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

2015 ◽  
Vol 87 (6) ◽  
pp. 3490-3497 ◽  
Author(s):  
Shunsuke Takahashi ◽  
Shinya Motooka ◽  
Tomohiro Usui ◽  
Shohei Kawasaki ◽  
Hidefumi Miyata ◽  
...  
Keyword(s):  
Single Molecule ◽  
Double Helix ◽  
Dna Double Helix

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

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.

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

2012 ◽  
Vol 116 (38) ◽  
pp. 11561-11569 ◽  
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

Stretched, Twisted, Supercoiled and Braided DNA

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.

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

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.

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

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.

scholarly journals Ionic strength modulates HU protein-induced DNA supercoiling

2021 ◽  
Author(s):  
Alexander Zhang ◽  
Yan Yan ◽  
Fenfei Leng ◽  
David Dunlap ◽  
Laura Finzi
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Magnetic Measurements ◽  
Double Helix ◽  
Bacterial Genome ◽  
Dna Supercoiling ◽  
Coli Strain ◽  
Dna Unwinding ◽  
Dna Compaction ◽  
Dna Double Helix

The histone-like protein from E. coli strain U93 (HU) is an abundant nucleoid-associated protein that contributes to the compaction of the bacterial genome as well as to the regulation of many of its transactions. Despite many years of investigations, the way and extent to which HU binding alters the DNA double helix and/or generates hierarchical structures using DNA as a scaffold is not completely understood. Here we combined single-molecule magnetic measurements with circular dichroism studies to monitor structural changes in the DNA-HU fiber as HU concentration was increased from 0 to 1000 nM under low and physiological monovalent salt conditions. We confirmed that DNA compaction correlated with HU concentration in a biphasic manner but DNA unwinding varied monotonically with HU concentration in 100 mM KCl. Instead, in more physiological 200 mM salt conditions, DNA compaction was monotonic while HU-induced DNA unwinding was negligible. Differential compaction and unwinding of DNA may be part of the response of bacteria to large variations in salt concentrations.

scholarly journals Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling

2020 ◽  
Vol 117 (11) ◽  
pp. 5853-5860 ◽  
Author(s):  
Ivan E. Ivanov ◽  
Addison V. Wright ◽  
Joshua C. Cofsky ◽  
Kevin D. Palacio Aris ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Genome Engineering ◽  
Double Helix ◽  
Energy Landscape ◽  
Dna Supercoiling ◽  
Seed Region ◽  
Loop Formation ◽  
A Genome ◽  
Dna Double Helix

The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.

scholarly journals Kinetics of dCas9 target search in Escherichia coli

Science ◽  
2017 ◽  
Vol 357 (6358) ◽  
pp. 1420-1424 ◽  
Author(s):  
Daniel Lawson Jones ◽  
Prune Leroy ◽  
Cecilia Unoson ◽  
David Fange ◽  
Vladimir Ćurić ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Single Molecule ◽  
Double Helix ◽  
Target Sequence ◽  
Chromosomal Dna ◽  
Guide Rna ◽  
A Cell ◽  
High Concentrations ◽  
Dna Double Helix

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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