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1.
EMBO J ; 39(18): e104185, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32705708

RESUMO

Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C-terminal region a previously unappreciated DNA-binding domain that exhibits specific binding to G-quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4-forming sequences, and exhibits partial redundancy with an adjacent PARP-binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX11. Loss of both Timeless and DDX11 causes epigenetic instability at G4-forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication-hindering G4 formation and ensures the prompt resolution of these structures by DDX11 to maintain processive DNA synthesis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , Quadruplex G , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Domínios Proteicos
2.
Mutagenesis ; 34(4): 341-354, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31348825

RESUMO

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.


Assuntos
Mutação , Estresse Oxidativo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Biomarcadores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ensaio Cometa , Dano ao DNA , Reparo do DNA , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Mutação/efeitos dos fármacos , Mutação/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Raios Ultravioleta
3.
Nucleic Acids Res ; 45(3): 1270-1280, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28180309

RESUMO

Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Sobrevivência Celular , Cromatina/metabolismo , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/genética , Relação Dose-Resposta à Radiação , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Raios Ultravioleta
5.
Hum Mutat ; 34(3): 481-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23255472

RESUMO

Nucleotide excision repair (NER) is the most flexible of all known DNA-repair mechanisms, and XPG is a 3'-endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP-G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP-G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV-irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV- but not oxidative-stress-induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.


Assuntos
Sobrevivência Celular/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adolescente , Alelos , Sequência de Aminoácidos , Brasil , Linhagem Celular , Clonagem Molecular , Síndrome de Cockayne/genética , Dano ao DNA/efeitos da radiação , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos da radiação , Heterozigoto , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Estresse Oxidativo/efeitos da radiação , Fenótipo , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de DNA , Raios Ultravioleta , Xeroderma Pigmentoso/genética , Adulto Jovem
6.
Leukemia ; 37(11): 2221-2230, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37752286

RESUMO

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, the prognosis of which varies according to the cytogenetic group. We characterized a rare chromosomal abnormality (del(8p), deletion of the short arm of chromosome 8) in the context of CLL. By comparing the largest cohort of del(8p) CLL to date (n = 57) with a non-del(8p) cohort (n = 155), del(8p) was significantly associated with a poor prognosis, a shorter time to first treatment, worse overall survival (OS), and a higher risk of Richter transformation. For patients treated with fludarabine-based regimens, the next-treatment-free survival and the OS were shorter in del(8p) cases (including those with mutated IGHV). One copy of the TNFRSF10B gene (coding a pro-apoptotic receptor activated by TRAIL) was lost in 91% of del(8p) CLL. TNFRSF10B was haploinsufficient in del(8p) CLL, and was involved in the modulation of fludarabine-induced cell death - as confirmed by our experiments in primary cells and in CRISPR-edited TNFRSF10B knock-out CLL cell lines. Lastly, del(8p) abrogated the synergy between fludarabine and TRAIL-induced apoptosis. Our results highlight del(8p)'s value as a prognostic marker and suggest that fit CLL patients (i.e. with mutated IGHV and no TP53 disruption) should be screened for del(8p) before the initiation of fludarabine-based treatment.


Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Aberrações Cromossômicas , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Prognóstico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Vidarabina/farmacologia , Vidarabina/uso terapêutico
7.
Front Immunol ; 13: 871766, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35432321

RESUMO

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is a B cell specific process required for the generation of specific and high affinity antibodies during the maturation of the immune response against foreign antigens. This process depends on the activity of both activation-induced cytidine deaminase (AID) and several DNA repair factors. AID-dependent SHM creates the full spectrum of mutations in Ig variable (V) regions equally distributed at G/C and A/T bases. In most mammalian cells, deamination of deoxycytidine into uracil during S phase induces targeted G/C mutagenesis using either direct replication of uracils or TLS mediated bypass, however only the machinery of activated B lymphocytes can generate A/T mutagenesis around AID-created uracils. The molecular mechanism behind the latter remains incompletely understood to date. However, the lack of a cellular model that reproduces both G/C and A/T mutation spectra constitutes the major hurdle to elucidating it. The few available B cell lines used thus far to study Ig SHM indeed undergo mainly G/C mutations, that make them inappropriate or of limited use. In this report, we show that in the Ramos cell line that undergoes constitutive G/C-biased SHM in culture, the low rate of A/T mutations is due to an imbalance in the ubiquitination/deubiquitination reaction of PCNA, with the deubiquitination reaction being predominant. The inhibition of the deubiquitinase complex USP1-UAF1 or the expression of constitutive fusion of ubiquitin to PCNA provides the missing clue required for DNA polymerase η recruitment and thereafter the introduction of A/T base pair (bp) mutations during the process of IgV gene diversification. This study reports the establishment of the first modified human B cell line that recapitulates the mechanism of SHM of Ig genes in vitro.


Assuntos
Imunoglobulina A , Hipermutação Somática de Imunoglobulina , Animais , Linhagem Celular , Humanos , Imunoglobulina A/genética , Mamíferos/metabolismo , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina
8.
Sci Rep ; 10(1): 1311, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992747

RESUMO

Somatic hypermutation of immunoglobulin genes is a highly mutagenic process that is B cell-specific and occurs during antigen-driven responses leading to antigen specificity and antibody affinity maturation. Mutations at the Ig locus are initiated by Activation-Induced cytidine Deaminase and are equally distributed at G/C and A/T bases. This requires the establishment of error-prone repair pathways involving the activity of several low fidelity DNA polymerases. In the physiological context, the G/C base pair mutations involve multiple error-prone DNA polymerases, while the generation of mutations at A/T base pairs depends exclusively on the activity of DNA polymerase η. Using two large cohorts of individuals with xeroderma pigmentosum variant (XP-V), we report that the pattern of mutations at Ig genes becomes highly enriched with large deletions. This observation is more striking for patients older than 50 years. We propose that the absence of Pol η allows the recruitment of other DNA polymerases that profoundly affect the Ig genomic landscape.


Assuntos
DNA Polimerase Dirigida por DNA/deficiência , Imunoglobulinas/genética , Deleção de Sequência , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Brasil , Estudos de Casos e Controles , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Ativação Enzimática , França , Frequência do Gene , Genótipo , Humanos , Pessoa de Meia-Idade , Mutação , Xeroderma Pigmentoso/genética
9.
Mol Metab ; 40: 101027, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32480041

RESUMO

OBJECTIVES: Apoptosis-Inducing Factor (AIF) is a protein involved in mitochondrial electron transport chain assembly/stability and programmed cell death. The relevant role of this protein is underlined because mutations altering mitochondrial AIF properties result in acute pediatric mitochondriopathies and tumor metastasis. By generating an original AIF-deficient mouse strain, this study attempted to analyze, in a single paradigm, the cellular and developmental metabolic consequences of AIF loss and the subsequent oxidative phosphorylation (OXPHOS) dysfunction. METHODS: We developed a novel AIF-deficient mouse strain and assessed, using molecular and cell biology approaches, the cellular, embryonic, and adult mice phenotypic alterations. Additionally, we conducted ex vivo assays with primary and immortalized AIF knockout mouse embryonic fibroblasts (MEFs) to establish the cell death characteristics and the metabolic adaptive responses provoked by the mitochondrial electron transport chain (ETC) breakdown. RESULTS: AIF deficiency destabilized mitochondrial ETC and provoked supercomplex disorganization, mitochondrial transmembrane potential loss, and high generation of mitochondrial reactive oxygen species (ROS). AIF-/Y MEFs counterbalanced these OXPHOS alterations by mitochondrial network reorganization and a metabolic reprogramming toward anaerobic glycolysis illustrated by the AMPK phosphorylation at Thr172, the overexpression of the glucose transporter GLUT-4, the subsequent enhancement of glucose uptake, and the anaerobic lactate generation. A late phenotype was characterized by the activation of P53/P21-mediated senescence. Notably, approximately 2% of AIF-/Y MEFs diminished both mitochondrial mass and ROS levels and spontaneously proliferated. These cycling AIF-/Y MEFs were resistant to caspase-independent cell death inducers. The AIF-deficient mouse strain was embryonic lethal between E11.5 and E13.5 with energy loss, proliferation arrest, and increased apoptotic levels. Contrary to AIF-/Y MEFs, the AIF KO embryos were unable to reprogram their metabolism toward anaerobic glycolysis. Heterozygous AIF+/- females displayed progressive bone marrow, thymus, and spleen cellular loss. In addition, approximately 10% of AIF+/- females developed perinatal hydrocephaly characterized by brain development impairment, meningeal fibrosis, and medullar hemorrhages; those mice died 5 weeks after birth. AIF+/- with hydrocephaly exhibited loss of ciliated epithelium in the ependymal layer. This phenotype was triggered by the ROS excess. Accordingly, it was possible to diminish the occurrence of hydrocephalus AIF+/- females by supplying dams and newborns with an antioxidant in drinking water. CONCLUSIONS: In a single knockout model and at 3 different levels (cell, embryo, and adult mice) we demonstrated that by controlling the mitochondrial OXPHOS/metabolism, AIF is a key factor regulating cell differentiation and fate. Additionally, by providing new insights into the pathological consequences of mitochondrial OXPHOS dysfunction, our new findings pave the way for novel pharmacological strategies.


Assuntos
Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Animais , Apoptose/fisiologia , Caspases/metabolismo , Respiração Celular , Feminino , Fibroblastos/metabolismo , Engenharia Genética/métodos , Glicólise/genética , Hidrocefalia/metabolismo , Masculino , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos/genética , Mitocôndrias/metabolismo , Modelos Animais , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo
10.
Mutat Res Genet Toxicol Environ Mutagen ; 836(Pt B): 127-142, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30442338

RESUMO

During DNA replication, forks may encounter unrepaired lesions that hamper DNA synthesis. Cells have universal strategies to promote damage bypass allowing cells to survive. DNA damage tolerance can be performed upon template switch or by specialized DNA polymerases, known as translesion (TLS) polymerases. Human cells count on more than eleven TLS polymerases and this work reviews the functions of some of these enzymes: Rev1, Pol η, Pol ι, Pol κ, Pol θ and Pol ζ. The mechanisms of damage bypass vary according to the lesion, as well as to the TLS polymerases available, and may occur directly at the fork during replication. Alternatively, the lesion may be skipped, leaving a single-stranded DNA gap that will be replicated later. Details of the participation of these enzymes are revised for the replication of damaged template. TLS polymerases also have functions in other cellular processes. These include involvement in somatic hypermutation in immunoglobulin genes, direct participation in recombination and repair processes, and contributing to replicating noncanonical DNA structures. The importance of DNA damage replication to cell survival is supported by recent discoveries that certain genes encoding TLS polymerases are induced in response to DNA damaging agents, protecting cells from a subsequent challenge to DNA replication. We retrace the findings on these genotoxic (adaptive) responses of human cells and show the common aspects with the SOS responses in bacteria. Paradoxically, although TLS of DNA damage is normally an error prone mechanism, in general it protects from carcinogenesis, as evidenced by increased tumorigenesis in xeroderma pigmentosum variant patients, who are deficient in Pol η. As these TLS polymerases also promote cell survival, they constitute an important mechanism by which cancer cells acquire resistance to genotoxic chemotherapy. Therefore, the TLS polymerases are new potential targets for improving therapy against tumors.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Resposta SOS em Genética
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