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1.
EMBO Rep ; 24(10): e56724, 2023 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-37664992

RESUMO

The centrosome is a cytoplasmic organelle with roles in microtubule organization that has also been proposed to act as a hub for cellular signaling. Some centrosomal components are required for full activation of the DNA damage response. However, whether the centrosome regulates specific DNA repair pathways is not known. Here, we show that centrosome presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection. Cells lacking centrosomes or depleted for CEP170 are, consequently, hypersensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting that changes in CEP170 can act as a mutation driver but could also be targeted to improve current oncological treatments.

2.
Nat Commun ; 12(1): 5512, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535666

RESUMO

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Hibridização de Ácido Nucleico , Edição de RNA , RNA/metabolismo , Adenosina Desaminase/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , DNA Helicases/metabolismo , Deleção de Genes , Genes Reporter , Instabilidade Genômica , Proteínas de Fluorescência Verde/metabolismo , Recombinação Homóloga/genética , Humanos , Enzimas Multifuncionais/metabolismo , Estabilidade Proteica , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/genética , Proteína de Replicação A/metabolismo
3.
Cancers (Basel) ; 14(1)2021 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-35008208

RESUMO

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are now a first-line maintenance treatment in ovarian cancer and have been approved in other cancer types, including breast, pancreatic and prostate. Despite their efficacy, and as is the case for other targeted therapies, resistance to PARPi has been reported clinically and is generating a growing patient population of unmet clinical need. Here, we discuss the mechanisms of resistance that have been described in pre-clinical models and focus on those that have been already identified in the clinic, highlighting the key challenges to fully characterise the clinical landscape of PARPi resistance and proposing ways of preventing and overcoming it.

4.
RNA ; 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33298529

RESUMO

In order to survive to the exposure of DNA damaging agents, cells activate a complex response that coordinates the cellular metabolism, cell cycle progression and DNA repair. Among many other events, recent evidence has described global changes in mRNA splicing in cells treated with genotoxic agents. Here, we explore further this DNA damage-dependent alternative splicing. Indeed, we show that both the splicing factor SF3B2 and the repair protein CtIP contribute to the global pattern of splicing both in cells treated or not to DNA damaging agents. Additionally, we focus on a specific DNA damage- and CtIP-dependent alternative splicing event of the helicase PIF1 and explore its relevance for the survival of cells upon exposure to ionizing radiation. Indeed, we described how the nuclear, active form of PIF1 is substituted by a splicing variant, named vPIF1, in a fashion that requires both the presence of DNA damage and CtIP. Interestingly, timely expression of vPIF1 is required for optimal survival to exposure to DNA damaging agents, but early expression of this isoform delays early events of the DNA damage response. On the contrary, expression of the full length PIF1 facilitates those early events, but increases the sensitivity to DNA damaging agents if the expression is maintained long-term.

5.
DNA Repair (Amst) ; 81: 102662, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31303544

RESUMO

DNA end resection is a critical step in the repair of DNA double strand breaks. It controls the way the lesion is going to be repaired, thus its regulation has a great importance in maintaining genomic stability. In this review, we focus in recent discoveries in the field that point to a modulation of resection by RNA molecules and RNA-related proteins. Moreover, we aim to reconcile contradictory reports on the positive or negative effect of DNA:RNA hybrids in the resection process.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Reparo do DNA por Junção de Extremidades , Eucariotos/genética , Eucariotos/metabolismo , Humanos , Hibridização de Ácido Nucleico , Reparo de DNA por Recombinação
6.
Nat Commun ; 10(1): 2135, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086179

RESUMO

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Recombinação Homóloga , Proteína de Replicação A/metabolismo , DNA/genética , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Ribonuclease H/metabolismo
7.
Adv Protein Chem Struct Biol ; 115: 95-134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30798939

RESUMO

Broken chromosomes are among the most complex and more difficult to repair DNA lesions. The loss of the continuity of the DNA molecule presents a challenge to the cells, thus the repair of DNA double strand breaks might lead to genomic alterations. Indeed, to minimize this threat to genomic integrity, different DNA repair pathways can act on a broken chromosome. The balance between them is tightly controlled, and it heavily depends on global and local cellular cues. In this chapter, we review our current understanding on the repair of DNA double strand breaks and focus in the regulation of the balance between alternative pathways. Most of this modulation takes place at the level of DNA end resection. Here, we focus mostly on the local signals that control the repair pathway choice, as the global cues have been extensively reviewed recently. We described epigenetic marks that either facilitate or inhibit DNA resection and homologous recombination, from histone marks and chromatin remodelers to non-coding RNA and RNA-related factors.


Assuntos
Quebra Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , DNA/genética , Humanos
9.
Cell Rep ; 24(12): 3262-3273.e4, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30232007

RESUMO

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression.


Assuntos
DNA Helicases/metabolismo , Quadruplex G , Reparo de DNA por Recombinação , Linhagem Celular Tumoral , DNA/química , DNA/genética , DNA/metabolismo , DNA Helicases/genética , Humanos , Ligação Proteica
10.
DNA Repair (Amst) ; 66-67: 11-23, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29705135

RESUMO

The appropriate repair of DNA double strand breaks is critical for genome maintenance. Thus, several cellular pathways collaborate to orchestrate a coordinated response. These include the repair of the breaks, which could be achieved by different mechanisms. A key protein involved in the regulation of the repair of broken chromosomes is CtIP. Here, we have found new partners of CtIP involved in the regulation of DNA break repair through affecting DNA end resection. We focus on the splicing complex SF3B and show that its depletion impairs DNA end resection and hampers homologous recombination. Functionally, SF3B controls CtIP function at, as least, two levels: by affecting CtIP mRNA levels and controlling CtIP recruitment to DNA breaks, in a way that requires ATM-mediated phosphorylation of SF3B2 at serine 289. Indeed, overexpression of CtIP rescues the resection defect caused by SF3B downregulation. Strikingly, other SF3B depletion phenotypes, such as impaired homologous recombination or cellular sensitivity to DNA damaging agents, are independent of CtIP levels, suggesting a more general role of SF3B in controlling the response to chromosome breaks.


Assuntos
Proteínas de Transporte/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Nucleares/metabolismo , Fatores de Processamento de RNA/metabolismo , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases , Humanos , Fosforilação
11.
J Colloid Interface Sci ; 521: 197-205, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29571101

RESUMO

The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency.


Assuntos
DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Interações Hidrofóbicas e Hidrofílicas , Tensoativos/química , Cátions , Etídio/química , Micelas , Estrutura Molecular , Relação Estrutura-Atividade , Propriedades de Superfície
12.
Nat Commun ; 9(1): 967, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29511213

RESUMO

BRCA1 is a tumor suppressor that regulates DNA repair by homologous recombination. Germline mutations in BRCA1 are associated with increased risk of breast and ovarian cancer and BRCA1 deficient tumors are exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, uncovering additional components of this DNA repair pathway is of extreme importance for further understanding cancer development and therapeutic vulnerabilities. Here, we identify EDC4, a known component of processing-bodies and regulator of mRNA decapping, as a member of the BRCA1-BRIP1-TOPBP1 complex. EDC4 plays a key role in homologous recombination by stimulating end resection at double-strand breaks. EDC4 deficiency leads to genome instability and hypersensitivity to DNA interstrand cross-linking drugs and PARP inhibitors. Lack-of-function mutations in EDC4 were detected in BRCA1/2-mutation-negative breast cancer cases, suggesting a role in breast cancer susceptibility. Collectively, this study recognizes EDC4 with a dual role in decapping and DNA repair whose inactivation phenocopies BRCA1 deficiency.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/metabolismo , Reparo do DNA , Proteínas/metabolismo , Proteína BRCA1/genética , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Recombinação Homóloga , Humanos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Proteínas/genética , Capuzes de RNA/genética , Capuzes de RNA/metabolismo
13.
Nucleic Acids Res ; 46(2): 730-747, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29253183

RESUMO

The DNA damage response (DDR) is an extensive signaling network that is robustly mobilized by DNA double-strand breaks (DSBs). The primary transducer of the DSB response is the protein kinase, ataxia-telangiectasia, mutated (ATM). Here, we establish nuclear poly(A)-binding protein 1 (PABPN1) as a novel target of ATM and a crucial player in the DSB response. PABPN1 usually functions in regulation of RNA processing and stability. We establish that PABPN1 is recruited to the DDR as a critical regulator of DSB repair. A portion of PABPN1 relocalizes to DSB sites and is phosphorylated on Ser95 in an ATM-dependent manner. PABPN1 depletion sensitizes cells to DSB-inducing agents and prolongs the DSB-induced G2/M cell-cycle arrest, and DSB repair is hampered by PABPN1 depletion or elimination of its phosphorylation site. PABPN1 is required for optimal DSB repair via both nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR), and specifically is essential for efficient DNA-end resection, an initial, key step in HRR. Using mass spectrometry analysis, we capture DNA damage-induced interactions of phospho-PABPN1, including well-established DDR players as well as other RNA metabolizing proteins. Our results uncover a novel ATM-dependent axis in the rapidly growing interface between RNA metabolism and the DDR.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas Nucleares/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Fosforilação , Proteína I de Ligação a Poli(A)/genética , Ligação Proteica , Mapas de Interação de Proteínas , Interferência de RNA
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