RESUMO
Supercoiling is a fundamental property of DNA that governs all strand opening reactions, including DNA replication, transcription, and homologous recombination. However, traditional genomic supercoiling assays are difficult and lack sensitivity. Building on prior assays using the DNA intercalator psoralen, we developed a supercoil mapping assay that is robust and sensitive to a wide range of supercoiling while requiring only commercially available reagents and common laboratory equipment. This method, psoralen affinity purification with genomic sequencing (Psora-seq), utilizes biotinylated psoralen and streptavidin-conjugated magnetic beads to facilitate efficient pull-down of psoralen-bound DNA, followed by deep sequencing to identify and quantify supercoiling at 1 kb resolution. Psora-seq overcomes two major bottlenecks associated with existing psoralen pull-down assays, inefficient photo-binding of psoralen-bound molecules, and poor recovery of cross-linked DNA.
Assuntos
DNA Super-Helicoidal , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Ficusina/química , Análise de Sequência de DNA/métodos , Mapeamento Cromossômico/métodos , Genômica/métodosRESUMO
Bidirectional DNA replication complexes initiated from the same origin remain colocalized in a factory configuration for part or all their lifetimes. However, there is little evidence that sister replisomes are functionally interdependent, and the consequence of factory replication is unknown. Here, we investigated the functional relationship between sister replisomes in Escherichia coli, which naturally exhibits both factory and solitary configurations in the same replication cycle. Using an inducible transcription factor roadblocking system, we found that blocking one replisome caused a significant decrease in overall progression and velocity of the sister replisome. Remarkably, progression was impaired only if the block occurred while sister replisomes were still in a factory configuration - blocking one fork had no significant effect on the other replisome when sister replisomes were physically separate. Disruption of factory replication also led to increased fork stalling and requirement of fork restart mechanisms. These results suggest that physical association between sister replisomes is important for establishing an efficient and uninterrupted replication program. We discuss the implications of our findings on mechanisms of replication factory structure and function, and cellular strategies of replicating problematic DNA such as highly transcribed segments.
Assuntos
Replicação do DNA , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Cromossomos/metabolismo , DNA , Proteínas de Ligação a DNA/metabolismoRESUMO
BACKGROUND: To harmonize assays for detection of HEV RNA, a World Health Organization International Standard (WHO IS) was established. The WHO IS represents the highest order standard for HEV RNA but is limited in quantity. Secondary standards are needed to limit the use of WHO IS and minimize the need to replace it. OBJECTIVE: Establish secondary standards for HEV NAAT assays and to calibrate these against the WHO IS. METHODS: Stocks of genotype 3 HEV were prepared using both cell lysates and cell culture supernatants to produce non-enveloped and quasi-enveloped virus stocks, respectively. Both stocks were heat-inactivated, diluted in negative human plasma, and lyophilized to produce two candidate secondary standards: HEV-RR (non-enveloped virus) and HEV-RR.1 (quasi-enveloped virus). Both candidate standards were characterized and calibrated against the WHO IS for HEV RNA in an international collaborative study. RESULTS: The collaborative study returned a total of 15 data sets, with different RNA extraction and amplification methods. The estimated mean values relative to the WHO IS (250,000 IU/ml) are 229,000 IU/ml and 355,000 IU/ml for HEV-RR and HEV-RR.1, respectively. CONCLUSION: We have established two secondary standards for HEV RNA calibrated against the WHO IS. These standards are non-infectious and stable under different storage temperatures.
Assuntos
Vírus da Hepatite E , Humanos , Vírus da Hepatite E/genética , RNA Viral/genética , Cooperação Internacional , Padrões de Referência , TecnologiaRESUMO
DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction - an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.
Assuntos
DNA Super-Helicoidal , Ficusina , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA/metabolismo , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Transcrição GênicaRESUMO
Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork.
Assuntos
Cromossomos Bacterianos , DnaB Helicases/metabolismo , Escherichia coli/genética , Instabilidade Genômica , Sistemas CRISPR-Cas , DNA Bacteriano/química , DNA Bacteriano/genética , DnaB Helicases/química , DnaB Helicases/genética , Escherichia coli/enzimologia , MutaçãoRESUMO
Fluorescence in situ hybridization (FISH) is a widely used technique to detect and localize specific DNA or RNA sequences in cells. Although supplanted in many ways by fluorescently labeled DNA binding proteins, FISH remains the only cytological method to examine many genetic loci at once (up to six), and can be performed in any cell type and genotype. These advantages have proved invaluable in studying the spatial relationships between chromosome regions and the dynamics of chromosome segregation in bacteria. A detailed protocol for DNA FISH in E. coli is described.
Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Hibridização in Situ Fluorescente/métodos , Segregação de Cromossomos , Análise Citogenética , Loci GênicosRESUMO
Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to 1 Mb from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing, show that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment and the role of programmed changes in chromosome structure in replication control are discussed.
Assuntos
Replicação do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Origem de Replicação , Membrana Celular/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Escherichia coli/química , Escherichia coli/metabolismoRESUMO
The well-conserved genes surrounding the E. coli replication origin, mioC and gidA, do not normally affect chromosome replication and have little known function. We report that mioC and gidA mutants exhibit a moderate cell division inhibition phenotype. Cell elongation is exacerbated by a fis deletion, likely owing to delayed replication and subsequent cell cycle stress. Measurements of replication initiation frequency and origin segregation indicate that mioC and gidA do not inhibit cell division through any effect on oriC function. Division inhibition is also independent of the two known replication/cell division checkpoints, SOS and nucleoid occlusion. Complementation analysis indicates that mioC and gidA affect cell division in trans, indicating their effect is at the protein level. Transcriptome analysis by RNA sequencing showed that expression of a cell division septum component, YmgF, is significantly altered in mioC and gidA mutants. Our data reveal new roles for the gene products of gidA and mioC in the division apparatus, and we propose that their expression, cyclically regulated by chromatin remodeling at oriC, is part of a cell cycle regulatory program coordinating replication initiation and cell division.
RESUMO
A green organic laboratory experiment was developed in which students synthesize a sensor for thiols using a microscale, solventless Diels-Alder reaction at room temperature or 37 °C. The molecular probe is easily purified by column chromatography in a Pasteur pipet and characterized by thin-layer chromatography and NMR spectroscopy. The thiol-reactive sensor becomes intensely fluorescent upon exposure to thiols from N-acetylcysteine, bovine serum albumin, or human hair (pretreated with a reducing agent to reveal cysteine thiols in α-keratin). This fluorescence is observable even with micrograms of probe.