RESUMEN
Targeting transcription replication conflicts, a major source of endogenous DNA double-stranded breaks and genomic instability could have important anticancer therapeutic implications. Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes. Through a rational drug design approach, we identified a small molecule PCNA inhibitor, AOH1996, which selectively kills cancer cells. AOH1996 enhances the interaction between PCNA and the largest subunit of RNA polymerase II, RPB1, and dissociates PCNA from actively transcribed chromatin regions, while inducing DNA double-stranded breaks in a transcription-dependent manner. Attenuation of RPB1 interaction with PCNA, by a point mutation in RPB1's PCNA-binding region, confers resistance to AOH1996. Orally administrable and metabolically stable, AOH1996 suppresses tumor growth as a monotherapy or as a combination treatment but causes no discernable side effects. Inhibitors of transcription replication conflict resolution may provide a new and unique therapeutic avenue for exploiting this cancer-selective vulnerability.
Asunto(s)
Cromatina , Neoplasias , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Neoplasias/tratamiento farmacológico , ADN , Replicación del ADNRESUMEN
INTRODUCTION: Cognitive-motor interference is the decrease in cognitive performance and/or physical performance occurring when a cognitive task and a physical task are performed concurrently (dual task) compared to when they are performed in isolation (single task). The aim of this study was to investigate the construct validity and test-retest reliability of two cognitive-motor interference tests in military contexts. MATERIALS AND METHODS: Twenty-two soldiers, officers, and cadets performed a 10-min loaded marching, a 10-min Psychomotor Vigilance Task, and the two tasks combined (visit 1). During visit 2, a 5-min running time trial, a 5-min Word Recall Task, and the two tasks combined. These tests were repeated by 20 participants after 2 weeks (visits 3 and 4). RESULTS: Significant impairments were shown on both running distance (P < .001) and number of words recalled (P = .004) in the dual-task condition compared to the single-task condition. Significantly shorter step length (P < .001) and higher step frequency (P < .001) were found during the loaded marching in the dual-task condition compared to the single-task condition. No significant differences were observed in mean reaction time (P = .402) and number of lapses (P = .479) during the Psychomotor Vigilance Task. Good-to-excellent reliability was found for all the cognitive and physical variables in both single- and dual-task conditions, except for the number of lapses. CONCLUSION: These findings suggest that the Running + Word Recall Task test is a valid and reliable dual-tasking test that could be used to assess cognitive-motor interference in military contexts.
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Cognición , Personal Militar , Humanos , Desempeño Psicomotor , Tiempo de Reacción , Reproducibilidad de los ResultadosRESUMEN
Peptides are increasingly being developed for use as therapeutics to treat many ailments, including cancer. Therapeutic peptides have the advantages of target specificity and low toxicity. The anticancer effects of a peptide can be the direct result of the peptide binding its intended target, or the peptide may be conjugated to a chemotherapy drug or radionuclide and used to target the agent to cancer cells. Peptides can be targeted to proteins on the cell surface, where the peptide-protein interaction can initiate internalization of the complex, or the peptide can be designed to directly cross the cell membrane. Peptides can induce cell death by numerous mechanisms including membrane disruption and subsequent necrosis, apoptosis, tumor angiogenesis inhibition, immune regulation, disruption of cell signaling pathways, cell cycle regulation, DNA repair pathways, or cell death pathways. Although using peptides as therapeutics has many advantages, peptides have the disadvantage of being easily degraded by proteases once administered and, depending on the mode of administration, often have difficulty being adsorbed into the blood stream. In this review, we discuss strategies recently developed to overcome these obstacles of peptide delivery and bioavailability. In addition, we present many examples of peptides developed to fight cancer.
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Neoplasias/tratamiento farmacológico , Péptidos/uso terapéutico , Péptidos de Penetración Celular/farmacología , Humanos , Modelos Biológicos , Nanopartículas/química , Péptidos/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma is a particularly difficult cancer to treat due to a lack of effective screening or treatment. Pancreatic cancer cells exhibit high proliferating cell nuclear antigen (PCNA) expression, which is associated with poor prognosis. PCNA, an important nuclear DNA replication and repair protein, regulates a myriad of proteins via the interdomain connector loop. Within this region, amino acids 126-133 are critical for PCNA interactions in cancer cells. Here, we investigate the ability of a decoy cell-penetrating peptide, R9-caPeptide, that mimics the interdomain connector loop region of PCNA to disrupt PCNA-protein interactions in pancreatic cancer cells. Our data suggest that R9-caPeptide causes dose-dependent toxicity in a panel of pancreatic cancer cell lines by inhibiting DNA replication fork progression and PCNA-regulated DNA repair, ultimately causing lethal DNA damage. Overall, these studies lay the foundation for novel therapeutic strategies that target PCNA in pancreatic cancer.
RESUMEN
We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase É (Pol É), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.
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ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Electroforesis/métodos , Complejos Multienzimáticos/análisis , Complejos Multienzimáticos/aislamiento & purificación , Antígenos Virales de Tumores/genética , Extractos Celulares/química , Línea Celular Tumoral , ADN Polimerasa I/aislamiento & purificación , ADN Polimerasa II/aislamiento & purificación , ADN Polimerasa III/aislamiento & purificación , Replicación del ADN , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , Células HeLa , Humanos , Antígeno Nuclear de Célula en Proliferación/análisis , Origen de Réplica/genética , Proteína de Replicación A/aislamiento & purificación , Proteína de Replicación C/aislamiento & purificación , Virus 40 de los Simios/genéticaRESUMEN
Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.
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Permeabilidad de la Membrana Celular , Neuroblastoma/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Humanos , Neuroblastoma/patología , Antígeno Nuclear de Célula en Proliferación/química , Unión Proteica/efectos de los fármacos , Puntos de Control de la Fase S del Ciclo Celular/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin-signaling pathways in Drosophila, demonstrating the utility of TIRFM of tagged sugar transporters to monitor signaling pathways in insects.
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Drosophila melanogaster/genética , Cuerpo Adiposo/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/fisiología , Androstadienos/farmacología , Animales , Animales Modificados Genéticamente , Membrana Celular/metabolismo , Dieta , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Cuerpo Adiposo/citología , Humanos , Antagonistas de Insulina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transporte de Proteínas , Transducción de Señal , WortmaninaRESUMEN
We designed Adaptive Neuromorphic Architecture (ANA) that self-adjusts its inherent parameters (for instance, the resonant frequency) naturally following the stimuli frequency. Such an architecture is required for brain-like engineered systems because some parameters of the stimuli (for instance, the stimuli frequency) are not known in advance. Such adaptivity comes from a circuit element with memory, namely mem-inductor or mem-capacitor (memristor's sisters), which is history-dependent in its behavior. As a hardware model of biological systems, ANA can be used to adaptively reproduce the observed biological phenomena in amoebae.
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Modelos Neurológicos , Redes Neurales de la Computación , Neuronas/fisiología , Neurociencias/instrumentación , Animales , Encéfalo/citología , HumanosRESUMEN
To describe the time course of cellular systems, we integrate ideas from thermodynamics and information theory to discuss the work needed to change the state of the cell. The biological example analyzed is experimental microarray transcription level oscillations of yeast in the different phases as characterized by oxygen consumption. Surprisal analysis was applied to identify groups of transcripts that oscillate in concert and thereby to compute changes in free energy with time. Three dominant transcript groups were identified by surprisal analysis. The groups correspond to the respiratory, early, and late reductive phases. Genes involved in ribosome biogenesis peaked at the respiratory phase. The work to prepare the state is shown to be the sum of the contributions of these groups. We paid particular attention to work requirements during ribosomal building, and the correlation with ATP levels and dissolved oxygen. The suggestion that cells in the respiratory phase likely build ribosomes, an energy intensive process, in preparation for protein production during the S phase of the cell cycle is validated by an experiment. Surprisal analysis thereby provided a useful tool for determining the synchronization of transcription events and energetics in a cell in real time.
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Relojes Biológicos , Regulación Fúngica de la Expresión Génica , Ribosomas/genética , Saccharomyces cerevisiae/genética , Simulación por Computador , Teoría de la Información , Modelos Biológicos , Consumo de Oxígeno , Ribosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Termodinámica , Transcripción GenéticaRESUMEN
The Drosophila insulin receptor (DInR) regulates a diverse array of biological processes including growth, axon guidance, and sugar homeostasis. Growth regulation by DInR is mediated by Chico, the Drosophila homolog of vertebrate insulin receptor substrate proteins IRS1-4. In contrast, DInR regulation of photoreceptor axon guidance in the developing visual system is mediated by the SH2-SH3 domain adaptor protein Dreadlocks (Dock). In vitro studies by others identified five NPXY motifs, one in the juxtamembrane region and four in the signaling C-terminal tail (C-tail), important for interaction with Chico. Here we used yeast two-hybrid assays to identify regions in the DInR C-tail that interact with Dock. These Dock binding sites were in separate portions of the C-tail from the previously identified Chico binding sites. To test whether these sites are required for growth or axon guidance in whole animals, a panel of DInR proteins, in which the putative Chico and Dock interaction sites had been mutated individually or in combination, were tested for their ability to rescue viability, growth and axon guidance defects of dinr mutant flies. Sites required for viability were identified. Unexpectedly, mutation of both putative Dock binding sites, either individually or in combination, did not lead to defects in photoreceptor axon guidance. Thus, either sites also required for viability are necessary for DInR function in axon guidance and/or there is redundancy built into the DInR/Dock interaction such that Dock is able to interact with multiple regions of DInR. We also found that simultaneous mutation of all five NPXY motifs implicated in Chico interaction drastically decreased growth in both male and female adult flies. These animals resembled chico mutants, supporting the notion that DInR interacts directly with Chico in vivo to control body size. Mutation of these five NPXY motifs did not affect photoreceptor axon guidance, segregating the roles of DInR in the processes of growth and axon guidance.
RESUMEN
Genetic and environmental factors are well-studied influences on phenotype; however, time is a variable that is rarely considered when studying changes in cellular phenotype. Time-resolved microarray data revealed genome-wide transcriptional oscillation in a yeast continuous culture system with â¼ 2 and â¼ 4 h periods. We mapped the global patterns of transcriptional oscillations into a 3D map to represent different cellular phenotypes of redox cycles. This map shows the dynamic nature of gene expression in that transcripts are ordered and coupled to each other through time and concentration space. Although cells differed in oscillation periods, transcripts involved in certain processes were conserved in a deterministic way. When oscillation period lengthened, the peak to trough ratio of transcripts increased and the fraction of cells in the unbudded (G0/G1) phase of the cell division cycle increased. Decreasing the glucose level in the culture medium was one way to increase the redox cycle, possibly from changes in metabolic flux. The period may be responding to lower glucose levels by increasing the fraction of cells in G1 and reducing S-phase gating so that cells can spend more time in catabolic processes. Our results support that gene transcripts are coordinated with metabolic functions and the cell division cycle.
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Genoma Fúngico , Fenotipo , Saccharomyces cerevisiae/genética , Transcripción Genética , Ciclo Celular , Fase G1 , Expresión Génica , Fase S , Saccharomyces cerevisiae/metabolismoRESUMEN
Insulin/Insulin-like growth factor signaling regulates homeostasis and growth in mammals, and is implicated in diseases from diabetes to cancer. In Drosophila melanogaster, as in other invertebrates, multiple Insulin-Like Peptides (DILPs) are encoded by a family of related genes. To assess DILPs' physiological roles, we generated small deficiencies that uncover single or multiple dilps, generating genetic loss-of-function mutations. Deletion of dilps1-5 generated homozygotes that are small, severely growth-delayed, and poorly viable and fertile. These animals display reduced metabolic activity, decreased triglyceride levels and prematurely activate autophagy, indicative of "starvation in the midst of plenty," a hallmark of Type I diabetes. Furthermore, circulating sugar levels are elevated in Df [dilp1-5] homozygotes during eating and fasting. In contrast, Df[dilp6] or Df[dilp7] animals showed no major metabolic defects. We discuss physiological differences between mammals and insects that may explain the unexpected survival of lean, 'diabetic' flies.
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Autofagia , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Insulina/genética , Animales , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Eliminación de Gen , Glucosa/metabolismo , Homocigoto , Triglicéridos/metabolismoRESUMEN
The finding of a genome-wide oscillation in transcription that gates cells into S phase and coordinates mitochondrial and metabolic functions has altered our understanding of how the cell cycle is timed and how stable cellular phenotypes are maintained. Here we present the evidence and arguments in support of the idea that everything oscillates, and the rationale for viewing the cell as an attractor from which deterministic noise can be tuned by appropriate coupling among the many feedback loops, or regulons, that make up the transcriptional-respiratory attractor cycle. The existence of this attractor also explains many of the dynamic macroscopic properties of the cell cycle and appears to be the timekeeping oscillator in both cell cycles and circadian rhythms. The path taken by this primordial oscillator in the course of differentiation or drug response may involve period-doubling behavior. Evidence for a relatively high-frequency timekeeping oscillator in yeast and mammalian cells comes from expression array analysis, and GC/MS in the case of yeast, and primarily from macroscopic measures of phase response to perturbation in the case of mammalian cells. Low-amplitude, genome-wide oscillations, a ubiquitous but often unrecognized attribute of phenotype, may be a source of seemingly intractable biological noise in microarray and proteomic studies. These oscillations in transcript and protein levels and the repeated cycles of synthesis and degradation they require, represent a high energy cost to the cell which must, from an evolutionary point of view, be recovered as essential information. We suggest that the information contained in this genome-wide oscillation is the dynamic code that organizes a stable phenotype from an otherwise passive genome.
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Relojes Biológicos/fisiología , Ciclo Celular , Regulación de la Expresión Génica , Procesos Estocásticos , Transcripción Genética , Comunicación Celular , Ritmo Circadiano/fisiología , Perfilación de la Expresión Génica , Genoma , FenotipoRESUMEN
Recent findings of a genome-wide oscillation involving the transcriptome of the budding yeast Saccharomyces cerevisiae suggest that the most promising path to an understanding of the cell as a dynamic system will proceed from carefully designed time-series sampling followed by the development of signal-processing methods suited to molecular biological datasets. When everything oscillates, conventional biostatistical approaches fall short in identifying functional relationships among genes and their transcripts. Worse, based as they are on steady-state assumptions, such approaches may be misleading. In this chapter, we describe the continuous gated synchrony system and the experiments leading to the concept of genome-wide oscillations, and suggest methods of analysis better suited to dissection of oscillating systems. Using a yeast continuous-culture system, the most precise and stable biological system extant, we explore analytical tools such as wavelet multiresolution decomposition, Fourier analysis, and singular value decomposition to uncover the dynamic architecture of phenotype.
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Biología Molecular/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proyectos de Investigación , Transducción de Señal , Genoma Fúngico , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
Perturbation of the gated-synchrony system in yeast with phenelzine, an antidepressant drug used in the treatment of affective disorders in humans, leads to a rapid lengthening in the period of the genome-wide transcriptional oscillation. The effect is a concerted, genome-scale change in expression that is first seen in genes maximally expressed in the late-reductive phase of the cycle, doubling the length of the reductive phase within two cycles after treatment. Clustering of genes based on their temporal patterns of expression yielded just three super clusters whose trajectories through time could then be mapped into a simple 3D figure. In contrast to transcripts in the late-reductive phase, most transcripts do not show transients in expression relative to others in their temporal cluster but change their period in a concerted fashion. Mapping the trajectories of the transcripts into low-dimensional surfaces that can be represented by simple systems of differential equations provides a readily testable model of the dynamic architecture of phenotype. In this system, period doubling may be a preferred pathway for phenotypic change. As a practical matter, low-amplitude, genome-wide oscillations, a ubiquitous but often unrecognized attribute of phenotype, could be a source of seemingly intractable biological noise in microarray studies.
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Genoma Fúngico/genética , Fenotipo , Saccharomyces cerevisiae/genética , Transcripción Genética/genética , División Celular , Modelos Genéticos , Oxígeno/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
Synaptic growth is essential for the development and plasticity of neural circuits. To identify molecular mechanisms regulating synaptic growth, we performed a gain-of-function screen for synapse morphology mutants at the Drosophila neuromuscular junction (NMJ). We isolated a B' regulatory subunit of protein phosphatase 2A (PP2A) that we have named well-rounded (wrd). Neuronal overexpression of wrd leads to overgrowth of the synaptic terminal. Endogenous Wrd protein is present in the larval nervous system and muscle and is enriched at central and neuromuscular synapses. wrd is required for normal synaptic development; in its absence, there are fewer synaptic boutons and there is a decrease in synaptic strength. wrd functions presynaptically to promote normal synaptic growth and postsynaptically to maintain normal levels of evoked transmitter release. In the absence of wrd, the presynaptic cytoskeleton is abnormal, with an increased proportion of unbundled microtubules. Reducing PP2A enzymatic activity also leads to an increase in unbundled microtubules, an effect enhanced by reducing wrd levels. Hence, wrd promotes the function of PP2A and is required for normal cytoskeletal organization, synaptic growth, and synaptic function at the Drosophila NMJ.
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Citoesqueleto/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Unión Neuromuscular/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2/metabolismo , Sinapsis/fisiología , Animales , Aumento de la Célula , Células Cultivadas , Activación Enzimática , Unión Neuromuscular/citología , Sinapsis/ultraestructura , UltrasonografíaRESUMEN
The large subunit of Saccharomyces cerevisiae DNA polymerase epsilon, Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase sigma (Pol sigma), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol sigma stimulates the polymerase activity of the Pol epsilon holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol epsilon, like Pol sigma, might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5 Delta mutants, like trf4 Delta mutants, are defective in DNA repair and sister chromatid cohesion.
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ADN Nucleotidiltransferasas/metabolismo , ADN Polimerasa II/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromátides/fisiología , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Nucleotidiltransferasas/química , ADN Nucleotidiltransferasas/genética , ADN Polimerasa II/química , ADN Polimerasa II/genética , Reparación del ADN , ADN de Hongos/biosíntesis , ADN de Hongos/genética , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Genes Fúngicos , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
The hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal-distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia.
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Cabello/citología , Mosaicismo , Animales , Linaje de la Célula , Quimera , Células Epiteliales/citología , Cabello/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB CRESUMEN
DNA polymerase epsilon (pol epsilon) is a multiple subunit complex consisting of at least four proteins, including catalytic Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol epsilon has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol epsilon complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol epsilon complex and truncated Pol2p (140 kDa), as was observed initially with the pol epsilon purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol epsilon is functionally identical to native pol epsilon purified from yeast and is therefore useful for biochemical characterization of the interactions of pol epsilon with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Pol2p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Pol2p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc.