RESUMEN
The computational design of synthetic DNA sequences with designer in vivo properties is gaining traction in the field of synthetic genomics. We propose here a computational method which combines a kinetic Monte Carlo framework with a deep mutational screening based on deep learning predictions. We apply our method to build regular nucleosome arrays with tailored nucleosomal repeat lengths (NRL) in yeast. Our design was validated in vivo by successfully engineering and integrating thousands of kilobases long tandem arrays of computationally optimized sequences which could accommodate NRLs much larger than the yeast natural NRL (namely 197 and 237 bp, compared to the natural NRL of â¼165 bp). RNA-seq results show that transcription of the arrays can occur but is not driven by the NRL. The computational method proposed here delineates the key sequence rules for nucleosome positioning in yeast and should be easily applicable to other sequence properties and other genomes.
Asunto(s)
Nucleosomas , Saccharomyces cerevisiae , Nucleosomas/metabolismo , Nucleosomas/genética , Nucleosomas/química , Saccharomyces cerevisiae/genética , Simulación por Computador , Método de Montecarlo , ADN/genética , ADN/química , ADN/metabolismo , Secuencia de Bases , Aprendizaje Profundo , Ensamble y Desensamble de CromatinaRESUMEN
G-quadruplexes (G4s) are nucleic acids secondary structures that may form in guanine-rich sequences, either intra or inter-molecularly. Ability of a primary sequence to form a G4 can be predicted computationally with an improving accuracy as well as tested in bulk using biophysical measurements. As a result, G4 density maps have been devised for a large number of genomes from all life kingdoms. Experimental validation of the formation of G4s in vivo however remains indirect and relies on their stabilization with small molecules, antibodies or proteins, or mutational studies, in order to measure downstream effects on gene expression or genome stability for example. Although numerous techniques exist to observe spontaneous formation of G4s in single-stranded DNA, observing G4 formation in double-stranded DNA (dsDNA) is more challenging. However, it is particularly relevant to understand if a given G4 sequence forms stably in a dsDNA context, if it is stable enough to dock proteins or pose a challenge to molecular motors such as helicases or polymerases. In essence, G4s can be a threat to genomic stability but carry as well as the potential to be elements of a structural language in the non-replicating genome. To study quantitatively the formation dynamics and stability of single intramolecular G4s embedded in dsDNA, we have adapted techniques of DNA manipulation under magnetic tweezers. This technique also allows to study encounters of molecular motors with G4 at a single molecule resolution, in order to gain insight into the specificity of G4 resolution by molecular motors, and its efficiency. The procedures described here include the design of the G4 substrate, the study of G4 formation probability and lifetime in dsDNA, as well as procedures to characterize the encounter between the Pif1 helicase and a G4 until G4 resolution. The procedures that we described here can easily be extended to the study of other G4s or molecular motors.
Asunto(s)
ADN , G-Cuádruplex , Humanos , ADN/metabolismo , ADN de Cadena Simple , Mutación , Inestabilidad Genómica , Fenómenos MagnéticosRESUMEN
G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in the presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in the presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.
Asunto(s)
G-Cuádruplex , Proteínas de Saccharomyces cerevisiae , ADN Helicasas/metabolismo , ADN/genética , ADN/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Recombinasas/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The artificial 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, application of this property in vivo has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro, analysis of nucleosome occupancy in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.
Asunto(s)
Nucleosomas , Saccharomyces cerevisiae , Secuencias Repetidas en Tándem , Ensamble y Desensamble de Cromatina , ADN de Hongos/genética , ADN de Hongos/metabolismo , Ingeniería Genética , Nucleosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencias Repetidas en Tándem/genéticaRESUMEN
The quest for small molecules that strongly bind to G-quadruplex-DNA (G4), so-called G4 ligands, has invigorated the G4 research field from its very inception. Massive efforts have been invested to discover or rationally design G4 ligands, evaluate their G4-interacting properties in vitro through a series of now widely accepted and routinely implemented assays, and use them as innovative chemical biology tools to interrogate cellular networks that might involve G4s. In sharp contrast, only uncoordinated efforts aimed at developing small molecules that destabilize G4s have been invested to date, even though it is now recognized that such molecular tools would have tremendous application in neurobiology as many genetic and age-related diseases are caused by an overrepresentation of G4s. Herein, we report on our efforts to develop in vitro assays to reliably identify molecules able to destabilize G4s. This workflow comprises the newly designed G4-unfold assay, adapted from the G4-helicase assay implemented with Pif1, as well as a series of biophysical and biochemical techniques classically used to study G4/ligand interactions (CD, UV-vis, PAGE, and FRET-melting), and a qPCR stop assay, adapted from a Taq-based protocol recently used to identify G4s in the genomic DNA of Schizosaccharomyces pombe. This unique, multipronged approach leads to the characterization of a phenylpyrrolocytosine (PhpC)-based G-clamp analog as a prototype of G4-disrupting small molecule whose properties are validated through many different and complementary in vitro evaluations.
Asunto(s)
ADN/química , Bibliotecas de Moléculas Pequeñas/química , G-Cuádruplex , Humanos , Ligandos , Estructura MolecularRESUMEN
G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA metabolic transactions. While many in vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in vivo in double-stranded DNA regions, where their formation is challenged by the complementary strand. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on G4 persistence. To address this, we designed a single-molecule assay allowing to measure G4 folding and persistence times in the presence of the complementary strand. We quantified both folding and unfolding rates of biologically relevant G4 sequences, such as the cMYC and cKIT oncogene promoters, human telomeres and an avian replication origin. We confirmed that G4s are found much more stable in tested replication origin and promoters than in human telomere repeats. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence time increased. Our assay opens new perspectives for the measurement of G4 dynamics in double-stranded DNA mimicking a replication fork, which is important to understand their role in DNA replication and gene regulation at a mechanistic level.
Asunto(s)
ADN/química , G-Cuádruplex , Animales , Pollos/genética , Dimerización , Humanos , Ligandos , Oncogenes , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Origen de Réplica , Telómero/químicaRESUMEN
DNA hemicatenanes (HCs) are four-way junctions in which one strand of a double-stranded helix is catenated with one strand of another double-stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, we sought to purify proteins capable of binding specifically HCs by fractionating nuclear extracts from HeLa cells. This approach identified three RNA-binding proteins: the Tudor-staphylococcal nuclease domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the paraspeckle protein component 1 and the splicing factor proline- and glutamine-rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in Escherichia coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited specificity for HCs, opening the interesting possibility of a link between the basic transcription machinery and HC structures via SND1.
Asunto(s)
Catenanos/metabolismo , ADN/genética , Endonucleasas/genética , Transcripción Genética , Animales , Catenanos/química , Cromosomas/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/metabolismo , Células HeLa , Humanos , Factor de Empalme Asociado a PTB/genética , Unión Proteica/genética , Proteínas de Unión al ARN/genética , Recombinación Genética/genéticaRESUMEN
DNA repeats constitute a large part of genomes of multicellular eucaryotes. For a longtime considered as junk DNA, their role in genome organization and tuning of gene expression is being increasingly documented. Synthetic biology has so far largely ignored DNA repeats as regulatory elements to manipulate functions in engineered genomes. The yeast Saccharomyces cerevisiae has been a workhorse of synthetic biology, owing to its genetic tractability. Here we demonstrate the ability to synthetize, in a simple manner, tandem DNA repeats of various size by Cas9-assisted oligonucleotide in vivo assembly in this organism. We show that long tandem DNA repeats of several kilobases can be assembled in one step for different monomer size and G/C content. The combinatorial nature of the approach allows exploring a wide variety of design for building synthetic tandem repeated DNA directly at a given locus in the Saccharomyces cerevisiae genome. This approach provides a simple way to incorporate tandem DNA repeat in synthetic genome designs to implement regulatory functions.
Asunto(s)
Sistemas CRISPR-Cas/genética , ADN/genética , Ingeniería Genética , Secuencias Repetidas en Tándem/genética , Genoma Fúngico/genética , Oligonucleótidos/genética , Saccharomyces cerevisiae/genética , Biología SintéticaRESUMEN
Understanding the effect of an ever-growing number of human variants detected by genome sequencing is a medical challenge. The yeast Saccharomyces cerevisiae model has held attention for its capacity to monitor the functional impact of missense mutations found in human genes, including the BRCA1 breast and ovarian cancer susceptibility gene. When expressed in yeast, the wild-type full-length BRCA1 protein forms a single nuclear aggregate and induces a growth inhibition. Both events are modified by pathogenic mutations of BRCA1. However, the biological processes behind these events in yeast remain to be determined. Here, we show that the BRCA1 nuclear aggregation and the growth inhibition are sensitive to misfolding effects induced by missense mutations. Moreover, misfolding mutations impair the nuclear targeting of BRCA1 in yeast cells and in a human cell line. In conclusion, we establish a connection between misfolding and nuclear transport impairment, and we illustrate that yeast is a suitable model to decipher the effect of misfolding mutations.
Asunto(s)
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Pliegue de Proteína , Saccharomyces cerevisiae/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Fluorescencia , Humanos , Modelos Biológicos , Mutación/genética , Señales de Localización Nuclear , Agregado de Proteínas , Dominios Proteicos , Estabilidad Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that G-quadruplex structures may act as 'knots' within genomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN Helicasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , G-Cuádruplex , RecQ Helicasas/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , ADN Helicasas/química , ADN Helicasas/genética , Replicación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Expresión Génica , Guanina/química , Guanina/metabolismo , Humanos , RecQ Helicasas/química , RecQ Helicasas/genética , Helicasa del Síndrome de WernerRESUMEN
The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.
Asunto(s)
Sistemas CRISPR-Cas/genética , Ingeniería de Proteínas , Reparación del ADN por Recombinación/genética , Cigoto/fisiología , Animales , Ratones , Ratones Endogámicos C57BL , Microinyecciones , Ratas , Ratas Sprague-DawleyRESUMEN
Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5' to 3' DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.
Asunto(s)
ADN Helicasas/análisis , Pruebas de Enzimas/métodos , G-Cuádruplex , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/metabolismo , Inhibidores Enzimáticos/farmacología , Fluorescencia , LigandosRESUMEN
In eukaryotes, the genome is packed into chromosomes, each consisting of large polymeric fibers made of DNA bound with proteins (mainly histones) and RNA molecules. The nature and precise 3D organization of this fiber has been a matter of intense speculations and debates. In the emerging picture, the local chromatin state plays a critical role in all fundamental DNA transactions, such as transcriptional control, DNA replication or repair. However, the molecular and structural mechanisms involved remain elusive. The purpose of this review is to give an overview of the tremendous efforts that have been made for almost 40 years to build physiologically relevant models of chromatin structure. The motivation behind building such models was to shift our representation and understanding of DNA transactions from a too simplistic 'naked DNA' view to a more realistic 'coated DNA' view, as a step towards a better framework in which to interpret mechanistically the control of genetic expression and other DNA metabolic processes. The field has evolved from a speculative point of view towards in vitro biochemistry and in silico modeling, but is still longing for experimental in vivo validations of the proposed structures or even proof of concept experiments demonstrating a clear role of a given structure in a metabolic transaction. The mere existence of a chromatin fiber as a relevant biological entity in vivo has been put into serious questioning. Current research is suggesting a possible reconciliation between theoretical studies and experiments, pointing towards a view where the polymorphic and dynamic nature of the chromatin fiber is essential to support its function in genome metabolism.
Asunto(s)
Cromatina/química , Animales , Cromatina/metabolismo , Humanos , Modelos Moleculares , Nucleosomas/química , Nucleosomas/metabolismo , Terminología como AsuntoRESUMEN
Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC-rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC(3) G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites' distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.
Asunto(s)
Aberraciones Cromosómicas , G-Cuádruplex , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Telómero/genética , Composición de Base , ADN Helicasas/genética , Replicación del ADN , Humanos , Repeticiones de Minisatélite/genética , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Saccharomyces cerevisiae/genética , Secuencias Repetidas en Tándem/genéticaRESUMEN
A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.
Asunto(s)
Proteína BRCA1/metabolismo , Bioensayo/métodos , Neoplasias de la Mama/fisiopatología , Núcleo Celular/ultraestructura , Genes BRCA1 , Cuerpos de Inclusión/metabolismo , Mutación , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteína BRCA1/química , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Núcleo Celular/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Fenotipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC(3) and Phen-DC(6), two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.
Asunto(s)
G-Cuádruplex/efectos de los fármacos , Repeticiones de Minisatélite/efectos de los fármacos , Fenantrolinas/farmacología , Compuestos de Quinolinio/farmacología , Saccharomyces cerevisiae/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Variación Genética , Humanos , Ligandos , Mutación , Fenantrolinas/química , Fenantrolinas/metabolismo , Compuestos de Quinolinio/química , Compuestos de Quinolinio/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Pif1p is the prototype member of a family of helicases that is highly conserved from yeast to humans. In yeast, Pif1p is involved in many aspects of the preservation of genome stability. In particular, Pif1p is involved in the maintenance of mitochondrial DNA and in the direct inhibition of telomerase at telomeres and double-stranded breaks. Here we describe methods to purify Pif1p and study in vitro its enzymatic properties and functional interaction with telomerase.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Telomerasa/antagonistas & inhibidores , Fraccionamiento Celular/métodos , Cromatografía/métodos , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Humanos , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.
Asunto(s)
ADN Helicasas/metabolismo , Inestabilidad Genómica , Repeticiones de Minisatélite , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alelos , Composición de Base , Secuencia de Bases , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Recombinante/química , ADN Recombinante/genética , ADN Recombinante/metabolismo , Humanos , Técnicas In Vitro , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Recombinasa Rad51/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
The catalytic subunit of yeast telomerase, Est2p, is a telomere associated throughout most of the cell cycle, while the Est1p subunit binds only in late S/G2 phase, the time of telomerase action. Est2p binding in G1/early S phase requires a specific interaction between telomerase RNA (TLC1) and Ku80p. Here, we show that in four telomerase-deficient strains (cdc13-2, est1A, tlc1-SD, and tlc1-BD), Est2p telomere binding was normal in G1/early S phase but reduced to about 40-50% of wild type levels in late S/G2 phase. Est1p telomere association was low in all four strains. Wild type levels of Est2p telomere binding in late S/G2 phase was Est1p-dependent and required that Est1p be both telomere-bound and associated with a stem-bulge region in TLC1 RNA. In three telomerase-deficient strains in which Est1p is not Est2p-associated (tlc1-SD, tlc1-BD, and est2A), Est1p was present at normal levels but its telomere binding was very low. When the G1/early S phase and the late S/G2 phase telomerase recruitment pathways were both disrupted, neither Est2p nor Est1p was telomere-associated. We conclude that reduced levels of Est2p and low Est1p telomere binding in late S/G2 phase correlated with an est phenotype, while a WT level of Est2p binding in G1 was not sufficient to maintain telomeres. In addition, even though Cdc13p and Est1p interact by two hybrid, biochemical and genetic criteria, this interaction did not occur unless Est1p was Est2p-associated, suggesting that Est1p comes to the telomere only as part of the holoenzyme. Finally, the G1 and late S/G2 phase pathways for telomerase recruitment are distinct and are likely the only ones that bring telomerase to telomeres in wild-type cells.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Telomerasa/metabolismo , Telómero/enzimología , Ciclo Celular , Unión Proteica , ARN/genética , ARN/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Telomerasa/genética , Telómero/genéticaRESUMEN
Pif1p is the prototypical member of the PIF1 family of DNA helicases, a subfamily of SFI helicases conserved from yeast to humans. Baker's yeast Pif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and may also have a general role in chromosomal replication by affecting Okazaki fragment maturation. Here we investigate the substrate preferences for Pif1p. The enzyme was preferentially active on RNA-DNA hybrids, as seen by faster unwinding rates on RNA-DNA hybrids compared to DNA-DNA hybrids. When using forked substrates, which have been shown previously to stimulate the enzyme, Pif1p demonstrated a preference for RNA-DNA hybrids. This preferential unwinding could not be correlated to preferential binding of Pif1p to the substrates that were the most readily unwound. Although the addition of the single-strand DNA-binding protein replication protein A (RPA) stimulated the helicase reaction on all substrates, it did not diminish the preference of Pif1p for RNA-DNA substrates. Thus, forked RNA-DNA substrates are the favored substrates for Pif1p in vitro. We discuss these findings in terms of the known biological roles of the enzyme.