Kinetics of dCas9 target search in Escherichia coli

How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

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(E)-N′-(4-Fluorobenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide

Molbank ◽

10.3390/m1058 ◽

2019 ◽

Vol 2019 (2) ◽

pp. M1058

Author(s):

Vinuta Kamat ◽

Rangappa Santosh ◽

Suresh Nayak

Keyword(s):

Escherichia Coli ◽

Double Helix ◽

Catalytic Amount ◽

Target Compound ◽

Addition Compound ◽

E Coli ◽

In Vitro Antibacterial Activity ◽

Ft Ir ◽

Dna Double Helix

5-methyl-2-(pyridin-3-yl)-1,3-thiazole-4-carbohydrazide (1) on treatment with 4-fluorobenzaldehyde in presence of catalytic amount of acetic acid, accessed the target compound (2) with the yield of 79%. The target compound was confirmed by 1H-NMR, 13C-NMR, FT-IR and LCMS. In vitro antibacterial activity against Staphylococcus aureus (S. Aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli), and Pseudomonas aeruginosa (P. aeruginosa) were carried out and compound 2 showed promising activity against B. subtilis. In addition, compound 2 was analyzed for DNA binding study. It revealed that compound 2 has a promising affinity towards DNA double helix.

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Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

Analytical Chemistry ◽

10.1021/acs.analchem.5b00044 ◽

2015 ◽

Vol 87 (6) ◽

pp. 3490-3497 ◽

Cited By ~ 4

Author(s):

Shunsuke Takahashi ◽

Shinya Motooka ◽

Tomohiro Usui ◽

Shohei Kawasaki ◽

Hidefumi Miyata ◽

...

Keyword(s):

Single Molecule ◽

Double Helix ◽

Dna Double Helix

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Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

10.1101/796003 ◽

2019 ◽

Author(s):

Hazal B. Kose ◽

Sherry Xie ◽

George Cameron ◽

Melania S. Strycharska ◽

Hasan Yardimci

Keyword(s):

Single Molecule ◽

Replication Fork ◽

Double Helix ◽

Dna Unwinding ◽

Helicase Activity ◽

Replicative Helicase ◽

Double Stranded Dna ◽

Order Of Magnitude ◽

Dna Strand ◽

Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

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Bulk and Single-Molecule Fluorescence Studies of the Saturation of the DNA Double Helix Using YOYO-3 Intercalator Dye

The Journal of Physical Chemistry B ◽

10.1021/jp303438d ◽

2012 ◽

Vol 116 (38) ◽

pp. 11561-11569 ◽

Cited By ~ 6

Author(s):

Sergio G. Lopez ◽

Maria J. Ruedas-Rama ◽

Salvador Casares ◽

Jose M. Alvarez-Pez ◽

Angel Orte

Keyword(s):

Single Molecule ◽

Double Helix ◽

Single Molecule Fluorescence ◽

Fluorescence Studies ◽

Dna Double Helix

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Stretched, Twisted, Supercoiled and Braided DNA

MRS Proceedings ◽

10.1557/proc-463-31 ◽

1996 ◽

Vol 463 ◽

Author(s):

John F. Marko

Keyword(s):

Single Molecule ◽

Double Helix ◽

Persistence Length ◽

Thermal Fluctuations ◽

Internal Dynamics ◽

Flexible Polymer ◽

Bending Elasticity ◽

Polymer Elasticity ◽

Dna Double Helix ◽

Semi Flexible Polymer

ABSTRACTThe DNA double helix is a semi-flexible polymer with twist rigidity. Its bending elasticity gives rise to entropie polymer elasticity, which can be precisely studied in single-molecule experiments. DNA's twist rigidity causes it to wrap around itself, or ‘supercoil’, when it is sufficiently twisted; thermal fluctuations destabilize supercoiling for DNAs twisted fewer than once per twist persistence length. Twisted DNAs under tension, braided DNAs, and the internal dynamics of supercoiled DNAs are discussed. The interplay between braiding and supercoiling free energy is argued to be important for the decatenation of duplicated DNAs in prokaryote cells.

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Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a)

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718686115 ◽

2018 ◽

Vol 115 (21) ◽

pp. 5444-5449 ◽

Cited By ~ 47

Author(s):

Digvijay Singh ◽

John Mallon ◽

Anustup Poddar ◽

Yanbo Wang ◽

Ramreddy Tippana ◽

...

Keyword(s):

Single Molecule ◽

Dna Cleavage ◽

Genome Engineering ◽

Fluorescence Analysis ◽

Dna Interaction ◽

Target Sequence ◽

Solution Ph ◽

Guide Rna ◽

Product Release ◽

Time Observation

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5′ guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

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Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

10.1101/205575 ◽

2017 ◽

Cited By ~ 7

Author(s):

Digvijay Singh ◽

John Mallon ◽

Anustup Poddar ◽

Yanbo Wang ◽

Ramreddy Tippana ◽

...

Keyword(s):

Single Molecule ◽

Dna Cleavage ◽

Genome Engineering ◽

Fluorescence Analysis ◽

Dna Interaction ◽

Target Sequence ◽

Solution Ph ◽

Guide Rna ◽

Product Release ◽

Time Observation

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We have used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologues. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9, they both bind any DNA in search of PAM (protospacer-adjacent motif) sequences, verifies the target sequence directionally from the PAM-proximal end and rapidly rejects any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ~16 bp for cleavage, Cpf1 requires ~ 17 bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions and 5’ guanine in guide-RNA differentially affected different Cpf1 orthologues. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

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High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.65.10.4363-4368.1999 ◽

1999 ◽

Vol 65 (10) ◽

pp. 4363-4368 ◽

Cited By ~ 88

Author(s):

Jong-il Choi ◽

Sang Yup Lee

Keyword(s):

Escherichia Coli ◽

Acetic Acid ◽

Propionic Acid ◽

Cell Concentration ◽

Feeding Strategy ◽

Fed Batch ◽

A Cell ◽

Fed Batch Culture ◽

High Concentrations ◽

High Level

ABSTRACT Fermentation strategies for production of high concentrations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] with different 3-hydroxyvalerate (3HV) fractions by recombinantEscherichia coli harboring the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes were developed. Fed-batch cultures of recombinant E. coli with the pH-stat feeding strategy facilitated production of high concentrations and high contents of P(3HB-co-3HV) in a chemically defined medium. When a feeding solution was added in order to increase the glucose and propionic acid concentrations to 20 g/liter and 20 mM, respectively, after each feeding, a cell dry weight of 120.3 g/liter and a relatively low P(3HB-co-3HV) content, 42.5 wt%, were obtained. Accumulation of a high residual concentration of propionic acid in the medium was the reason for the low P(3HB-co-3HV) content. An acetic acid induction strategy was used to stimulate the uptake and utilization of propionic acid. When a fed-batch culture and this strategy were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 141.9 g/liter, 88.1 g/liter, 62.1 wt%, and 15.3 mol%, respectively. When an improved nutrient feeding strategy, acetic acid induction, and oleic acid supplementation were used, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 203.1 g/liter, 158.8 g/liter, 78.2 wt%, and 10.6 mol%, respectively; this resulted in a high level of productivity, 2.88 g of P(3HB-co-3HV)/liter-h.

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Sequence Specific Force Curves Measured by Mechanically Opening the DNA Double Helix

MRS Proceedings ◽

10.1557/proc-489-3 ◽

1997 ◽

Vol 489 ◽

Author(s):

U. Bockelmann ◽

B. Essevaz-Roulet ◽

F. Heslot

Keyword(s):

Single Molecule ◽

Double Helix ◽

Force Sensor ◽

Optical Microscope ◽

Microscope Slide ◽

Characteristic Variation ◽

Force Curves ◽

Dna Double Helix ◽

Glass Microscope Slide ◽

Glass Microscope

AbstractUsing techniques of molecular biology, we have designed a molecular construction which allows to attach the two complementary strands of one end of a single molecule of bacteriophage λ DNA separately to a glass microscope slide and a microscopic bead. A soft microneedle acting as a force sensor is chemically attached to the bead and its deflection is measured by an optical microscope. Keeping the base of the force lever fixed, the glass slide is displaced slowly, leading to a progressive opening of the double helix. The force measured during the opening process shows a characteristic variation which is related to the sequence of the bases along the DNA molecule. We present a brief summary of the present state of our work.

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A hold-and-feed mechanism drives directional DNA loop extrusion by condensin

10.1101/2021.10.29.466147 ◽

2021 ◽

Author(s):

Indra A Shaltiel ◽

Sumanjit Datta ◽

Léa Lecomte ◽

Markus Hassler ◽

Marc Kschonsak ◽

...

Keyword(s):

Electron Microscopy ◽

Single Molecule ◽

Protein Complexes ◽

Double Helix ◽

Power Stroke ◽

Reaction Cycle ◽

Cryo Electron Microscopy ◽

Condensin Complex ◽

Dna Loop ◽

Dna Double Helix

SMC protein complexes structure genomes by extruding DNA loops, but the molecular mechanism that underlies their activity has remained unknown. We show that the active condensin complex entraps the bases of a DNA loop in two separate chambers. Single-molecule and cryo-electron microscopy provide evidence for a power-stroke movement at the first chamber that feeds DNA into the SMC-kleisin ring upon ATP binding, while the second chamber holds on upstream of the same DNA double helix. Unlocking the strict separation of 'motor' and 'anchor' chambers turns condensin from a one-sided into a bidirectional DNA loop extruder. We conclude that the orientation of two topologically bound DNA segments during the course of the SMC reaction cycle determines the directionality of DNA loop extrusion.

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