RESUMEN
Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR-Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.
Asunto(s)
Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Enfermedades Genéticas Congénitas/genética , Repeticiones de Microsatélite/genética , Edición Génica , Humanos , Mutación/genética , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Repeticiones de Trinucleótidos/genéticaRESUMEN
Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.
Asunto(s)
Roturas del ADN de Doble Cadena , Distrofia Miotónica/genética , Recombinación Genética , Repeticiones de Trinucleótidos/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Deleción Cromosómica , Cromosomas Fúngicos/genética , Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP)/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Conversión Génica/genética , Genoma Humano/genética , Humanos , Distrofia Miotónica/patología , Proteína Recombinante y Reparadora de ADN Rad52/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
BACKGROUND: Double-strand break repair (DSBR) is a highly regulated process involving dozens of proteins acting in a defined order to repair a DNA lesion that is fatal for any living cell. Model organisms such as Saccharomyces cerevisiae have been used to study the mechanisms underlying DSBR, including factors influencing its efficiency such as the presence of distinct combinations of microsatellites and endonucleases, mainly by bulk analysis of millions of cells undergoing repair of a broken chromosome. Here, we use a microfluidic device to demonstrate in yeast that DSBR may be studied at a single-cell level in a time-resolved manner, on a large number of independent lineages undergoing repair. RESULTS: We used engineered S. cerevisiae cells in which GFP is expressed following the successful repair of a DSB induced by Cas9 or Cpf1 endonucleases, and different genetic backgrounds were screened to detect key events leading to the DSBR efficiency. Per condition, the progenies of 80-150 individual cells were analyzed over 24 h. The observed DSBR dynamics, which revealed heterogeneity of individual cell fates and their contributions to global repair efficacy, was confronted with a coupled differential equation model to obtain repair process rates. Good agreement was found between the mathematical model and experimental results at different scales, and quantitative comparisons of the different experimental conditions with image analysis of cell shape enabled the identification of three types of DSB repair events previously not recognized: high-efficacy error-free, low-efficacy error-free, and low-efficacy error-prone repair. CONCLUSIONS: Our analysis paves the way to a significant advance in understanding the complex molecular mechanism of DSB repair, with potential implications beyond yeast cell biology. This multiscale and multidisciplinary approach more generally allows unique insights into the relation between in vivo microscopic processes within each cell and their impact on the population dynamics, which were inaccessible by previous approaches using molecular genetics tools alone.
Asunto(s)
Microfluídica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Reparación del ADN , Diferenciación Celular , EndonucleasasRESUMEN
Trinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders. Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract. The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts. Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract. In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared. Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Terapia Genética/métodos , Repeticiones de Trinucleótidos/genética , ADN/genética , ADN/metabolismo , Endonucleasas/metabolismo , Terapia Genética/tendencias , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Humanos , Modelos Genéticos , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.
Asunto(s)
ADN Helicasas/genética , Replicación del ADN , ADN de Hongos/genética , Genoma Fúngico , Antígeno Nuclear de Célula en Proliferación/genética , Recombinasa Rad51/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Alelos , Fragilidad Cromosómica , ADN Helicasas/metabolismo , ADN de Hongos/metabolismo , Electroforesis en Gel Bidimensional , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Inestabilidad Genómica , Secuencias Invertidas Repetidas , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Repeticiones de TrinucleótidosRESUMEN
Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.
Asunto(s)
Endonucleasas/metabolismo , Terapia Genética , Repeticiones de Trinucleótidos , Animales , Endonucleasas/clasificación , Endonucleasas/uso terapéutico , Inestabilidad Genómica , Humanos , Ingeniería de Proteínas , Especificidad por Sustrato , Expansión de Repetición de TrinucleótidoRESUMEN
BACKGROUND: The opportunistic pathogen Candida glabrata is a member of the Saccharomycetaceae yeasts. Like its close relative Saccharomyces cerevisiae, it underwent a whole-genome duplication followed by an extensive loss of genes. Its genome contains a large number of very long tandem repeats, called megasatellites. In order to determine the whole replication program of the C. glabrata genome and its general chromosomal organization, we used deep-sequencing and chromosome conformation capture experiments. RESULTS: We identified 253 replication fork origins, genome wide. Centromeres, HML and HMR loci, and most histone genes are replicated early, whereas natural chromosomal breakpoints are located in late-replicating regions. In addition, 275 autonomously replicating sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition. Analysis of ARSs allowed us to identify a 17-bp consensus, similar to the S. cerevisiae ARS consensus sequence but slightly more constrained. Megasatellites are not in close proximity to replication origins or termini. Using chromosome conformation capture, we also show that early origins tend to cluster whereas non-subtelomeric megasatellites do not cluster in the yeast nucleus. CONCLUSIONS: Despite a shorter cell cycle, the C. glabrata replication program shares unexpected striking similarities to S. cerevisiae, in spite of their large evolutionary distance and the presence of highly repetitive large tandem repeats in C. glabrata. No correlation could be found between the replication program and megasatellites, suggesting that their formation and propagation might not be directly caused by replication fork initiation or termination.
Asunto(s)
Candida glabrata/genética , Cromosomas Fúngicos , Replicación del ADN , Genoma Fúngico , Ciclo Celular/genética , Genes FúngicosRESUMEN
Centrifugal elutriation discriminates cells according to their sedimentation coefficients, generating homogeneous samples well suited for genomic comparative approaches. It can, for instance, isolate G1 daughter cells from a Saccharomyces cerevisiae unsynchronized population, alleviating ageing and cell-cycle biases when conducting genome-wide/single-cell studies. The present report describes a straightforward and robust procedure to determine whether a cell population of virtually any yeast species can be efficiently elutriated, while offering solutions to optimize success. This approach was used to characterize elutriation parameters and S-phase progression of four yeast species (S. cerevisiae, Candida glabrata, Lachancea kluyveri and Pichia sorbitophila) and could theoretically be applied to any culture of single, individual cells.
Asunto(s)
Centrifugación/métodos , Fase G1 , Micología/métodos , Saccharomyces cerevisiae/citología , División Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/aislamiento & purificación , Levaduras/citología , Levaduras/genética , Levaduras/aislamiento & purificaciónRESUMEN
Megasatellites are large DNA tandem repeats, originally described in Candida glabrata, in protein-coding genes. Most of the genes in which megasatellites are found are of unknown function. In this work, we extended the search for megasatellites to 20 additional completely sequenced fungal genomes and extracted 216 megasatellites in 203 out of 142,121 genes, corresponding to the most exhaustive description of such genetic elements available today. We show that half of the megasatellites detected encode threonine-rich peptides predicted to be intrinsically disordered, suggesting that they may interact with several partners or serve as flexible linkers. Megasatellite motifs were clustered into several families. Their distribution in fungal genes shows that different motifs are found in orthologous genes and similar motifs are found in unrelated genes, suggesting that megasatellite formation or spreading does not necessarily track the evolution of their host genes. Altogether, these results suggest that megasatellites are created and lost during evolution of fungal genomes, probably sharing similar functions, although their primary sequences are not necessarily conserved.
Asunto(s)
Ascomicetos/genética , Basidiomycota/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Secuencias Repetidas en Tándem , Secuencia de Aminoácidos , Ascomicetos/clasificación , Basidiomycota/clasificación , Evolución Molecular , Proteínas Fúngicas/clasificación , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Treonina/genéticaRESUMEN
Megasatellites are a new family of long tandem repeats, recently discovered in the yeast Candida glabrata. Compared to shorter tandem repeats, such as minisatellites, megasatellite motifs range in size from 135 to more than 300 bp, and allow calculation of evolutionary distances between individual motifs. Using divergence based on nucleotide substitutions among similar motifs, we determined the smallest distance between two motifs, allowing their subsequent clustering. Motifs belonging to the same cluster are recurrently found in different megasatellites located on different chromosomes, showing transfer of genetic information between megasatellites. In comparison, evolution of the few similar tandem repeats in Saccharomyces cerevisiae FLO genes mainly involves subtelomeric homologous recombination. We estimated selective constraints acting on megasatellite motifs and their host genes, and found that motifs are under strong purifying selection. Surprisingly, motifs inserted within pseudogenes are also under purifying selection, whereas the pseudogenes themselves evolve neutrally. We propose that megasatellite motifs propagate by a combination of three different molecular mechanisms: (i) gene duplication, (ii) ectopic homologous recombination and (iii) transfer of motifs from one megasatellite to another one. These mechanisms actively cooperate to create new megasatellites, that may play an important role in the adaptation of Candida glabrata to its human host.
Asunto(s)
Candida glabrata/genética , Evolución Molecular , Saccharomyces cerevisiae/genética , Secuencias Repetidas en Tándem , Análisis por Conglomerados , Genes Fúngicos , Genoma Fúngico , SeudogenesRESUMEN
Since formation of the first proto-eukaryotes, gene repertoire and genome complexity have significantly increased. Among genetic elements responsible for this increase are tandem repeats. Here we describe a genome-wide analysis of large tandem repeats, called megasatellites, in 58 vertebrate genomes. Two bursts occurred, one after the radiation between Agnatha and Gnathostomata fishes and the second one in therian mammals. Megasatellites are enriched in subtelomeric regions and frequently encoded in genes involved in transcription regulation, intracellular trafficking, and cell membrane metabolism, reminiscent of what is observed in fungus genomes. The presence of many introns within young megasatellites suggests that an exon-intron DNA segment is first duplicated and amplified before accumulation of mutations in intronic parts partially erases the megasatellite in such a way that it becomes detectable only in exons. Our results suggest that megasatellite formation and evolution is a dynamic and still ongoing process in vertebrate genomes.
Asunto(s)
Evolución Molecular , Vertebrados , Animales , Exones/genética , Genoma Fúngico , Intrones/genética , Mamíferos/genética , Vertebrados/genéticaRESUMEN
Megasatellites are large tandem repeats found in all fungal genomes but especially abundant in the opportunistic pathogen Candida glabrata. They are encoded in genes involved in cell-cell interactions, either between yeasts or between yeast and human cells. In the present work, we have been using an iterative genetic system to delete several Candida glabrata megasatellite-containing genes and found that 2 of them were positively involved in adhesion to epithelial cells, whereas 3 genes negatively controlled adhesion. Two of the latter, CAGL0B05061g or CAGL0A04851g, were also negative regulators of yeast-to-yeast adhesion, making them central players in controlling Candida glabrata adherence properties. Using a series of synthetic Saccharomyces cerevisiae strains in which the FLO1 megasatellite was replaced by other tandem repeats of similar length but different sequences, we showed that the capacity of a strain to flocculate in liquid culture was unrelated to its capacity to adhere to epithelial cells or to invade agar. Finally, to understand how megasatellites were initially created and subsequently expanded, an experimental evolution system was set up, in which modified yeast strains containing different megasatellite seeds were grown in bioreactors for more than 200 generations and selected for their ability to sediment at the bottom of the culture tube. Several flocculation-positive mutants were isolated. Functionally relevant mutations included general transcription factors as well as a 230-kbp segmental duplication.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Candida glabrata/genética , Floculación , Genoma Fúngico , Humanos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Megasatellites are DNA tandem arrays made of large motifs; they were discovered in the yeast Candida glabrata. They are widespread in this species (40 copies) but are not found in any other hemiascomycete so far, raising the intriguing question of their origin. They are found mainly in genes encoding cell wall products, suggesting that megasatellites were selected for a function linked to cell-cell adhesion or to pathogenicity. Their putative role in promoting genome rearrangements by interfering with DNA replication will also be discussed.
Asunto(s)
Candida glabrata/genética , ADN Satélite/genética , Secuencias Repetidas en Tándem/genética , Candida glabrata/patogenicidad , ADN de Hongos/genética , ADN Satélite/clasificación , ADN Satélite/fisiología , Evolución Molecular , Genoma Fúngico , Modelos BiológicosRESUMEN
Trinucleotide repeats are a peculiar class of microsatellites whose expansions are responsible for approximately 30 human neurological or developmental disorders. The molecular mechanisms responsible for these expansions in humans are not totally understood, but experiments in model systems such as yeast, transgenic mice, and human cells have brought evidence that the mismatch repair machinery is involved in generating these expansions. The present review summarizes, in the first part, the role of mismatch repair in detecting and fixing the DNA strand slippage occurring during microsatellite replication. In the second part, key molecular differences between normal microsatellites and those that show a bias toward expansions are extensively presented. The effect of mismatch repair mutants on microsatellite expansions is detailed in model systems, and in vitro experiments on mismatched DNA substrates are described. Finally, a model presenting the possible roles of the mismatch repair machinery in microsatellite expansions is proposed.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Repeticiones de Microsatélite , Expansión de Repetición de Trinucleótido , Animales , ADN/metabolismo , Reparación del ADN , Genotipo , Humanos , Meiosis , Ratones , Ratones Transgénicos , Mitosis , Proteínas MutL/genética , Proteínas MutS/genética , Recombinación Genética , Saccharomyces cerevisiae , Schizosaccharomyces , Repeticiones de TrinucleótidosRESUMEN
Duplex DNA naturally folds into a right-handed double helix in physiological conditions. Some sequences of unusual base composition may nevertheless form alternative structures, as was shown for many repeated sequences in vitro However, evidence for the formation of noncanonical structures in living cells is difficult to gather. It mainly relies on genetic assays demonstrating their function in vivo or through genetic instability reflecting particular properties of such structures. Efforts were made to reveal their existence directly in a living cell, mainly by generating antibodies specific to secondary structures or using chemical ligands selected for their affinity to these structures. Among secondary structure-forming DNAs are G-quadruplexes, human fragile sites containing minisatellites, AT-rich regions, inverted repeats able to form cruciform structures, hairpin-forming CAG/CTG triplet repeats, and triple helices formed by homopurine-homopyrimidine GAA/TTC trinucleotide repeats. Many of these alternative structures are involved in human pathologies, such as neurological or developmental disorders, as in the case of trinucleotide repeats, or cancers triggered by translocations linked to fragile sites. This review will discuss and highlight evidence supporting the formation of alternative DNA structures in vivo and will emphasize the role of the mismatch repair machinery in binding mispaired DNA duplexes, triggering genetic instability.
Asunto(s)
Emparejamiento Base/genética , ADN/genética , G-Cuádruplex , Animales , Línea Celular Tumoral , Sitios Frágiles del Cromosoma/genética , Reparación de la Incompatibilidad de ADN/genética , Células HeLa , Humanos , Repeticiones de Minisatélite/genética , Inversión de Secuencia/genética , Repeticiones de Trinucleótidos/genéticaRESUMEN
Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.
Asunto(s)
Evolución Molecular , Genes Fúngicos/genética , Genoma Fúngico , Levaduras/clasificación , Levaduras/genética , Cromosomas Fúngicos/genética , Secuencia Conservada/genética , Duplicación de Gen , Datos de Secuencia Molecular , ARN Ribosómico/genética , ARN de Transferencia/genética , Proteínas de Saccharomyces cerevisiae/genética , Sintenía/genética , Secuencias Repetidas en Tándem/genéticaRESUMEN
Minisatellites are DNA tandem repeats that are found in all sequenced genomes. In the yeast Saccharomyces cerevisiae, they are frequently encountered in genes encoding cell wall proteins. Minisatellites present in the completely sequenced genome of the pathogenic yeast Candida glabrata were similarly analyzed, and two new types of minisatellites were discovered: minisatellites that are composed of two different intermingled repeats (called compound minisatellites), and minisatellites containing unusually long repeated motifs (126-429 bp). These long repeat minisatellites may reach unusual length for such elements (up to 10 kb). Due to these peculiar properties, they have been named 'megasatellites'. They are found essentially in genes involved in cell-cell adhesion, and could therefore be involved in the ability of this opportunistic pathogen to colonize the human host. In addition to megasatellites, found in large paralogous gene families, there are 93 minisatellites with simple shorter motifs, comparable to those found in S. cerevisiae. Most of the time, these minisatellites are not conserved between C. glabrata and S. cerevisiae, although their host genes are well conserved, raising the question of an active mechanism creating minisatellites de novo in hemiascomycetes.
Asunto(s)
Candida glabrata/genética , Genes Fúngicos , Repeticiones de Minisatélite , Secuencia de Aminoácidos , Adhesión Bacteriana/genética , Candida glabrata/patogenicidad , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genoma Fúngico , Datos de Secuencia Molecular , Virulencia/genéticaRESUMEN
Trinucleotide repeats are a peculiar class of microsatellites involved in many neurological as well as developmental disorders. Their propensity to generate very large expansions over time is supposedly due to their capacity to form specific secondary structures, such as imperfect hairpins, triple helices, or G-quadruplexes. These unusual structures were proposed to trigger expansions in vivo. Here, I review known technical issues linked to these structures, such as slippage during polymerase chain reaction and aberrant migration of long trinucleotide repeats during agarose gel electrophoresis. Our current understanding of interactions between trinucleotide repeat secondary structures and the mismatch-repair machinery is also quickly reviewed, and critical questions relevant to these interactions are addressed.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Repeticiones de Trinucleótidos , Electroforesis en Gel de Agar , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Expansión de Repetición de TrinucleótidoRESUMEN
Physical separation of branched DNA from linear molecules is based on the difference of mobility of linear versus branched DNA during two-dimensional agarose gel electrophoresis. Structured DNA migrates as slower species when compared to linear DNA of similar molecular weight. Metabolic processes such as S phase replication or double strand-break repair may generate branched DNA molecules. Trinucleotide repeats are naturally prone to form secondary structures that can modify their migration through an agarose gel matrix. These structures may also interfere in vivo with replication, by slowing down replication-fork progression, transiently stalling forks, possibly leading to secondary structure such as Holliday junctions or hemicatenanes. Alternatively, reversed replication forks may occur following fork stalling, disrupting replication dynamics and modifying DNA migration on agarose gel. So although two-dimensional agarose gel electrophoresis theoretically allows to resolve a mixture of structured DNA molecules and quantify them by radioactive hybridization, its practical application to trinucleotide repeats faces some serious technical challenges.
Asunto(s)
ADN/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Repeticiones de Trinucleótidos , ADN/química , Replicación del ADN , Electroforesis en Gel Bidimensional , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/genéticaRESUMEN
Cells can repair a double-strand break (DSB) by homologous recombination if a homologous sequence is provided as a template. This can be achieved by classical gene conversion (with or without crossover) or by single-strand annealing (SSA) between two direct repeat sequences flanking the DSB. To initiate SSA, single-stranded regions are needed adjacent to the break, extending up to the direct repeats in such a way that complementary strands can anneal to each other to repair the DSB. In the present protocol, we describe a GFP reporter assay in Saccharomyces cerevisiae allowing for the quantification of nuclease efficacy at inducing a DSB, by monitoring the reconstitution of a functional GFP gene whose expression can be rapidly quantified by flow cytometry.