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1.
Nat Commun ; 10(1): 5611, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31819057

RESUMO

Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. It is, however, unclear whether organisms have evolved general mechanism(s) to deal with this damage. Here we show that the mRNA-surveillance pathway of no-go decay and the associated ribosome-quality control are activated in response to nucleobase alkylation and oxidation. Our findings reveal that these processes are important for clearing chemically modified mRNA and the resulting aberrant-protein products. In the absence of Xrn1, the level of damaged mRNA significantly increases. Furthermore, deletion of LTN1 results in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. This accumulation is accompanied by Hel2-dependent regulatory ubiquitylation of ribosomal proteins. Collectively, our data highlight the burden of chemically damaged mRNA on cellular homeostasis and suggest that organisms evolved mechanisms to counter their accumulation.


Assuntos
Estresse Oxidativo , Ribossomos/metabolismo , 4-Nitroquinolina-1-Óxido/metabolismo , Alquilação , Adutos de DNA/metabolismo , Dano ao DNA , Células HEK293 , Humanos , Metanossulfonato de Metila/farmacologia , Mutação/genética , Oxirredução , Peptídeos/metabolismo , Polirribossomos/metabolismo , Agregados Proteicos , Quinolonas/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
2.
An Acad Bras Cienc ; 91(3): e20180655, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576914

RESUMO

This study evaluated 24 patients with lung cancer (CA) and 23 individuals with no smoking history or cancer in the family and without respiratory disease in childhood (CO). Peripheral blood lymphocytes was used to perform alkaline comet assay and to assess DNA damage as well as to evaluate methyl methane sulfonate (MMS) DNA repair after one hour and three hours at 37 ºC. The percentage of residual damage (RD) after three hours of MMS treatment, for each patient was assessed. The majority of patients were in the CA group, male patients, former smokers, with a history of smoking for 15 years and without associated comorbidities. Alkaline and residual damages were higher in the CA group when compared to controls (alkaline damage P = 0.015 and RD P = 0.05). After one hour of MMS treatment the DNA damage of the CA increased indicating failure to repair it, compared to the controls, and after three hours DNA repair was observed in both groups. Patients with lung cancer are mostly men, former smokers and with more than 15 years of tobacco consumption, undergoing chemotherapy, have high rates of DNA damage and deficiency in their ability to repair against induced damage when compared to controls.


Assuntos
Dano ao DNA , Reparo do DNA , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Idoso , Antineoplásicos Alquilantes/farmacologia , Estudos de Casos e Controles , Ensaio Cometa/métodos , Estudos Transversais , Feminino , Humanos , Linfócitos/efeitos dos fármacos , Masculino , Metanossulfonato de Metila/farmacologia , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Fumar/efeitos adversos , Fatores de Tempo
3.
BMC Plant Biol ; 19(1): 364, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426748

RESUMO

BACKGROUND: Poly (ADP-ribosyl) ation (PARylation) is an important posttranslational modification that regulates DNA repair, gene transcription, stress responses and developmental processes in multicellular organisms. Poly (ADP-ribose) polymerase (PARP) catalyzes PARylation by consecutively adding ADP-ribose moieties from NAD+ to the amino acid receptor residues on target proteins. Arabidopsis has three canonical PARP members, and two of these members, AtPARP1 and AtPARP2, have been demonstrated to be bona fide poly (ADP-ribose) polymerases and to regulate DNA repair and stress response processes. However, it remains unknown whether AtPARP3, a member that is highly expressed in seeds, has similar biochemical activity to that of AtPARP1 and AtPARP2. Additionally, although both the phylogenetic relationships and structural similarities indicate that AtPARP1 and AtPARP2 correspond to animal PARP1 and PARP2, respectively, two previous studies have indicated that AtPARP2, and not AtPARP1, accounts for most of the PARP activity in Arabidopsis, which is contrary to the knowledge that PARP1 is the predominant PARP in animals. RESULTS: In this study, we obtained both in vitro and in vivo evidence demonstrating that AtPARP3 does not act as a typical PARP in Arabidopsis. Domain swapping and point mutation assays indicated that AtPARP3 has lost NAD+-binding capability and is inactive. In addition, our results showed that AtPARP1 was responsible for most of the PARP enzymatic activity in response to the DNA damage-inducing agents zeocin and methyl methanesulfonate (MMS) and was more rapidly activated than AtPARP2, which supports that AtPARP1 remains the predominant PARP member in Arabidopsis. AtPARP1 might first become activated by binding to damaged sites, and AtPARP2 is then poly (ADP-ribosyl) ated by AtPARP1 in vivo. CONCLUSIONS: Collectively, our biochemical and genetic analysis results strongly support the notion that AtPARP3 has lost poly (ADP-ribose) polymerase activity in plants and performs different functions from those of AtPARP1 and AtPARP2. AtPARP1, instead of AtPARP2, plays the predominant role in PAR synthesis in both seeds and seedlings. These data bring new insights into our understanding of the physiological functions of plant PARP family members.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Dano ao DNA/genética , Reparo do DNA/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerases/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Bleomicina/farmacologia , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo
4.
DNA Repair (Amst) ; 82: 102675, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31450087

RESUMO

The pathogenesis of colorectal cancer (CRC) involves different mechanisms, such as genomic and microsatellite instabilities. Recently, a contribution of the base excision repair (BER) pathway in CRC pathology has been emerged. In this context, the involvement of APE1 in the BER pathway and in the transcriptional regulation of genes implicated in tumor progression strongly correlates with chemoresistance in CRC and in more aggressive cancers. In addition, the APE1 interactome is emerging as an important player in tumor progression, as demonstrated by its interaction with Nucleophosmin (NPM1). For these reasons, APE1 is becoming a promising target in cancer therapy and a powerful prognostic and predictive factor in several cancer types. Thus, specific APE1 inhibitors have been developed targeting: i) the endonuclease activity; ii) the redox function and iii) the APE1-NPM1 interaction. Furthermore, mutated p53 is a common feature of advanced CRC. The relationship between APE1 inhibition and p53 is still completely unknown. Here, we demonstrated that the inhibition of the endonuclease activity of APE1 triggers p53-mediated effects on cell metabolism in HCT-116 colon cancer cell line. In particular, the inhibition of the endonuclease activity, but not of the redox function or of the interaction with NPM1, promotes p53 activation in parallel to sensitization of p53-expressing HCT-116 cell line to genotoxic treatment. Moreover, the endonuclease inhibitor affects mitochondrial activity in a p53-dependent manner. Finally, we demonstrated that 3D organoids derived from CRC patients are susceptible to APE1-endonuclease inhibition in a p53-status correlated manner, recapitulating data obtained with HCT-116 isogenic cell lines. These findings suggest the importance of further studies aimed at testing the possibility to target the endonuclease activity of APE1 in CRC.


Assuntos
Neoplasias do Colo/patologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Metanossulfonato de Metila/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Proteína Supressora de Tumor p53/genética
5.
J Biol Chem ; 294(37): 13629-13637, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31320474

RESUMO

The Mag1 and Tpa1 proteins from budding yeast (Saccharomyces cerevisiae) have both been reported to repair alkylation damage in DNA. Mag1 initiates the base excision repair pathway by removing alkylated bases from DNA, and Tpa1 has been proposed to directly repair alkylated bases as does the prototypical oxidative dealkylase AlkB from Escherichia coli However, we found that in vivo repair of methyl methanesulfonate (MMS)-induced alkylation damage in DNA involves Mag1 but not Tpa1. We observed that yeast strains without tpa1 are no more sensitive to MMS than WT yeast, whereas mag1-deficient yeast are ∼500-fold more sensitive to MMS. We therefore investigated the substrate specificity of Mag1 and found that it excises alkylated bases that are known AlkB substrates. In contrast, purified recombinant Tpa1 did not repair these alkylated DNA substrates, but it did exhibit the prolyl hydroxylase activity that has also been ascribed to it. A comparison of several of the kinetic parameters of Mag1 and its E. coli homolog AlkA revealed that Mag1 catalyzes base excision from known AlkB substrates with greater efficiency than does AlkA, consistent with an expanded role of yeast Mag1 in repair of alkylation damage. Our results challenge the proposal that Tpa1 directly functions in DNA repair and suggest that Mag1-initiated base excision repair compensates for the absence of oxidative dealkylation of alkylated nucleobases in budding yeast. This expanded role of Mag1, as compared with alkylation repair glycosylases in other organisms, could explain the extreme sensitivity of Mag1-deficient S. cerevisiae toward alkylation damage.


Assuntos
Proteínas de Transporte/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alquilantes , Alquilação/genética , Proteínas de Transporte/genética , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/genética , DNA Fúngico/metabolismo , Remoção de Radical Alquila/genética , Endodesoxirribonucleases/genética , Escherichia coli/metabolismo , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Mutação , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Especificidade por Substrato
6.
Res Microbiol ; 170(4-5): 171-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30953691

RESUMO

Living organisms have developed specific defence mechanisms to counteract hostile environmental conditions. Alkylation stress response mechanisms also occur in Mycobacterium tuberculosis (MTB) the pathogen responsible for tuberculosis. The effect of alkylating agents on the cellular growth of MTB was investigated using methyl methanesulfonate (MMS) as methyl donor demonstrating that limited doses of alkylating agents might affect MTB cell viability. A global investigation of Mycobacterium smegmatis response to alkylating stress was then pursued by differential proteomics to identify the most affected cellular pathways. Quantitative analysis of proteomic profiles demonstrated that most of the proteins upregulated in the presence of alkylating agents are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we identified the GlmU bifunctional enzyme as a possible factor involved in biofilm production. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the M. smegmatis defence mechanisms.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Metanossulfonato de Metila/farmacologia , Complexos Multienzimáticos/metabolismo , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium tuberculosis/crescimento & desenvolvimento , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Alquilação , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/genética , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Ácido N-Acetilneuramínico/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
7.
J Biol Chem ; 294(23): 9295-9307, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-30948509

RESUMO

Interest in pharmacological agents capable of increasing cellular NAD+ concentrations has stimulated investigations of nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). NR and NMN require large dosages for effect. Herein, we describe synthesis of dihydronicotinamide riboside (NRH) and the discovery that NRH is a potent NAD+ concentration-enhancing agent, which acts within as little as 1 h after administration to mammalian cells to increase NAD+ concentrations by 2.5-10-fold over control values. Comparisons with NR and NMN show that in every instance, NRH provides greater NAD+ increases at equivalent concentrations. NRH also provides substantial NAD+ increases in tissues when administered by intraperitoneal injection to C57BL/6J mice. NRH substantially increases NAD+/NADH ratio in cultured cells and in liver and no induction of apoptotic markers or significant increases in lactate levels in cells. Cells treated with NRH are resistant to cell death caused by NAD+-depleting genotoxins such as hydrogen peroxide and methylmethane sulfonate. Studies to identify its biochemical mechanism of action showed that it does not inhibit NAD+ consumption, suggesting that it acts as a biochemical precursor to NAD+ Cell lysates possess an ATP-dependent kinase activity that efficiently converts NRH to the compound NMNH, but independent of Nrk1 or Nrk2. These studies identify a putative new metabolic pathway to NAD+ and a potent pharmacologic agent for NAD+ concentration enhancement in cells and tissues.


Assuntos
Apoptose/efeitos dos fármacos , NAD/metabolismo , Niacinamida/análogos & derivados , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Humanos , Peróxido de Hidrogênio/farmacologia , Injeções Intraperitoneais , Ácido Láctico/metabolismo , Fígado/metabolismo , Masculino , Metanossulfonato de Metila/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , NAD/análise , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Niacinamida/administração & dosagem , Niacinamida/síntese química , Niacinamida/farmacologia
8.
mSphere ; 4(2)2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894431

RESUMO

DNA damage-induced Rad51 focus formation is the hallmark of homologous recombination-mediated DNA repair. Earlier, we reported that Rad51 physically interacts with Hsp90, and under the condition of Hsp90 inhibition, it undergoes proteasomal degradation. Here, we show that the dynamic interaction between Rad51 and Hsp90 is crucial for the DNA damage-induced nuclear function of Rad51. Guided by a bioinformatics study, we generated a single mutant of Rad51, which resides at the N-terminal domain, outside the ATPase core domain. The mutant with an E to L change at residue 108 (Rad51E108L) was predicted to bind more strongly with Hsp90 than the wild-type (Rad51WT). A coimmunoprecipitation study demonstrated that there exists a distinct difference between the in vivo associations of Rad51WT-Hsp90 and of Rad51E108L-Hsp90. We found that upon DNA damage, the association between Rad51WT and Hsp90 was significantly reduced compared to that in the undamaged condition. However, the mutant Rad51E108L remained tightly associated with Hsp90 even after DNA damage. Consequently, the recruitment of Rad51E108L to the double-stranded broken ends was reduced significantly. The E108L-rad51 strain manifested severe sensitivity toward methyl methanesulfonate (MMS) and a complete loss of gene conversion efficiency, a phenotype similar to that of the Δrad51 strain. Previously, some of the N-terminal domain mutants of Rad51 were identified in a screen for a Rad51 interaction-deficient mutant; however, our study shows that Rad51E108L is not defective either in the self-interaction or its interaction with the members of the Rad52 epistatic group. Our study thus identifies a novel mutant of Rad51 which, owing to its greater association with Hsp90, exhibits a severe DNA repair defect.IMPORTANCE Rad51-mediated homologous recombination is the major mechanism for repairing DNA double-strand break (DSB) repair in cancer cells. Thus, regulating Rad51 activity could be an attractive target. The sequential assembly and disassembly of Rad51 to the broken DNA ends depend on reversible protein-protein interactions. Here, we discovered that a dynamic interaction with molecular chaperone Hsp90 is one such regulatory event that governs the recruitment of Rad51 onto the damaged DNA. We uncovered that Rad51 associates with Hsp90, and upon DNA damage, this complex dissociates to facilitate the loading of Rad51 onto broken DNA. In a mutant where such dissociation is incomplete, the occupancy of Rad51 at the broken DNA is partial, which results in inefficient DNA repair. Thus, it is reasonable to propose that any small molecule that may alter the dynamics of the Rad51-Hsp90 interaction is likely to impact DSB repair in cancer cells.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Choque Térmico HSP90/metabolismo , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Animais , Biologia Computacional , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Metanossulfonato de Metila/farmacologia , Camundongos , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/efeitos dos fármacos
9.
J Basic Microbiol ; 59(3): 302-313, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30614541

RESUMO

Δlon mutant of Escherichia coli becomes hypersensitive to DNA damaging agents and over-produce capsule due to stabilization of the Lon substrates, namely, SulA and RcsA, respectively. These phenotypes were earlier found to be suppressed in Δlon ssrA::cat/pUC4 K and Δlon faa (DnaJ, G232D) strains, called as "Alp" strains. We observed that a plasmid carrying an E. coli chromosomal fragment harboring few genes, a heat shock gene htpY and a portion of dnaK capable of encoding truncated N-terminal ATPase domain (244 aa) could suppress lon mutant phenotypes. Deletion of htpY did not affect the efficiency of suppression. Clones expressing DnaK' (244 aa) peptide alone could suppress both Δlon phenotypes in copy number dependent manner. Inactivation of clpQ did not affect the MMSR phenotype of Δlon strain carrying dnaK' clones indicating that ClpYQ protease does not degrade SulA. We hypothesize that the high levels of defective DnaK'-DnaJ chaperone complex formed in these strains might lead to aggregation of SulA and RcsA and, thereby the suppression of Δlon phenotypes. Systematic deletion analysis of dnaK' revealed that, ∼220 aa N-terminal DnaK peptide is required for suppression of cps-lac over-expression and ∼169 aa peptide is enough for the suppression of MMSS phenotype of Δlon mutant.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico HSP70/metabolismo , Protease La/genética , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/química , Expressão Gênica , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Metanossulfonato de Metila/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Plasmídeos/genética , Plasmídeos/metabolismo , Protease La/deficiência , Deleção de Sequência
10.
DNA Repair (Amst) ; 74: 1-16, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30639951

RESUMO

Cells respond to DNA damage by activating cell cycle checkpoints, arresting cell division or DNA replication while damage is repaired. In Saccharomyces cerevisiae, activation of the checkpoint kinase Rad53 leads to cell cycle arrest, with Rad53 deactivation required for proper resumption of the cell cycle. Rtt107 is a S. cerevisiae protein that acts as a scaffold in the response to DNA damage, and rtt107Δ mutants exhibit prolonged activation of Rad53 when subjected to replication stress. This phenotype has been attributed to checkpoint dampening, wherein an Rtt107-Slx4-Dpb11 interaction limits formation of a Rad9-Dpb11 complex that promotes Rad53 activation. However, we found that the rtt107Δ mutant contains higher levels of DNA damage during replication stress, presenting an alternative possible cause of Rad53 hyperactivation. We therefore sought to address the relevance of checkpoint dampening to the Rad53 hyperactivation phenotype of the rtt107Δ mutant by using a rad9-ST462,474AA allele that specifically disrupts Rad9-Dpb11 interaction. Incorporation of the rad9-ST462,474AA allele slightly suppressed the rtt107Δ mutant's DNA damage sensitivity phenotypes, while having little effect on Rad53 hyperactivation. This indicated that in the context of acute replication stress, Rad53 hyperactivation in the rtt107Δ mutant did not primarily result from Rad9-Dpb11 interaction. A H2A-S129A mutation, which generally reduces Rad9-mediated Rad53 activation, led to more robust suppression of rtt107Δ mutant phenotypes. Suppression of rtt107Δ mutant DNA damage sensitivity by the H2A-S129A or the rad9-ST462,474AA alleles required intact DNA damage tolerance pathways, indicating a reliance of the rtt107Δ mutant on tolerance pathways for reasons other than misregulation of Rad53 activity. Collectively, this work proposed a revised model of Rad53 hyperactivation after acute replicative stress in the rtt107Δ mutant, in which this phenotype was primarily a consequence of excess DNA damage.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Dano ao DNA , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Metanossulfonato de Metila/farmacologia , Mutação , Proteínas Nucleares/genética , Fenótipo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética
11.
New Phytol ; 222(3): 1380-1391, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30636294

RESUMO

Double-stranded breaks can be repaired by different mechanisms such as homologous recombination (HR), classical nonhomologous end joining (C-NHEJ) and alternative end joining (Alt-EJ). Polymerase Q (POLQ) has been proposed to be the main factor involved in Alt-EJ-mediated DNA repair. Here we describe the role of POLQ in DNA repair and gene targeting in Physcomitrella patens. The disruption of the POLQ gene does not influence the genetic stability of P. patens nor its development. The polq mutant shows the same sensitivity as wild-type towards most of the genotoxic agents tested (ultraviolet (UV), methyl methanesulfonate (MMS) and cisplatin) with the notable exception of bleomycin for which it shows less sensitivity than the wild-type. Furthermore, we show that POLQ is involved in the repair of CRISPR-Cas9-induced double-stranded breaks in P. patens. We also demonstrate that POLQ is a potential competitor and/or inhibitor of the HR repair pathway. This finding has a consequence in terms of genetic engineering, as in the absence of POLQ the frequency of gene targeting is significantly increased and the number of clean two-sided HR-mediated insertions is enhanced. Therefore, the control of POLQ activity in plants could be a useful strategy to optimize the tools of genome engineering for plant breeding.


Assuntos
Bryopsida/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Sequência de Bases , Bleomicina/farmacologia , Bryopsida/efeitos dos fármacos , Bryopsida/efeitos da radiação , Cisplatino/farmacologia , Reparo do DNA por Junção de Extremidades , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Metanossulfonato de Metila/farmacologia , Mutação/genética , Taxa de Mutação , Fenótipo , Raios Ultravioleta
12.
Sci China Life Sci ; 62(8): 1078-1086, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30465232

RESUMO

The anticancer therapies with the joint treatment of a histone deacetylase (HDAC) inhibitor and a DNA-damaging approach are actively under clinical investigations, but the underlying mechanism is unclear. Histone homeostasis is critical to genome stability, transcriptional accuracy, DNA repair process, senescence, and survival. We have previously demonstrated that the HDAC inhibitor, trichostatin A (TSA), could promote the degradation of the core histones induced by γ-radiation or the DNAalkylating agent methyl methanesulfonate (MMS) in non-cancer cells, including mouse spermatocyte and embryonic fibroblast cell lines. In this study, we found that the joint treatment by TSA and MMS induced the death of the cultured cancer cells with an additive effect, but induced degradation of the core histones synergistically in these cells. We then analyzed various combinations of other HDAC inhibitors, including suberoylanilide hydroxamic acid and valproate sodium, with MMS or other DNAdamaging agents, including etoposide and camptothecin. Most of these combined treatments induced cell death additively, but all the tested combinations induced degradation of the core histones synergistically. Meanwhile, we showed that cell cycle arrest might not be a primary consequence for the joint treatment of TSA and MMS. Given that clinic treatments of cancers jointly with an HDAC inhibitor and a DNA-damaging approach often show synergistic effects, histone degradation might more accurately underlie the synergistic effects of these joint treatments in clinic applications than other parameters, such as cell death and cell cycle arrest. Thus, our studies might suggest that the degradation of the core histones can serve as a new target for the development of cancer therapies.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacocinética , Histonas/metabolismo , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Camptotecina/metabolismo , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Quimioterapia Combinada , Etoposídeo/metabolismo , Etoposídeo/farmacologia , Raios gama , Inibidores de Histona Desacetilases/metabolismo , Homeostase , Humanos , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Metanossulfonato de Metila/metabolismo , Metanossulfonato de Metila/farmacologia , Camundongos , Ácido Valproico/metabolismo , Ácido Valproico/farmacologia , Vorinostat/metabolismo , Vorinostat/farmacologia
13.
J Plant Physiol ; 233: 20-30, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30576929

RESUMO

CROWDED NUCLEI (CRWN) family in Arabidopsis consists of four members, CRWN1 to CRWN4. It has been previously reported that the CRWN proteins are involved in the control of nuclear morphology and degradation of ABI5. In this study, however, we discover that CRWN-family proteins are not only involved in attenuating responsiveness to abscisic acid (ABA), but also implicated in inhibiting reactive oxygen species (ROS) production and DNA damage induced by genotoxic agent methyl methanesulfonate (MMS). Our results demonstrate that three crwn double mutants, i.e. crwn1 crwn3, crwn2 crwn3, and crwn2 crwn4, show slightly earlier leaf senescence, enhanced leaf cell death, and obvious overaccumulation of ROS under regular growth conditions. When treated with 0.15 µM ABA or 0.01% MMS, two double mutants, crwn1 crwn3 and crwn2 crwn3, exhibit significant decreased germination rates as well as leaf opening and greening rates. Moreover, subsequent investigations indicate that the MMS treatment strongly inhibits the growth of crwn mutant seedlings, while this inhibition is substantially relieved by imidazole (IMZ); by contrast, DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has no effect on relief of the growth inhibition. Further studies reveal that under 0.01% MMS treatment conditions, crwn mutants, especially the three double mutants, accumulate more ROS compared to Col-0, and their genomic DNA suffers from more severe DNA damage relative to Col-0, which is indicated by significantly higher 8-oxo-7-hydrodeoxyguanosine (8-oxo dG) content as observed in the crwn mutants. Altogether, these data clearly demonstrate that the CRWN-family proteins play important roles in diminishing ROS accumulation and protecting genomic DNA against excessive oxidative damage caused by MMS.


Assuntos
Proteínas de Arabidopsis/fisiologia , Dano ao DNA , Proteínas Nucleares/fisiologia , Estresse Oxidativo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Morte Celular/fisiologia , Dano ao DNA/efeitos dos fármacos , DNA de Plantas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Proteínas Nucleares/genética , Estresse Oxidativo/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Espécies Reativas de Oxigênio/metabolismo
14.
Mutagenesis ; 33(4): 283-289, 2018 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-30204902

RESUMO

Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, 'ImageStream X' series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0-5 µg/ml), Carbendazim (0-1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0-6.3 µg/ml) for a period of 1.5-2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish 'ground truth' cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the 'gold standard' light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of 'gold standard' manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay.


Assuntos
Citometria de Fluxo/métodos , Linfócitos/fisiologia , Testes para Micronúcleos/métodos , Benzimidazóis/farmacologia , Carbamatos/farmacologia , Linhagem Celular , Núcleo Celular/fisiologia , Citocinese/fisiologia , Dano ao DNA/fisiologia , Humanos , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia
15.
Free Radic Biol Med ; 129: 97-106, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30223018

RESUMO

A genetic analysis of synthetic lethal interactions in yeast revealed that the mutation of SOD1, encoding an antioxidant enzyme that scavenges superoxide anion radical, impaired the growth of a set of mutants defective in homologous recombination (HR) pathway. Hence, SOD1 inhibition has been proposed as a promising approach for the selective killing of HR-deficient cancer cells. However, we show that the deletion of RAD51 and SOD1 is not synthetic lethal but displays considerably slow growth and synergistic sensitivity to both reactive oxygen species (ROS)- and DNA double-strand break (DSB)-generating drugs in the budding yeast Saccharomyces cerevisiae. The function of Sod1 in regard to Rad51 is dependent on Ccs1, a copper chaperone for Sod1. Sod1 deficiency aggravates genomic instability in conjunction with the absence of Rad51 by inducing DSBs and an elevated mutation frequency. Inversely, lack of Rad51 causes a Sod1 deficiency-derived increase of intracellular ROS levels. Taken together, our results indicate that there is a significant and specific crosstalk between two major cellular damage response pathways, ROS signaling and DSB repair, for cell survival.


Assuntos
DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Instabilidade Genômica/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Superóxido Dismutase-1/genética , 4-Nitroquinolina-1-Óxido/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/metabolismo , Recombinação Homóloga , Peróxido de Hidrogênio/farmacologia , Hidroxiureia/farmacologia , Metanossulfonato de Metila/farmacologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Paraquat/farmacologia , Fleomicinas/farmacologia , Quinolonas/farmacologia , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/deficiência
16.
Nucleic Acids Res ; 46(17): 9027-9043, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30102394

RESUMO

Nucleases play important roles in nucleic acid metabolism. Some archaea encode a conserved protein known as Hef-associated nuclease (HAN). In addition to its C-terminal DHH nuclease domain, HAN also has three N-terminal domains, including a DnaJ-Zinc-finger, ribosomal protein S1-like, and oligonucleotide/oligosaccharide-binding fold. To further understand HAN's function, we biochemically characterized the enzymatic properties of HAN from Pyrococcus furiosus (PfuHAN), solved the crystal structure of its DHH nuclease domain, and examined its role in DNA repair. Our results show that PfuHAN is a Mn2+-dependent 3'-exonuclease specific to ssDNA and ssRNA with no activity on blunt and 3'-recessive double-stranded DNA. Domain truncation confirmed that the intrinsic nuclease activity is dependent on the C-terminal DHH nuclease domain. The crystal structure of the DHH nuclease domain adopts a trimeric topology, with each subunit adopting a classical DHH phosphoesterase fold. Yeast two hybrid assay confirmed that the DHH domain interacts with the IDR peptide of Hef nuclease. Knockout of the han gene or its C-terminal DHH nuclease domain in Haloferax volcanii resulted in increased sensitivity to the DNA damage reagent MMS. Our results imply that HAN nuclease might be involved in repairing stalled replication forks in archaea.


Assuntos
Proteínas Arqueais/química , Reparo do DNA , DNA de Cadeia Simples/química , Exonucleases/química , Pyrococcus furiosus/enzimologia , RNA Arqueal/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cátions Bivalentes , Clonagem Molecular , Cristalografia por Raios X , Quebras de DNA de Cadeia Simples , Dano ao DNA , Replicação do DNA , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Expressão Gênica , Haloferax volcanii/química , Haloferax volcanii/efeitos dos fármacos , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Cinética , Manganês/química , Manganês/metabolismo , Metanossulfonato de Metila/farmacologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Pyrococcus furiosus/química , Pyrococcus furiosus/efeitos dos fármacos , Pyrococcus furiosus/genética , RNA Arqueal/genética , RNA Arqueal/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
17.
Oxid Med Cell Longev ; 2018: 7820890, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29849914

RESUMO

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, and this class of drugs has been studied as protective agents against DNA damages. Alkylating agents (AAs) are able to induce alkylation in macromolecules, causing DNA damage, as DNA methylation. Our objective was to evaluate atorvastatin (AVA) antimutagenic, cytoprotective, and antigenotoxic potentials against DNA lesions caused by AA. AVA chemopreventive ability was evaluated using antimutagenicity assays (Salmonella/microsome assay), cytotoxicity, cell cycle, and genotoxicity assays in HepG2 cells. The cells were cotreated with AVA and the AA methyl methanesulfonate (MMS) or cyclophosphamide (CPA). Our datum showed that AVA reduces the alkylation-mediated DNA damage in different in vitro experimental models. Cytoprotection of AVA at low doses (0.1-1.0 µM) was observed after 24 h of cotreatment with MMS or CPA at their LC50, causing an increase in HepG2 survival rates. After all, AVA at 10 µM and 25 µM had decreased effect in micronucleus formation in HepG2 cells and restored cell cycle alterations induced by MMS and CPA. This study supports the hypothesis that statins can be chemopreventive agents, acting as antimutagenic, antigenotoxic, and cytoprotective components, specifically against alkylating agents of DNA.


Assuntos
Atorvastatina/farmacologia , Ciclofosfamida/farmacologia , Dano ao DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Alquilantes/química , Alquilantes/farmacologia , Alquilação , Atorvastatina/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Núcleo Celular/química , Núcleo Celular/efeitos dos fármacos , Ciclofosfamida/química , Células Hep G2 , Humanos , Metanossulfonato de Metila/química , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética
18.
Hum Mol Genet ; 27(13): 2306-2317, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29668892

RESUMO

XRCC1 is an essential scaffold protein for base excision repair (BER) and helps to maintain genomic stability. XRCC1 has been indicated as a substrate for small ubiquitin-like modifier modification (SUMOylation); however, how XRCC1 SUMOylation is regulated in cells and how SUMOylated XRCC1 regulates BER activity are not well understood. Here, we show that SUMOylation of XRCC1 is regulated in cells under methyl-methanesulfonate (MMS) treatment and facilitates BER. Poly(ADP-ribose) polymerase 1 (PARP1) is activated by MMS immediately and synthesizes poly(ADP-ribose) (PAR), which in turn promotes recruitment of SUMO E3 TOPORS to XRCC1 and facilitates XRCC1 SUMOylation. A SUMOylation-defective mutant of XRCC1 had lower binding activity for DNA polymerase beta (POLB) and was linked to a lower capacity for repair of MMS-induced DNA damages. Our study therefore identified a pathway in which DNA damage-induced poly(ADP-ribosyl)ation (PARylation) promotes SUMOylation of XRCC1, which leads to more efficient recruitment of POLB to complete BER.


Assuntos
DNA Polimerase beta/genética , Poli ADP Ribosilação/genética , Sumoilação/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Oxirredutases do Álcool/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Humanos , Metanossulfonato de Metila/farmacologia , Poli(ADP-Ribose) Polimerase-1/genética , Ligação Proteica/genética
19.
J Bacteriol ; 200(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29610212

RESUMO

In Vibrio cholerae, high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase (tag) was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of tag that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of tag expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. In vitro binding assays and heterologous host expression studies show that VpsT acts directly at the tag promoter in response to c-di-GMP to induce tag expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in V. cholerae extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade.IMPORTANCEVibrio cholerae is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in V. cholerae between a sessile biofilm-forming state and a motile lifestyle, both of which are important during V. cholerae environmental persistence and human infections. Here, we report that in V. cholerae c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in V. cholerae.


Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Reparo do DNA , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Vibrio cholerae/genética , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Metanossulfonato de Metila/farmacologia , Vibrio cholerae/fisiologia
20.
Nucleic Acids Res ; 46(10): 5061-5074, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29635344

RESUMO

The Set2 methyltransferase and its target, histone H3 lysine 36 (H3K36), affect chromatin architecture during the transcription and repair of DNA double-stranded breaks. Set2 also confers resistance against the alkylating agent, methyl methanesulfonate (MMS), through an unknown mechanism. Here, we show that Schizosaccharomyces pombe (S. pombe) exhibit MMS hypersensitivity when expressing a set2 mutant lacking the catalytic histone methyltransferase domain or a H3K36R mutant (reminiscent of a set2-null mutant). Set2 acts synergistically with base excision repair factors but epistatically with nucleotide excision repair (NER) factors, and determines the timely nuclear accumulation of the NER initiator, Rhp23, in response to MMS. Set2 facilitates Rhp23 recruitment to chromatin at the brc1 locus, presumably to repair alkylating damage and regulate the expression of brc1+ in response to MMS. Set2 also show epistasis with DNA damage checkpoint proteins; regulates the activation of Chk1, a DNA damage response effector kinase; and acts in a similar functional group as proteins involved in homologous recombination. Consistently, Set2 and H3K36 ensure the dynamicity of Rhp54 in DNA repair foci formation after MMS treatment. Overall, our results indicate a novel role for Set2/H3K36me in coordinating the recruitment of DNA repair machineries to timely manage alkylating damage.


Assuntos
Alquilantes/farmacologia , Reparo do DNA/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epistasia Genética , Regulação Fúngica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Lisina/metabolismo , Metanossulfonato de Metila/farmacologia , Metilação/efeitos dos fármacos , Domínios Proteicos , Proteínas de Schizosaccharomyces pombe/genética
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