RTEL1 suppressed the telomere shortening and growth defect, confirming the causal role of the RTEL1 mutations in HHS and demonstrating the essential function of human RTEL1 in telomere protection and elongation. Finally, we show that human RTEL1 interacts with the shelterin protein TRF1, providing a potential recruitment mechanism of RTEL1 to telomeres.


Assuntos
DNA Helicases/genética , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Deficiência Intelectual/genética , Microcefalia/genética , Mutação , Telômero/genética , Animais , Sequência de Bases , Western Blotting , Proliferação de Células , Células Cultivadas , DNA Helicases/metabolismo , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Saúde da Família , Feminino , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Expressão Gênica , Instabilidade Genômica/genética , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Camundongos , Microcefalia/metabolismo , Microcefalia/patologia , Linhagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Encurtamento do Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo
4.
Nat Commun ; 15(1): 2210, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472229

RESUMO

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.


Assuntos
Proteínas Quinases , Transdução de Sinais , Proteínas Quinases/metabolismo , Fosforilação , Proteína de Replicação A/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Replicação do DNA , Dano ao DNA , DNA de Cadeia Simples , Reparo do DNA
5.
Chromosoma ; 121(6): 613-27, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23052336

RESUMO

In most eukaryotes, telomeres are composed of tandem arrays of species-specific DNA repeats ending with a G-rich 3' overhang. In budding yeast, Cdc13 binds this overhang and recruits Ten1-Stn1 and the telomerase protein Est1 to protect (cap) and elongate the telomeres, respectively. To dissect and study the various pathways employed to cap and maintain the telomere end, we engineered telomerase to incorporate Tetrahymena telomeric repeats (G4T2) onto the telomeres of the budding yeast Kluyveromyces lactis. These heterologous repeats caused telomere-telomere fusions, cell cycle arrest at G2/M, and severely reduced viability--the hallmarks of telomere uncapping. Fusing Cdc13 or Est1 to universal minicircle sequence binding protein (UMSBP), a small protein that binds the single-stranded G4T2 repeats, rescued the cell viability and restored telomere capping, but not telomerase-mediated telomere maintenance. Surprisingly, Cdc13-UMSBP-mediated telomere capping was dependent on the homologous recombination factor Rad52, while Est1-UMSBP was not. Thus, our results distinguish between two, redundant, telomere capping pathways.


Assuntos
Proteínas Fúngicas/metabolismo , Kluyveromyces/genética , Telômero/genética , Telômero/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Clonagem Molecular , Reparo do DNA por Junção de Extremidades/genética , Proteínas Fúngicas/genética , Kluyveromyces/citologia , Kluyveromyces/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Tetrahymena/genética
6.
Trends Cell Biol ; 31(10): 843-855, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34183232

RESUMO

The nucleus is a dynamic environment containing chromatin, membraneless organelles, and specialized molecular structures at the nuclear membrane. Within the spectrum of DNA repair activities are observations of increased mobility of damaged chromatin and the displacement of DNA lesions to specific nuclear environments. Here, we focus on the role that nuclear-specific filamentous actin plays in mobilizing damaged chromatin in response to DNA double-strand breaks and replication stress. We also examine nuclear pore complexes and promyelocytic leukemia-nuclear bodies as specialized platforms for homology-directed repair. The literature suggests an emerging model where specific types of DNA lesions are subjected to nuclear-derived forces that mobilize damaged chromatin and promote interaction with repair hubs to facilitate specialized repair reactions.


Assuntos
Condensados Biomoleculares , Cromatina , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Humanos , Corpos Nucleares
7.
Nat Cell Biol ; 22(12): 1460-1470, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33257806

RESUMO

Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.


Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Reparo do DNA/genética , Replicação do DNA/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Animais , Antineoplásicos/farmacologia , Carboplatina/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Polimerização , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
8.
Prog Biophys Mol Biol ; 147: 17-25, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30991055

RESUMO

DNA replication plays a central role in genome health. Deleterious alteration of replication dynamics, or "replication stress", is a key driver of genome instability and oncogenesis. The replication stress response is regulated by the ATR kinase, which functions to mitigate replication abnormalities through coordinated efforts that arrest the cell cycle and repair damaged replication forks. mTOR kinase regulates signaling networks that control cell growth and metabolism in response to environmental cues and cell stress. In this review, we discuss interconnectivity between the ATR and mTOR pathways, and provide putative mechanisms for mTOR engagement in DNA replication and the replication stress response. Finally, we describe how connectivity between mTOR and replication stress may be exploited for cancer therapy.


Assuntos
Replicação do DNA , Serina-Treonina Quinases TOR/metabolismo , Animais , Dano ao DNA , Humanos
9.
Nat Commun ; 10(1): 4224, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31530811

RESUMO

Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death in replication stressed cells results in distinct cellular outcomes depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and cancer genome evolution.


Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Replicação do DNA , Mitose , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Telômero/metabolismo , Morte Celular , Linhagem Celular Tumoral , Humanos , Neoplasias/genética , Neoplasias/fisiopatologia , Telômero/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
10.
Mol Cell Oncol ; 3(4): e1183743, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27652327

RESUMO

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations, including aneuploidy, during culture. Recently, we identified a replication stress-based mechanism leading to ongoing chromosomal instability in aneuploid hPSCs that may also operate during the initiation of instability in diploid cells.

11.
Cell Stem Cell ; 18(2): 253-61, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26669899

RESUMO

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations such as aneuploidy in culture. These aberrations progressively increase over time and may compromise the properties and clinical utility of the cells. The underlying mechanisms that drive initial genomic instability and its continued progression are largely unknown. Here, we show that aneuploid hPSCs undergo DNA replication stress, resulting in defective chromosome condensation and segregation. Aneuploid hPSCs show altered levels of actin cytoskeletal genes controlled by the transcription factor SRF, and overexpression of SRF rescues impaired chromosome condensation and segregation defects in aneuploid hPSCs. Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate during initiation of instability in diploid cells.


Assuntos
Aberrações Cromossômicas , Replicação do DNA , Instabilidade Genômica , Células-Tronco Pluripotentes/metabolismo , Estresse Fisiológico , Citoesqueleto de Actina/genética , Actinas/metabolismo , Anáfase , Aneuploidia , Segregação de Cromossomos/genética , Replicação do DNA/genética , Diploide , Regulação para Baixo/genética , Humanos , Metáfase , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Estresse Fisiológico/genética , Transcrição Gênica
12.
EMBO Mol Med ; 7(9): 1138-52, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26197802

RESUMO

Chromosomal instability in early cancer stages is caused by replication stress. One mechanism by which oncogene expression induces replication stress is to drive cell proliferation with insufficient nucleotide levels. Cancer development is driven by alterations in both genetic and environmental factors. Here, we investigated whether replication stress can be modulated by both genetic and non-genetic factors and whether the extent of replication stress affects the probability of neoplastic transformation. To do so, we studied the effect of folate, a micronutrient that is essential for nucleotide biosynthesis, on oncogene-induced tumorigenicity. We show that folate deficiency by itself leads to replication stress in a concentration-dependent manner. Folate deficiency significantly enhances oncogene-induced replication stress, leading to increased DNA damage and tumorigenicity in vitro. Importantly, oncogene-expressing cells, when grown under folate deficiency, exhibit a significantly increased frequency of tumor development in mice. These findings suggest that replication stress is a quantitative trait affected by both genetic and non-genetic factors and that the extent of replication stress plays an important role in cancer development.


Assuntos
Carcinogênese/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Ácido Fólico/metabolismo , Instabilidade Genômica/efeitos dos fármacos , Oncogenes/efeitos dos fármacos , Animais , Camundongos
13.
PLoS One ; 4(5): e5666, 2009 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-19461895

RESUMO

BACKGROUND: Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency. METHODOLOGY/PRINCIPAL FINDINGS: We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3' overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types. CONCLUSIONS/SIGNIFICANCE: Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease.


Assuntos
Disceratose Congênita/fisiopatologia , Telômero/metabolismo , Adulto , Células Sanguíneas/metabolismo , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Disceratose Congênita/genética , Disceratose Congênita/patologia , Ativação Enzimática , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Masculino , Mutação/genética , Linhagem , RNA/metabolismo , Irmãos , Pele/enzimologia , Pele/patologia , Síndrome , Telomerase/metabolismo
14.
Neurodegener Dis ; 2(1): 16-27, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16908999

RESUMO

Peripheral acetylcholine levels notably control the synthesis in macrophages of pro-inflammatory cytokines; however, it remains unclear whether this peripheral regulatory pathway affects central nervous system neurons. To explore the interrelationship between neuronal cholinergic homeostasis and peripheral inflammatory responses in primates, we used spinal cord sections from cynomolgus monkeys after 7 days oral or intravenous treatment with Monarsen oligonucleotide. Monarsen is an antisense oligonucleotide 3'-protected by 2'-oxymethylation, which was proved to induce selective destruction of the stress-induced acetylcholinesterase splice variant AChE-R mRNA. Handling stress predictably suppressed neuronal choline acetyl transferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in all treated monkeys. In Monarsen-treated animals, we further observed suppression of stress-induced increases in plasma AChE activities. Corresponding decreases in AChE-R mRNA were seen in spinal cord neurons, associated with parallel decline patterns in the mRNA encoding for the splice factor SC35 (the levels of which co-increase with those of AChE-R) as well as in the neuronal pro-inflammatory interleukins IL-1beta and IL-6. The antisense effects showed direct dose dependence and were inversely associated with neuronal cell size. These findings suggest a causal association between neuronal cholinergic allostasis and inflammatory reactions in primates and support the peripheral use of RNA-targeted intervention with AChE-R accumulation for the management of both stress and inflammatory responses.


Assuntos
Mielite/metabolismo , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/antagonistas & inibidores , Medula Espinal/metabolismo , Estresse Fisiológico/metabolismo , Acetilcolina/metabolismo , Acetilcolinesterase/biossíntese , Acetilcolinesterase/genética , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Animais , Colina O-Acetiltransferase/efeitos dos fármacos , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Macaca fascicularis , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Mielite/tratamento farmacológico , Mielite/fisiopatologia , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/uso terapêutico , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiopatologia , Estresse Fisiológico/complicações , Estresse Fisiológico/fisiopatologia , Resultado do Tratamento , Proteínas Vesiculares de Transporte de Acetilcolina/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismo
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