RESUMEN
Activating transcription factor 3 (ATF3) is a highly regulated protein that is implicated in a wide range of pathological conditions including inflammation and transformation. Transcription from the ATF3 gene is induced by several stress-induced signaling pathways, including amino acid limitation (amino acid response, AAR) and ER stress (unfolded protein response, UPR). Induction of ATF3 transcription by these pathways is mediated by ATF4 and cJUN recruitment to enhancer elements within the ATF3 gene. Although a canonical promoter (promoter A) has been studied by numerous laboratories, a second promoter activity (promoter A1), 43â¯kb upstream of the first, has been reported to respond to stress-induced signaling and to be critical for ATF3 expression in certain transformed cells. The results of the present study show that in normal human hepatocytes and HepG2 human hepatoma cells both basal as well as AAR- and UPR-induced transcription occurs almost exclusively from promoter A. This selectivity between the two promoters correlated with increased binding of ATF4, recruitment of RNA polymerase II, and the expected histone modifications in the promoter A region of the gene. Time course studies of ATF3 transcription activity revealed that the temporal kinetics for ATF3 induction differ between the AAR and UPR, with the former being more transient than the latter. Collectively, the results document that ATF3 expression in normal and transformed human liver originates from the canonical promoter A that responds to multiple stress signals.
Asunto(s)
Factor de Transcripción Activador 3/genética , Aminoácidos/metabolismo , Estrés del Retículo Endoplásmico/genética , Hepatocitos/metabolismo , Regiones Promotoras Genéticas/genética , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Células Cultivadas , Células Hep G2 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Elementos de Respuesta/genética , Transcripción Genética , Respuesta de Proteína Desplegada/genéticaRESUMEN
Amino acid (AA) deprivation in mammalian cells activates a collection of signaling cascades known as the AA response (AAR), which is characterized by transcriptional induction of stress-related genes, including FBJ murine osteosarcoma viral oncogene homolog (cFOS). The present study established that the signaling mechanism underlying the AA-dependent transcriptional regulation of the cFOS gene in HepG2 human hepatocellular carcinoma cells is independent of the classic GCN2-eIF2-ATF4 pathway. Instead, a RAS-RAF-MEK-ERK cascade mediates AAR signaling to the cFOS gene. Increased cFOS transcription is observed from 4-24 h after AAR-activation, exhibiting little or no overlap with the rapid and transient increase triggered by the well-known serum response. Furthermore, serum is not required for the AA-responsiveness of the cFOS gene and no phosphorylation of promoter-bound serum response factor (SRF) is observed. The ERK-phosphorylated transcription factor E-twenty six-like (p-ELK1) is increased in its association with the cFOS promoter after activation of the AAR. This research identified cFOS as a target of the AAR and further highlights the importance of AA-responsive MAPK signaling in HepG2 cells.
Asunto(s)
Aminoácidos/deficiencia , Carcinoma Hepatocelular/genética , Genes fos/genética , Neoplasias Hepáticas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Factor de Transcripción Activador 4/fisiología , Aminoácidos/farmacología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Células Cultivadas , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteínas Serina-Treonina Quinasas/fisiología , Activación Transcripcional/efectos de los fármacosRESUMEN
DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the Saccharomyces cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the ß-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway.
Asunto(s)
Adenosina Trifosfato/química , Antígeno Nuclear de Célula en Proliferación/química , Proteína de Replicación C/química , Saccharomyces cerevisiae/química , Adenosina Trifosfato/metabolismo , Unión Competitiva , Catálisis , Cumarinas/química , Cumarinas/metabolismo , ADN/química , ADN/metabolismo , Cinética , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Moleculares , Mutación , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3'OH (3'DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3'DNA, fluorescent ß and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5'phosphate (5'P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5'DNA). The 5'P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3'DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5'DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3'DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Adenosina Trifosfatasas/metabolismo , ADN/química , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismo , ARN/metabolismo , Proteína de Replicación A/metabolismo , Proteína de Replicación C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Moldes GenéticosRESUMEN
Excision repair processes are essential to maintain genome stability. A decrease in efficiency and fidelity of these pathways at regions of the genome that can assume non-canonical DNA structures has been proposed as a possible mechanism to explain the increased mutagenesis and consequent diseased state frequently associated with these sites. Here we describe the development of a FRET-based approach to monitor the presence of G quadruplex (G4) DNA, a non-canonical DNA structure formed in runs of guanines, in damage-containing single-stranded and double-stranded DNA. Using this approach, we directly show for the first time that the presence within the G4 structure of an abasic site, the most common lesion spontaneously generated during cellular metabolism, decreases the efficiency of human AP endonuclease activity and that this effect is mostly the result of a decreased enzymatic activity and not of decreased binding of the enzyme to the damaged site. This approach can be generally applied to dissecting the biochemistry of DNA repair at non-canonical DNA structures.
Asunto(s)
Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , G-Cuádruplex , ADN/química , ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Genes myc , Polietilenglicoles/químicaRESUMEN
Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the Saccharomyces cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All mutants reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps, suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of proliferating cell nuclear antigen in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading.
Asunto(s)
ADN de Hongos/química , ADN Polimerasa Dirigida por ADN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Secuencias de Aminoácidos , ADN de Hongos/biosíntesis , ADN de Hongos/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Mutación Puntual , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Escherichia coli γ complex clamp loader functions to load the ß sliding clamp onto sites of DNA replication and repair. The clamp loader uses the energy of ATP binding and hydrolysis to drive conformational changes allowing for ß binding and opening, DNA binding, and then release of the ß·DNA complex. Although much work has been done studying the sliding clamp and clamp loader mechanism, kinetic analysis of the events following ߷γ complex·DNA formation is not complete. Using fluorescent clamp closing and release assays, we show that ß closing is faster than ß release, indicating that γ complex closes ß before releasing it around DNA. Using a fluorescent ATP hydrolysis assay, we show that there is a burst of ATP hydrolysis before ß closing and that ß release may be the rate-limiting step in the overall clamp loading reaction. The combined use of these fluorescent assays provides a unique perspective into the E. coli clamp loader by providing a measure of the relative timing of different events in the clamp loading reaction, helping to elucidate the complicated clamp loading mechanism.
Asunto(s)
ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Adenosina Trifosfato/metabolismo , HidrólisisRESUMEN
Molecular events in the clamp-loading reaction pathway of DNA replication are revealed by new crystal structures of bacteriophage T4 clamp loader-clamp-DNA complexes that capture two distinct conformations with the clamp open and closed.
Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriófago T4/genética , ADN Viral/biosíntesis , ADN Viral/química , Escherichia coli/genética , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds ß first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.