Correction for Duprey and Groisman, "FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling In Vivo".
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ABSTRACT:
SUBMITTER: Duprey A
PROVIDER: S-EPMC10790772 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Correction for Duprey and Groisman, "FEDS: a Novel Fluorescence-Based High-Throughput Method for Measuring DNA Supercoiling <i>In Vivo</i>".
Duprey Alexandre A Groisman Eduardo A EA
mBio 20231207 1
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Project description:DNA supercoiling (DS) is essential for life because it controls critical processes, including transcription, replication, and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here, we report a method for high-throughput measurement of bacterial DNA supercoiling in vivoFluorescent evaluation of DNA supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling and the gene for a red fluorescent protein transcribed by a constitutive promoter as the internal standard. Using FEDS, we uncovered single-cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low-mean/high-variance DNA supercoiling subpopulation rather than from a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases.IMPORTANCE DNA represents the chemical support of genetic information in all forms of life. In addition to its linear sequence of nucleotides, it bears critical information in its structure. This information, called DNA supercoiling, is central to all fundamental DNA processes, such as transcription and replication, and defines cellular physiology. Unlike reading of a nucleotide sequence, DNA supercoiling determinations have been laborious. We have now developed a method for rapid measurement of DNA supercoiling and established its utility by identifying a novel regulator of DNA supercoiling in the bacterium Salmonella enterica as well as behaviors that could not have been discovered with current methods.
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Project description:DNA supercoiling is essential for life because it controls critical processes, including transcription, replication and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here we report a method for high-throughput measurement of bacterial DNA supercoiling in vivo. Fluorescent Evaluation of DNA Supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling, and the gene for a red fluorescent protein transcribed by a constitutive promoter as internal standard. Using FEDS, we uncovered single cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low mean/high variance DNA supercoiling subpopulation, rather than a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases.
2020-07-02 | GSE137586 | GEO
Project description:Novel fluorescence-based high-throughput method uncovers single-cell DNA supercoiling heterogeneity
| PRJNA565972 | ENA
Project description:Understanding the thermodynamics of DNA motifs is important for prediction and design of probes and primers, but melt curve analyses are low-throughput and produce inaccurate results for motifs such as bulges and mismatches. Here, we developed a new, accurate and high-throughput method for measuring DNA motif thermodynamics called TEEM (Toehold Exchange Energy Measurement). It is a refined framework of comparing two toehold exchange reactions, which are competitive strand displacement between oligonucleotides. In a single experiment, TEEM can measure over 1000 ΔG° values with standard error of roughly 0.05 kcal/mol.
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Project description: Not available
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Project description:Transitions in DNA structure have the capacity to regulate genes, but have been poorly characterised in eukaryotes due to a lack of appropriate techniques. One important example is DNA supercoiling, which can directly regulate transcription initiation, elongation and coordinated expression of neighbouring genes. DNA supercoiling is the over- or under-winding of the DNA double helix, which occurs as a consequence of polymerase activity and is modulated by topoisomerase activity [5]. To map the distribution of DNA supercoiling in nuclei, we developed biotinylated 4,5,8-trimethylpsoralen (bTMP) pull-down to preferentially enrich for under-wound DNA. Here we describe in detail the experimental design, quality controls and analyses associated with the study by Naughton et al. [13] that characterised for the first time the large-scale distribution of DNA supercoiling in human cells (GEO: GSE43488 and GSE43450GSE43488GSE43450).
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Project description:BackgroundCytokinins are one kind of phytohormones essential for plant growth, development and stress responses. In the past half century, significant progresses have been made in the studies of cytokinin signal transduction and metobolic pathways, but the mechanism of cytokinin translocation is poorly understood. Arabidopsis (Arabidopsis thaliana) response regulator 5 (ARR5) is a type-A response factor in cytokinin signaling which is induced by cytokinins and has been used as a reporter gene for the endogenous cytokinins in Arabidopsis. Here, we report a fluorescence-based high-throughput method to screen cytokinin translocation mutants using an ethyl methyl sulfone (EMS) mutagenesis library generated with ARR5::eGFP transgenic plants.ResultsThe seedlings with enhanced green fluorescent protein (GFP) signal in roots were screened in a luminescence imaging system (LIS) in large scale to obtain mutants with over-accumulated cytokinins in roots. The selected mutants were confirmed under a fluorescence microscopy and then performed phenotypic analysis. In this way, we obtained twelve mutants with elevated GFP signal in the roots and further found three of them displayed reduced GFP signal in the aerial tissues. Two of the mutants were characterized and proved to be the atabcg14 allelic mutants which are defective in the long-distance translocation of root-synthesized cytokinins.ConclusionsWe provide a strategy for screening mutants defective in cytokinin translocation, distribution or signaling. The strategy can be adapted to establish a system for screening mutants defective in other hormone transporters or signaling components using a fluorescence reporter.
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Project description:Current cell-based assays for determining the functional properties of high-density lipoproteins (HDL) have limitations. We report here the development of a new, robust fluorometric cell-free biochemical assay that measures HDL lipid peroxidation (HDLox) based on the oxidation of the fluorochrome Amplex Red. HDLox correlated with previously validated cell-based (r = 0.47, p<0.001) and cell-free assays (r = 0.46, p<0.001). HDLox distinguished dysfunctional HDL in established animal models of atherosclerosis and Human Immunodeficiency Virus (HIV) patients. Using an immunoaffinity method for capturing HDL, we demonstrate the utility of this novel assay for measuring HDLox in a high throughput format. Furthermore, HDLox correlated significantly with measures of cardiovascular diseases including carotid intima media thickness (r = 0.35, p<0.01) and subendocardial viability ratio (r = -0.21, p = 0.05) and physiological parameters such as metabolic and anthropometric parameters (p<0.05). In conclusion, we report the development of a new fluorometric method that offers a reproducible and rapid means for determining HDL function/quality that is suitable for high throughput implementation.
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Project description:DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes (Escherichia coli and Mycobacterium smegmatis DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes (E. coli DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, K M, V max, and k cat. We found that k cat for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5'-triphosphate binding to E. coli DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors.
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Project description:Single-molecule studies of protein-DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains-a method for organizing arrays of DNA molecules on a fluid lipid bilayer-has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900,000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3-a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments.
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