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1.
Nat Commun ; 11(1): 4124, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807787

RESUMO

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Reparo do DNA/fisiologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Células HCT116 , Humanos , Immunoblotting , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nanomedicina/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
2.
Nat Commun ; 11(1): 3503, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665547

RESUMO

DNA replication timing is tightly regulated during S-phase. S-phase length is determined by DNA synthesis rate, which depends on the number of active replication forks and their velocity. Here, we show that E2F-dependent transcription, through E2F6, determines the replication capacity of a cell, defined as the maximal amount of DNA a cell can synthesise per unit time during S-phase. Increasing or decreasing E2F-dependent transcription during S-phase increases or decreases replication capacity, and thereby replication rates, thus shortening or lengthening S-phase, respectively. The changes in replication rate occur mainly through changes in fork speed without affecting the number of active forks. An increase in fork speed does not induce replication stress directly, but increases DNA damage over time causing cell cycle arrest. Thus, E2F-dependent transcription determines the DNA replication capacity of a cell, which affects the replication rate, controlling the time it takes to duplicate the genome and complete S-phase.


Assuntos
Cromatina/metabolismo , Replicação do DNA/fisiologia , Western Blotting , Cromatina/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Replicação do DNA/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Fase S/genética , Fase S/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Mutat Res ; 853: 503194, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32522349

RESUMO

Obesity is associated with elevated cancer risk, which may be represented by elevated genomic damage. Oxidative stress plays a key role in obesity related detrimental health consequences including DNA oxidation damage. The elevated cancer risk in obesity may be a consequence. Weight loss has been shown to reduce genomic damage, but the role of oxidative stress in that has not been clarified. The aim of this study is therefore to investigate the influence of bariatric surgery induced weight loss on DNA oxidation damage in morbidly obese subjects. For this aim, we used cryopreserved peripheral blood mononuclear cells in the FPG comet assay. Advanced protein oxidation products and 3-nitrotyrosine were measured as oxidative and nitrative protein stress markers. Furthermore, expression of oxidative stress related proteins HSP70 and Nrf2 as well as mitochondrial enzyme citrate synthase and NADPH oxidase subunit p22 phox were analysed. Our findings revealed significantly reduced DNA strand breaks, but DNA base oxidation was not reduced. We observed significant reduction in plasma AOPPs and 3-nitrotyrosine, which indicated an improvement in oxidative/nitrative stress. However, expression of HSP70 and Nrf2 were not altered after weight loss. In addition, expression of citrate synthase and p22 phox were also unaltered. Overall, bariatric surgery induced significant reduction in excess body weight and improved the patients' health status, including reduced DNA strand breaks and slightly improved antioxidant status in some of the investigated endpoints, while cellular ROS formation and DNA oxidation damage stayed unaltered. This complex situation may be due to combined beneficial effects of weight loss and burdening of the body with fat breakdown products. In the future, collecting samples two years after surgery, when patients have been in a weight plateau for some time, might be a promising approach.


Assuntos
Dano ao DNA/fisiologia , Estresse Oxidativo/fisiologia , Perda de Peso/fisiologia , Adulto , Antioxidantes/metabolismo , Cirurgia Bariátrica/métodos , Ensaio Cometa/métodos , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Obesidade/metabolismo , Obesidade/cirurgia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
4.
Psychopharmacology (Berl) ; 237(7): 1909-1915, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32529266

RESUMO

RATIONALE: Depression is a major mental disorder affecting millions of people worldwide. Serotonin and norepinephrine reuptake inhibitors (SNRIs) are one of the antidepressant drugs prescribed for depression treatment. OBJECTIVE AND METHOD: There are many contradiction studies about the adverse effect and genotoxicity of SNRIs. So here, based on the guidelines proposed at the PRISMA statement, we performed a quantitative systematic review by searching international electronic databases (PubMed, Scopus, Embase, and Web of Science) for published documents on SSNRIs and their genotoxicity effects. RESULTS: The database searches retrieved 336 records, 18 of which met the inclusion criteria. Evaluation of the selected articles showed that a total of 9 articles were appropriate for final review. Most of these studies (78%) reported positive results for the genotoxicity of SNRIs CONCLUSION: Finally, we can conclude that these drugs have the potential to damage DNA.


Assuntos
Antidepressivos/efeitos adversos , Dano ao DNA/efeitos dos fármacos , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/genética , Inibidores da Recaptação de Serotonina e Norepinefrina/efeitos adversos , Antidepressivos/farmacologia , Dano ao DNA/fisiologia , Transtorno Depressivo/metabolismo , Humanos , Norepinefrina/antagonistas & inibidores , Norepinefrina/genética , Norepinefrina/metabolismo , Serotonina/genética , Serotonina/metabolismo , Inibidores de Captação de Serotonina/efeitos adversos , Inibidores de Captação de Serotonina/farmacologia , Inibidores da Recaptação de Serotonina e Norepinefrina/farmacologia
5.
Nat Commun ; 11(1): 2950, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528002

RESUMO

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.


Assuntos
DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Humanos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estrutura Secundária de Proteína , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
6.
Am J Physiol Cell Physiol ; 319(1): C45-C63, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32374675

RESUMO

Numerous age-dependent alterations at the molecular, cellular, tissue and organ systems levels underlie the pathophysiology of aging. Herein, the focus is upon the secreted protein thrombospondin-1 (TSP1) as a promoter of aging and age-related diseases. TSP1 has several physiological functions in youth, including promoting neural synapse formation, mediating responses to ischemic and genotoxic stress, minimizing hemorrhage, limiting angiogenesis, and supporting wound healing. These acute functions of TSP1 generally require only transient expression of the protein. However, accumulating basic and clinical data reinforce the view that chronic diseases of aging are associated with accumulation of TSP1 in the extracellular matrix, which is a significant maladaptive contributor to the aging process. Identification of the relevant cell types that chronically produce and respond to TSP1 and the molecular mechanisms that mediate the resulting maladaptive responses could direct the development of therapeutic agents to delay or revert age-associated maladies.


Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Trombospondina 1/biossíntese , Trombospondina 1/genética , Animais , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/terapia , Dano ao DNA/fisiologia , Humanos , Doenças Musculoesqueléticas/genética , Doenças Musculoesqueléticas/metabolismo , Doenças Musculoesqueléticas/terapia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Transdução de Sinais/fisiologia , Trombospondina 1/antagonistas & inibidores , Cicatrização/fisiologia
7.
Nat Commun ; 11(1): 2147, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358495

RESUMO

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.


Assuntos
Antígeno Nuclear de Célula em Proliferação/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Ensaio Cometa , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Imunofluorescência , Instabilidade Genômica/genética , Instabilidade Genômica/fisiologia , Células HEK293 , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Ubiquitinação/genética , Ubiquitinação/fisiologia
8.
Nat Commun ; 11(1): 2169, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358516

RESUMO

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Animais , Caenorhabditis elegans/genética , Dano ao DNA/genética , Reparo do DNA/genética , Genômica/métodos , Humanos , Mutação/genética , Mutação Puntual/genética
9.
PLoS Pathog ; 16(4): e1008481, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298394

RESUMO

Reactive oxygen species (ROS) production is one of the earliest responses when plants percept pathogens and acts as antimicrobials to block pathogen entry. However, whether and how pathogens tolerate ROS stress remains elusive. Here, we report the chromatin remodeling in Verticillium dahliae, a soil-borne pathogenic fungus that causes vascular wilts of a wide range of plants, facilitates the DNA damage repair in response to plant ROS stress. We identified VdDpb4, encoding a histone-fold protein of the ISW2 chromatin remodeling complex in V. dahliae, is a virulence gene. The reduced virulence in wild type Arabidopsis plants arising from VdDpb4 deletion was impaired in the rbohd mutant plants that did not produce ROS. Further characterization of VdDpb4 and its interacting protein, VdIsw2, an ATP-dependent chromatin-remodeling factor, we show that while the depletion of VdIsw2 led to the decondensing of chromatin, the depletion of VdDpb4 resulted in a more compact chromatin structure and affected the VdIsw2-dependent transcriptional effect on gene expression, including genes involved in DNA damage repair. A knockout mutant of either VdDpb4 or VdIsw2 reduced the efficiency of DNA repair in the presence of DNA-damaging agents and virulence during plant infection. Together, our data demonstrate that VdDpb4 and VdIsw2 play roles in maintaining chromatin structure for positioning nucleosomes and transcription regulation, including genes involved in DNA repair in response to ROS stress during development and plant infection.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Verticillium/genética , Arabidopsis/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Verticillium/patogenicidade , Virulência
10.
Cell Mol Life Sci ; 77(20): 4031-4047, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32318759

RESUMO

Fate determination in self-renewal and differentiation of hematopoietic stem and progenitor cells (HSCs and HPCs) is ultimately controlled by gene expression, which is profoundly influenced by the global and local chromatin state. Cellular metabolism directly influences the chromatin state through the dynamic regulation of the enzymatic activities that modify DNA and histones, but also generates genotoxic metabolites that can damage DNA and thus pose threat to the genome integrity. On the other hand, mechanisms modulating the chromatin state impact metabolism by regulating the expression and activities of key metabolic enzymes. Moreover, through regulating either DNA damage response directly or expression of genes involved in this process, chromatin modulators play active and crucial roles in guarding the genome integrity, breaching of which results in defective HSPC function. Therefore, HSPC function is regulated by the dynamic and two-way interactions between metabolism and chromatin. Here, we review recent advances that provide a chromatin perspective on the major impacts the metabolic and genotoxic factors can have on HSPC function and fate determination.


Assuntos
Cromatina/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/fisiologia , Dano ao DNA/fisiologia , Histonas/metabolismo , Humanos
11.
Proc Natl Acad Sci U S A ; 117(17): 9338-9348, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284409

RESUMO

Oxidation of guanine generates several types of DNA lesions, such as 8-oxoguanine (8OG), 5-guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These guanine-derived oxidative DNA lesions interfere with both replication and transcription. However, the molecular mechanism of transcription processing of Gh and Sp remains unknown. In this study, by combining biochemical and structural analysis, we revealed distinct transcriptional processing of these chemically related oxidized lesions: 8OG allows both error-free and error-prone bypass, whereas Gh or Sp causes strong stalling and only allows slow error-prone incorporation of purines. Our structural studies provide snapshots of how polymerase II (Pol II) is stalled by a nonbulky Gh lesion in a stepwise manner, including the initial lesion encounter, ATP binding, ATP incorporation, jammed translocation, and arrested states. We show that while Gh can form hydrogen bonds with adenosine monophosphate (AMP) during incorporation, this base pair hydrogen bonding is not sufficient to hold an ATP substrate in the addition site and is not stable during Pol II translocation after the chemistry step. Intriguingly, we reveal a unique structural reconfiguration of the Gh lesion in which the hydantoin ring rotates ∼90° and is perpendicular to the upstream base pair planes. The perpendicular hydantoin ring of Gh is stabilized by noncanonical lone pair-π and CH-π interactions, as well as hydrogen bonds. As a result, the Gh lesion, as a functional mimic of a 1,2-intrastrand crosslink, occupies canonical -1 and +1 template positions and compromises the loading of the downstream template base. Furthermore, we suggest Gh and Sp lesions are potential targets of transcription-coupled repair.


Assuntos
Guanidinas/química , Guanosina/análogos & derivados , Hidantoínas/química , RNA Polimerase II/metabolismo , Compostos de Espiro/química , Pareamento de Bases , DNA/química , DNA/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Guanidinas/metabolismo , Guanina/metabolismo , Guanosina/química , Guanosina/metabolismo , Hidantoínas/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Purinas/metabolismo , RNA Polimerase II/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Compostos de Espiro/metabolismo , Transcrição Genética/fisiologia , Ativação Transcricional/fisiologia
12.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118716, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32275931

RESUMO

RNA editing has emerged as a novel mechanism in cancer progression. The double stranded RNA-specific adenosine deaminase (ADAR) modifies the expression of an important proportion of genes involved in cell cycle control, DNA damage response (DDR) and transcriptional processing, suggesting an important role of ADAR in transcriptome regulation. Despite the phenotypic implications of ADAR deregulation in several cancer models, the role of ADAR on DDR and proliferation in breast cancer has not been fully addressed. Here, we show that ADAR expression correlates significantly with clinical outcomes and DDR, cell cycle and proliferation mRNAs of previously reported edited transcripts in breast cancer patients. ADAR's knock-down in a breast cancer cell line produces stability changes of mRNAs involved in DDR and DNA replication. Breast cancer cells with reduced levels of ADAR show a decreased viability and an increase in apoptosis, displaying a significant decrease of their DDR activation, compared to control cells. These results suggest that ADAR plays an important role in breast cancer progression through the regulation of mRNA stability and expression of those genes involved in proliferation and DDR impacting the viability of breast cancer cells.


Assuntos
Adenosina Desaminase/metabolismo , Neoplasias da Mama/metabolismo , Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Transcriptoma , Adenosina Desaminase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Células MCF-7 , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética
13.
Plant Mol Biol ; 103(3): 321-340, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32277429

RESUMO

Plants live in constantly changing and often unfavorable or stressful environments. Environmental changes induce biotic and abiotic stress, which, in turn, may cause genomic DNA damage. Hence, plants simultaneously suffer abiotic/biotic stress and DNA damage. However, little information is available on the signaling crosstalk that occurs between DNA damage and abiotic/biotic stresses. Arabidopsis thaliana SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) is a pivotal transcription factor that regulates thousands of genes in response to DNA double-strand break (DSB), and we recently reported that SOG1 has a role in immune responses. In the present study, the effects of SOG1 overexpression on the DNA damage and immune responses were examined. Results found that SOG1 overexpression enhances the regulation of numerous downstream genes. Relative to the wild type plants, then, DNA damage responses were observed to be strongly induced. SOG1 overexpression also upregulates chitin (a major components of fungal cell walls) responsive genes in the presence of DSBs, implying that pathogen defense response is activated by DNA damage via SOG1. Further, SOG1 overexpression enhances fungal resistance. These results suggest that SOG1 regulates crosstalk between DNA damage response and the immune response and that plants have evolved a sophisticated defense network to contend with environmental stress.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Dano ao DNA/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Fatores de Transcrição/metabolismo , Apoptose/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , DNA de Plantas , Regulação da Expressão Gênica de Plantas/imunologia , Folhas de Planta/citologia , Ligação Proteica , Estresse Fisiológico , Fatores de Transcrição/genética
14.
Chemosphere ; 253: 126643, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32278190

RESUMO

Tembotrione is a rather novel pesticide, usually used for post-emergence weed control. Even though its use is rapidly growing, it is not followed by an adequate flow of scientific evidence regarding its toxicity towards non-target organisms. We evaluated the potential of low doses of tembotrione to induce oxidative stress and cytogenetic damage in blood and brain cells of adult male Wistar rats. Parameters of lipid peroxidation, glutathione levels, activities of antioxidant enzymes and primary DNA damage were assessed following 28-day repeated oral exposure to doses comparable with the currently proposed health-based reference values. The results of the alkaline comet assay showed that such low doses of tembotrione have the potency to inflict primary DNA damage in both peripheral blood leukocytes and brain of treated rats, even with only slight changes in the oxidative biomarker levels. The DNA damage in blood and brain cells of Wistar rats significantly increased at all applied doses, suggesting that tembotrione genotoxicity is mainly a result of direct interaction with DNA while the induction of oxidative stress responses contributes to DNA instability in a lesser extent. The findings of the present study call for further research using other sensitive biomarkers of effect and different exposure scenarios.


Assuntos
Cicloexanonas/toxicidade , Dano ao DNA/fisiologia , Herbicidas/toxicidade , Sulfonas/toxicidade , Animais , Antioxidantes/farmacologia , Encéfalo/efeitos dos fármacos , Ensaio Cometa , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Testes de Toxicidade
15.
Nat Commun ; 11(1): 1356, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170071

RESUMO

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.


Assuntos
DNA/química , DNA/metabolismo , Imagem Individual de Molécula/métodos , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Biofísica/métodos , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Microscopia de Força Atômica , Ligação Proteica , Raios Ultravioleta
16.
PLoS Genet ; 16(3): e1008654, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150542

RESUMO

Mistranslation is typically deleterious for cells, although specific mistranslated proteins can confer a short-term benefit in a particular environment. However, given its large overall cost, the prevalence of high global mistranslation rates remains puzzling. Altering basal mistranslation levels of Escherichia coli in several ways, we show that generalized mistranslation enhances early survival under DNA damage, by rapidly activating the SOS response. Mistranslating cells maintain larger populations after exposure to DNA damage, and thus have a higher probability of sampling critical beneficial mutations. Both basal and artificially increased mistranslation increase the number of cells that are phenotypically tolerant and genetically resistant under DNA damage; they also enhance survival at high temperature. In contrast, decreasing the normal basal mistranslation rate reduces cell survival. This wide-ranging stress resistance relies on Lon protease, which is revealed as a key effector that induces the SOS response in addition to alleviating proteotoxic stress. The new links between error-prone protein synthesis, DNA damage, and generalised stress resistance indicate surprising coordination between intracellular stress responses and suggest a novel hypothesis to explain high global mistranslation rates.


Assuntos
Sobrevivência Celular/genética , Biossíntese de Proteínas/genética , Resposta SOS em Genética/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação/genética , Mutação/fisiologia , Protease La/genética , Protease La/metabolismo
17.
Nat Commun ; 11(1): 1477, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198385

RESUMO

In the model organism Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Imagem Óptica/métodos , Células Procarióticas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Biofísica , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA , DNA Bacteriano/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Fatores de Transcrição/metabolismo , Raios Ultravioleta
18.
Nat Rev Nephrol ; 16(6): 352-364, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32157251

RESUMO

Tonicity-responsive enhancer-binding protein (TonEBP), which is also known as nuclear factor of activated T cells 5 (NFAT5), was discovered 20 years ago as a transcriptional regulator of the cellular response to hypertonic (hyperosmotic salinity) stress in the renal medulla. Numerous studies since then have revealed that TonEBP is a pleiotropic stress protein that is involved in a range of immunometabolic diseases. Some of the single-nucleotide polymorphisms (SNPs) in TONEBP introns are cis-expression quantitative trait loci that affect TONEBP transcription. These SNPs are associated with increased risk of type 2 diabetes mellitus, diabetic nephropathy, inflammation, high blood pressure and abnormal plasma osmolality, indicating that variation in TONEBP expression might contribute to these phenotypes. In addition, functional studies have shown that TonEBP is involved in the pathogenesis of rheumatoid arthritis, atherosclerosis, diabetic nephropathy, acute kidney injury, hyperlipidaemia and insulin resistance, autoimmune diseases (including type 1 diabetes mellitus and multiple sclerosis), salt-sensitive hypertension and hepatocellular carcinoma. These pathological activities of TonEBP are in contrast to the protective actions of TonEBP in response to hypertonicity, bacterial infection and DNA damage induced by genotoxins. An emerging theme is that TonEBP is a stress protein that mediates the cellular response to a range of pathological insults, including excess caloric intake, inflammation and oxidative stress.


Assuntos
Doenças Autoimunes/metabolismo , Dano ao DNA/fisiologia , Fatores de Transcrição NFATC/metabolismo , Estresse Fisiológico/fisiologia , Artrite Reumatoide/metabolismo , Aterosclerose/metabolismo , Infecções Bacterianas/metabolismo , Carcinoma Hepatocelular/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Proteínas de Choque Térmico , Humanos , Hiperlipidemias/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Resistência à Insulina , Neoplasias Hepáticas/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/fisiologia , Obesidade/metabolismo , Estresse Oxidativo/fisiologia , Polimorfismo de Nucleotídeo Único , Estresse Salino/fisiologia , Viroses/metabolismo
19.
Cell ; 180(6): 1228-1244.e24, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32142649

RESUMO

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.


Assuntos
Reparo do DNA/fisiologia , RNA Polimerase II/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Polimerase II/genética , Ubiquitinação
20.
PLoS One ; 15(3): e0226860, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119683

RESUMO

The mitochondrial theory of aging attributes much of the aging process to mitochondrial DNA damage. The polymerase gamma (PolG) mutant mouse was designed to evaluate this theory and thus carries a mutated proofreading region of polymerase gamma (D257A) that exclusively transcribes the mitochondrial genome. As a result, PolGD257A mice accumulate mitochondrial DNA (mtDNA) mutations that lead to premature aging, as evidenced by hair loss, weight loss, kyphosis, increased rates of apoptosis, organ damage, and an early death, occurring around 12 months of age. Research has shown that exercise decreases skeletal muscle mtDNA mutations and normalizes protein levels in PolG mice. However, brain mtDNA changes with exercise in PolG mice have not been studied. We found no effects of exercise on mtDNA mutations or copy number in either the brain or liver of PolG mice, despite changes to body mass. Our results suggest that mitochondrial mutations play little role in exercise-brain interactions in the PolG model of accelerated aging. In addition to evaluating the effect of exercise on mtDNA outcomes, we also implemented novel methods for both extracting mtDNA and measuring mtDNA mutations, with aims for improving the efficiency and accuracy of these methods.


Assuntos
Senilidade Prematura/prevenção & controle , Dano ao DNA/fisiologia , Polimerase do DNA Mitocondrial/genética , DNA Mitocondrial/genética , Condicionamento Físico Animal/fisiologia , Senilidade Prematura/genética , Senilidade Prematura/patologia , Senilidade Prematura/fisiopatologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Variações do Número de Cópias de DNA , Polimerase do DNA Mitocondrial/metabolismo , DNA Mitocondrial/isolamento & purificação , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Humanos , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação
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