RESUMEN
Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.
Asunto(s)
ARN Largo no Codificante/metabolismo , Telómero/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Células A549 , Bleomicina/farmacología , G-Cuádruplex , Humanos , Hibridación Fluorescente in Situ , Microscopía Fluorescente , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Interferencia de ARN , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Recombinasas/antagonistas & inhibidores , Recombinasas/genética , Recombinasas/metabolismo , Telómero/química , Proteína 1 de Unión a Repeticiones Teloméricas/antagonistas & inhibidores , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/antagonistas & inhibidores , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The DNA replication machinery encounters problems at numerous genomic regions that are inherently difficult to replicate. These genomic regions include telomeres, which contain repetitive DNA and telomere-binding proteins. If not properly regulated, replication of such genomic regions can result in DNA damage, leading to genomic instability. Studies implicated a role of Timeless-related proteins at difficult-to-replicate genomic regions, including telomeres. However, how these proteins maintain telomeres was elusive. In a recent report, we described the role of Swi1, a Timeless-related protein, in telomere maintenance in fission yeast. We demonstrated that Swi1 is required for proper replication of repeat DNA sequences at telomeres. We also showed that Swi1-deficient cells utilize recombination-based ALT (alternative lengthening of telomeres)-like mechanisms to maintain telomeres in the absence of telomerase. Here, we highlight these findings and present additional data to discuss the role of Swi1Timeless in telomere protection and ALT prevention.
Asunto(s)
Telómero/genética , Telómero/metabolismo , Proteínas Portadoras , Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Genoma , Genómica , Unión Proteica , Recombinasas/antagonistas & inhibidores , Recombinasas/metabolismo , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Telomerasa/metabolismo , Homeostasis del TelómeroRESUMEN
Among DNA damages, double-strand breaks (DSBs) are one of the most harmful lesions to a cell. Failure in DSB repair could lead to genomic instability and cancer. Homologous recombination (HR) and nonhomologous end joining (NHEJ) are major DSB repair pathways in higher eukaryotes. It is known that expression of DSB repair genes is altered in various cancers. Activation of DSB repair genes is one of the reasons for chemo- and radioresistance. Therefore, targeting DSB repair is an attractive strategy to eliminate cancer. Besides, therapeutic agents introduce breaks in the genome as an intermediate. Therefore, blocking the residual repair using inhibitors can potentiate the efficacy of cancer treatment. In this review, we discuss the importance of targeting DSB repair pathways for the treatment of cancer. Recent advances in the development of DSB repair inhibitors and their clinical relevance are also addressed.
Asunto(s)
Reparación del ADN , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Recombinasas/antagonistas & inhibidores , Recombinasas/metabolismo , Recombinación Genética/efectos de los fármacos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mus81 resolvase and Sgs1 helicase have well-established roles in mitotic DNA repair. Moreover, Mus81 is part of a minor crossover (CO) pathway in the meiosis of budding yeast, plants and vertebrates. The major pathway depends on meiosis-specific synaptonemal complex (SC) formation, ZMM proteins and the MutLγ complex for CO-directed resolution of joint molecule (JM)-recombination intermediates. Sgs1 has also been implicated in this pathway, although it may mainly promote the non-CO outcome of meiotic repair. We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions. Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena. Sgs1 may exert functions similar to those in other eukaryotes. However, we propose an additional role in supporting homologous CO formation by promoting homologous over intersister interactions. Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast. We propose that in these two organisms, which independently lost the SC during evolution, the basal set of mitotic repair proteins is sufficient for executing meiotic recombination.
Asunto(s)
Endodesoxirribonucleasas/fisiología , Meiosis/genética , RecQ Helicasas/fisiología , Recombinasas/fisiología , Recombinación Genética , Núcleo Celular/enzimología , Cromátides , Segregación Cromosómica , ADN/química , ADN/metabolismo , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Mutación , Interferencia de ARN , RecQ Helicasas/análisis , RecQ Helicasas/antagonistas & inhibidores , Recombinasas/análisis , Recombinasas/antagonistas & inhibidores , Complejo Sinaptonémico , Tetrahymena thermophila/enzimología , Tetrahymena thermophila/genéticaRESUMEN
The antibacterial activity and mechanism of silver nanoparticles (Ag-NPs) on Staphylococcus aureus ATCC 6538P were investigated in this study. The experiment results showed the minimum bactericidal concentration (MBC) of Ag-NPs to S. aureus was 20 µg/ml. Moreover, when bacteria cells were exposed to 50 µg/ml Ag-NPs for 6 h, the cell DNA was condensed to a tension state and could have lost their replicating abilities. When S. aureus cells were exposed to 50 µg/ml Ag-NPs for 12 h, the cell wall was breakdown, resulting in the release of the cellular contents into the surrounding environments, and finally became collapsed. And Ag-NPs could reduce the enzymatic activity of respiratory chain dehydrogenase. Furthermore, the proteomic analysis showed that the expression abundance of some proteins was changed in the treated bacterial cell with Ag-NPs, formate acetyltransferase increased 5.3-fold in expression abundance, aerobic glycerol-3-phosphate dehydrogenase decreased 6.5-fold, ABC transporter ATP-binding protein decreased 6.2-fold, and recombinase A protein decreased 4.9-fold.
Asunto(s)
Antibacterianos/farmacología , Nanopartículas del Metal/química , Plata/farmacología , Staphylococcus aureus/efectos de los fármacos , Acetiltransferasas/metabolismo , Antibacterianos/química , Pared Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glicerolfosfato Deshidrogenasa/antagonistas & inhibidores , Glicerolfosfato Deshidrogenasa/metabolismo , Pruebas de Sensibilidad Microbiana , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Proteómica , Recombinasas/antagonistas & inhibidores , Recombinasas/metabolismo , Plata/química , Staphylococcus aureus/citología , Staphylococcus aureus/enzimologíaRESUMEN
Lyme disease, the most common vector-borne zoonosis in North America, is caused by the spirochetal pathogen Borrelia burgdorferi. The telomere resolvase encoded by this organism (ResT) promotes the formation of covalently closed hairpin ends on the linear DNA molecules of B. burgdorferi through a two-step transesterification. ResT is essential for survival and is therefore an attractive target for the development of highly specific antiborrelial drugs. To identify ResT inhibitors, a novel fluorescence-based high-throughput assay was developed and used to screen a library of 27,520 small-molecule drug-like compounds. Six confirmed inhibitors of ResT, with 50% inhibitory concentrations between 2 and 10 muM, were identified. The inhibitors were characterized further and were grouped into three distinct classes based on their inhibitory features. The high-throughput screening assay developed in this paper, along with the six inhibitory compounds identified, provides a starting point for the future development of novel antiborrelial drugs as well as small-molecule inhibitors that will be helpful for the further dissection of the reaction mechanism.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Borrelia burgdorferi/efectos de los fármacos , Borrelia burgdorferi/enzimología , Inhibidores Enzimáticos/farmacología , Enfermedad de Lyme/microbiología , Recombinasas/antagonistas & inhibidores , Animales , HumanosRESUMEN
DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Recombinasas/antagonistas & inhibidores , Proteínas Arqueales/clasificación , Proteínas Arqueales/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/genética , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Estructura Cuaternaria de Proteína , Recombinasas/metabolismo , Alineación de Secuencia , Sulfolobus solfataricus/enzimología , Sulfolobus solfataricus/genéticaRESUMEN
FimB and FimE are site-specific recombinases, part of the lambda integrase family, and invert a 314 bp DNA switch that controls the expression of type 1 fimbriae in Escherichia coli. FimB and FimE differ in their activity towards the fim switch, with FimB catalysing inversion in both directions in comparison to the higher-frequency but unidirectional on-to-off recombination catalysed by FimE. Previous work has demonstrated that FimB, but not FimE, recombination is completely inhibited in vitro and in vivo by a regulator, PapB, expressed from a distinct fimbrial locus. The aim of this work was to investigate differences between FimB and FimE activity by exploiting the differential inhibition demonstrated by PapB. The research focused on genetic changes to the fim switch that alter recombinase binding and its structural context. FimB and FimE still recombined a switch in which the majority of fimS DNA was replaced with a larger region of non-fim DNA. This demonstrated a minimal requirement for FimB and FimE recombination of the Fim binding sites and associated inverted repeats. With the original leucine-responsive regulatory protein (Lrp) and integration host factor (IHF)-dependent structure removed, PapB was now able to inhibit both recombinases. The relative affinities of FimB and FimE were determined for the four 'half sites'. This analysis, along with the effect of extensive swaps and duplications of the half sites on recombination frequency, demonstrated that FimB recruitment and therefore subsequent activity was dependent on a single half site and its context, whereas FimE recombination was less stringent, being able to interact initially with two half sites with equally high affinity. While increasing FimB recombination frequencies failed to overcome PapB repression, mutations made in recombinase binding sites resulted in inhibition of FimE recombination by PapB. Overall, the data support a model in which the recombinases differ in loading order and co-operative interactions. PapB exploits this difference and FimE becomes susceptible when its normal loading is restricted or changed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Integrasas/metabolismo , Recombinasas/metabolismo , Recombinación Genética , Secuencia de Bases , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Integrasas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Recombinasas/antagonistas & inhibidores , Recombinasas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded (ss) DNA-dependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1Delta or rad54Delta mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.