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1.
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
2.
STAR Protoc ; 3(1): 101212, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35265860

RESUMO

Alternative lengthening of telomeres (ALT) is a homologous recombination-based telomere maintenance mechanism. It is active in approximately 10-15% of cancers. We present a DNA-fiber protocol, combining YOYO-1 staining of genomic DNA, telomere fluorescence in situ hybridization (FISH), and EdU labeling of nascent DNA, to measure telomere extension events in ALT cancer cells. The protocol can be used to delineate ALT-mediated telomere extension. For complete details on the use and execution of this protocol, please refer to Barroso-Gonzalez et al. (2021).


Assuntos
Neoplasias , Imagem Individual de Molécula , DNA/genética , Hibridização in Situ Fluorescente/métodos , Neoplasias/genética , Telômero/genética , Homeostase do Telômero/genética
3.
Cell Rep ; 37(10): 110088, 2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34879271

RESUMO

Alternative lengthening of telomeres (ALT) is a telomere-elongation mechanism observed in ∼15% of cancer subtypes. Current models indicate that ALT is mediated by homology-directed repair mechanisms. By disrupting MSH6 gene expression, we show that the deficiency of MutSα (MSH2/MSH6) DNA mismatch repair complex causes striking telomere hyperextension. Mechanistically, we show MutSα is specifically recruited to telomeres in ALT cells by associating with the proliferating-cell nuclear antigen (PCNA) subunit of the ALT telomere replisome. We also provide evidence that MutSα counteracts Bloom (BLM) helicase, which adopts a crucial role in stabilizing hyper-extended telomeres and maintaining the survival of MutSα-deficient ALT cancer cells. Lastly, we propose a model in which MutSα deficiency impairs heteroduplex rejection, leading to premature initiation of telomere DNA synthesis that coincides with an accumulation of telomere variant repeats (TVRs). These findings provide evidence that the MutSα DNA mismatch repair complex acts to restrain unwarranted ALT.


Assuntos
DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Neoplasias/enzimologia , Ácidos Nucleicos Heteroduplexes/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Células HeLa , Humanos , Modelos Genéticos , Proteína 2 Homóloga a MutS/genética , Neoplasias/genética , Neoplasias/patologia , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Telômero/genética
4.
Sci Rep ; 11(1): 13195, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162976

RESUMO

Chromatin organization within the nuclear volume is essential to regulate many aspects of its function and to safeguard its integrity. A key player in this spatial scattering of chromosomes is the nuclear envelope (NE). The NE tethers large chromatin domains through interaction with the nuclear lamina and other associated proteins. This organization is perturbed in cells from Hutchinson-Gilford progeria syndrome (HGPS), a genetic disorder characterized by premature aging features. Here, we show that HGPS-related lamina defects trigger an altered 3D telomere organization with increased contact sites between telomeres and the nuclear lamina, and an altered telomeric chromatin state. The genome-wide replication timing signature of these cells is perturbed, with a shift to earlier replication for regions that normally replicate late. As a consequence, we detected a higher density of replication forks traveling simultaneously on DNA fibers, which relies on limiting cellular dNTP pools to support processive DNA synthesis. Remarkably, increasing dNTP levels in HGPS cells rescued fragile telomeres, and improved the replicative capacity of the cells. Our work highlights a functional connection between NE dysfunction and telomere homeostasis in the context of premature aging.


Assuntos
Cromatina/ultraestrutura , Desoxirribonucleotídeos/metabolismo , Lamina Tipo A/fisiologia , Lâmina Nuclear/patologia , Progéria/genética , Homeostase do Telômero/genética , Telômero/patologia , Adulto , Animais , Células Cultivadas , Senescência Celular/genética , Dano ao DNA , Replicação do DNA , Fibroblastos , Genes Reporter , Proteínas de Fluorescência Verde , Código das Histonas , Humanos , Recém-Nascido , Lamina Tipo A/análise , Lamina Tipo A/deficiência , Lamina Tipo A/genética , Lamina Tipo B/análise , Camundongos , Camundongos Knockout , Progéria/patologia , Proteínas Recombinantes de Fusão/metabolismo , Pele/patologia
5.
Front Cell Dev Biol ; 8: 493, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612998

RESUMO

Telomeres are repetitive regions of DNA bound by specialized proteins at the termini of linear chromosomes that prevent the natural chromosome ends from being recognized as DNA double strand breaks. Telomeric DNA is gradually eroded with each round of cell division, resulting in the accumulation of critically short or dysfunctional telomeres that eventually trigger cellular senescence. Consequently, telomere length is indicative of the proliferative capacity of a cell. Multiple methods exist to measure telomere length and telomere content, but a simple and reliable technique to accurately measure individual telomere lengths is currently lacking. We have developed the Telomere length Combing Assay (TCA) to measure telomere length on stretched DNA fibers. We used TCA to measure telomere erosion in primary human fibroblasts, and to detect telomere lengthening in response to activation of telomere maintenance pathways. TCA was also used to accurately measure telomere length in healthy individuals, and to identify critically short telomeres in patients with telomere biology disorders. TCA is performed on isolated DNA, negating the need for cycling cells. TCA is amenable to semi-automated image analysis, and can be fully automated using the Genomic Vision molecular combing platform. This not only precludes sampling bias, but also provides the potential for high-throughput applications and clinical development. TCA is a simple and versatile technique to measure the distribution of individual telomere lengths in a cell population, offering improved accuracy, and more detailed biological insight for telomere length measurement applications.

6.
Nat Commun ; 10(1): 5345, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31745078

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

7.
Nat Commun ; 10(1): 2252, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138797

RESUMO

The collapse of stalled replication forks is a major driver of genomic instability. Several committed mechanisms exist to resolve replication stress. These pathways are particularly pertinent at telomeres. Cancer cells that use Alternative Lengthening of Telomeres (ALT) display heightened levels of telomere-specific replication stress, and co-opt stalled replication forks as substrates for break-induced telomere synthesis. FANCM is a DNA translocase that can form independent functional interactions with the BLM-TOP3A-RMI (BTR) complex and the Fanconi anemia (FA) core complex. Here, we demonstrate that FANCM depletion provokes ALT activity, evident by increased break-induced telomere synthesis, and the induction of ALT biomarkers. FANCM-mediated attenuation of ALT requires its inherent DNA translocase activity and interaction with the BTR complex, but does not require the FA core complex, indicative of FANCM functioning to restrain excessive ALT activity by ameliorating replication stress at telomeres. Synthetic inhibition of FANCM-BTR complex formation is selectively toxic to ALT cancer cells.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Linhagem Celular Tumoral , Replicação do DNA , Células HCT116 , Células HEK293 , Células HeLa , Humanos
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