RESUMO
Arsenic is a well-established human carcinogen of poorly understood mechanism of genotoxicity. It is generally accepted that arsenic acts indirectly by generating oxidative DNA damage that can be converted to replication-dependent DNA double-strand breaks (DSBs), as well as by interfering with DNA repair pathways and DNA methylation. Here we show that in budding yeast arsenic also causes replication and transcription-independent DSBs in all phases of the cell cycle, suggesting a direct genotoxic mode of arsenic action. This is accompanied by DNA damage checkpoint activation resulting in cell cycle delays in S and G2/M phases in wild type cells. In G1 phase, arsenic activates DNA damage response only in the absence of the Yku70-Yku80 complex which normally binds to DNA ends and inhibits resection of DSBs. This strongly indicates that DSBs are produced by arsenic in G1 but DNA ends are protected by Yku70-Yku80 and thus invisible for the checkpoint response. Arsenic-induced DSBs are processed by homologous recombination (HR), as shown by Rfa1 and Rad52 nuclear foci formation and requirement of HR proteins for cell survival during arsenic exposure. We show further that arsenic greatly sensitizes yeast to phleomycin as simultaneous treatment results in profound accumulation of DSBs. Importantly, we observed a similar response in fission yeast Schizosaccharomyces pombe, suggesting that the mechanisms of As(III) genotoxicity may be conserved in other organisms.
Assuntos
Arsênio/toxicidade , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Replicação do DNA/genética , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Recombinação Homóloga/efeitos dos fármacos , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genéticaRESUMO
ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Anfotericina B/farmacologia , Antifúngicos/farmacologia , Fosfatidilserinas/fisiologia , Saccharomyces cerevisiae/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Depsipeptídeos/farmacologia , Expressão Gênica , Células HeLa , Humanos , Camundongos , Natamicina/farmacologia , Fosfatidilserinas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Esfingolipídeos/fisiologiaRESUMO
The Saccharomyces cerevisiae Uls1 belongs to the Swi2-Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here, we examine a physiological role of Uls1 and report for the first time its involvement in response to replication stress. We found that deletion of ULS1 in cells lacking RAD52 caused a synthetic growth defect accompanied by prolonged S phase and aberrant cell morphology. uls1Δ also progressed slower through S phase upon MMS treatment and took longer to resolve replication intermediates during recovery. This suggests an important function for Uls1 during replication stress. Consistently, cells lacking Uls1 and endonuclease Mus81 were more sensitive to HU, MMS and CPT than single mus81Δ. Interestingly, deletion of ULS1 attenuated replication stress-related defects in sgs1Δ, such as sensitivity to HU and MMS while increasing the level of PCNA ubiquitination and Rad53 phosphorylation. Importantly, Uls1 interactions with Mus81 and Sgs1 were dependent on its helicase domain. We propose that Uls1 directs a subset of DNA structures arising during replication into the Sgs1-dependent pathway facilitating S phase progression. Thus, in the absence of Uls1 other modes of replication fork processing and repair are employed.
Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , Replicação do DNA , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Adenosina Trifosfatases/genética , Dano ao DNA , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Deleção de Genes , Proteína Rad52 de Recombinação e Reparo de DNA/genética , RecQ Helicases/genética , Fase S , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico , Ubiquitina-Proteína Ligases/genéticaRESUMO
Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum ß-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with "phiKMV-like viruses". Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.
Assuntos
Bacteriófagos/isolamento & purificação , Klebsiella pneumoniae/virologia , Podoviridae/isolamento & purificação , Esgotos/virologia , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Genoma Viral , Especificidade de Hospedeiro , Dados de Sequência Molecular , Filogenia , Podoviridae/classificação , Podoviridae/genética , Podoviridae/fisiologia , Proteínas Virais/genéticaRESUMO
The Saccharomyces cerevisiae Yhl035p/Vmr1p is an ABC transporter of the MRP subfamily that is conserved in all post Whole Genome Duplication species. The deletion of the YHL035 gene caused growth sensitivity to several amphiphilic drugs such as cycloheximide, 2,4-dichlorophenoxyacetic acid, 2,4-dinitrophenol as well as to cadmium and other toxic metals. Vmr1p-GFP was located in the vacuolar membrane. The ATP-dependent transport of a DNP-S-glutathione conjugate was reduced in a vesicular fraction from the VMR1 deletant. The energy-dependent efflux of rhodamine 6G was increased by VMR1 deletion. Growth sensitivity to cadmium of the VMR1-deleted strain was more pronounced in glycerol/ethanol than in glucose-grown cells. The VMR1 promoter had higher activity when grown in glycerol/ethanol compared with glucose. In glucose, the VMR1 promoter was activated by the deletion of the glucose-dependent repressor ADR1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Farmacorresistência Fúngica Múltipla , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Vacúolos/enzimologia , Ácido 2,4-Diclorofenoxiacético/toxicidade , 2,4-Dinitrofenol/toxicidade , Transportadores de Cassetes de Ligação de ATP/genética , Antifúngicos/toxicidade , Cádmio/toxicidade , Cicloeximida/toxicidade , Deleção de Genes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
The stress-activated kinase Hog1p mediates arsenic tolerance by decreasing arsenite influx through the aquaglyceroporin Fps1p in Saccharomyces cerevisiae. Unexpectedly, we found that overexpression of FPS1 increased arsenite tolerance suggesting a physiological role of Fps1p in arsenic detoxification. Consistently, during arsenite treatment transcription of FPS1 gene was strongly upregulated, while Fps1p was not degraded and remained localized to the plasma membrane. Moreover, deletion of FPS1 gene resulted in arsenate sensitivity. Finally, transport experiments revealed that Fps1p in concert with the arsenite transporter Acr3p mediates arsenite efflux.
Assuntos
Arsenitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aquagliceroporinas/genética , Aquagliceroporinas/metabolismo , Arsênio/farmacologia , Transporte Biológico , Western Blotting , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360-375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59-66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive reaction (red emission). This suggests that even one protein may be responsible for favouring stronger interactions between Pseudomonas aeruginosa cells and cationic liposomal formulations (PC:Chol:DOTAP 3:4:3).
Assuntos
Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Parede Celular/efeitos dos fármacos , Colesterol/farmacologia , Ácidos Graxos Monoinsaturados/farmacologia , Fosfatidilcolinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Antibacterianos/administração & dosagem , Antibacterianos/química , Cátions , Parede Celular/metabolismo , Colesterol/química , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos Monoinsaturados/química , Corantes Fluorescentes , Interações Hidrofóbicas e Hidrofílicas , Lipopolissacarídeos/metabolismo , Lipossomos , Microscopia de Fluorescência , Fosfatidilcolinas/química , Pseudomonas aeruginosa/metabolismo , Compostos de Amônio Quaternário/química , Eletricidade EstáticaRESUMO
The mitochondrial genomes of some Phaseolus species contain a fragment of chloroplast trnA gene intron, named pvs-trnA for its location within the Phaseolus vulgaris sterility sequence (pvs). The purpose of this study was to determine the type of transfer (intracellular or horizontal) that gave rise to pvs-trnA. Using a PCR approach we could not find the respective portion of the trnA gene as a part of pvs outside the Phaseolus genus. However, a BLAST search revealed longer fragments of trnA present in the mitochondrial genomes of some Citrus species, Helianthus annuus and Zea mays. Basing on the identity or near-identity between these mitochondrial sequences and their chloroplast counterparts we concluded that they had relocated from chloroplasts to mitochondria via recent, independent, intracellular DNA transfers. In contrast, pvs-trnA displayed a relatively higher sequence divergence when compared with its chloroplast counterpart from Phaseolus vulgaris. Alignment of pvs-trnA with corresponding trnA fragments from 35 plant species as well as phylogenetic analysis revealed that pvs-trnA grouped with non-eudicot sequences and was well separated from all Fabales sequences. In conclusion, we propose that pvs-trnA arose via horizontal transfer of a trnA intron fragment from chloroplast of a non-eudicot plant to Phaseolus mitochondria. This is the first example of horizontal transfer of a chloroplast sequence to the mitochondrial genome in higher plants.