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 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.

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 Heterogeneity in Nucleosome Spacing Governs Chromatin Elasticity

2019 ◽  
Author(s):  
Bruno Beltran ◽  
Deepti Kannan ◽  
Quinn MacPherson ◽  
Andrew J. Spakowitz
Keyword(s):  
Time Scales ◽  
Living Cell ◽  
Double Helix ◽  
Chromatin Organization ◽  
Chromatin Loop ◽  
Loop Formation ◽  
Dominant Source ◽  
Wormlike Chain ◽  
Dna Double Helix ◽  
Nucleosome Binding

Within a living cell, the myriad of proteins that bind DNA introduce heterogeneously spaced kinks into an otherwise semiflexible DNA double helix. To investigate the effects of heterogeneous nucleosome binding on chromatin organization, we extend the wormlike chain (WLC) model to include statistically spaced, rigid kinks. On time scales where nucleosome positions are fixed, we find that the probability of chromatin loop formation can differ by up to six orders of magnitude between two sets of nucleosome positions drawn from the same distribution. On longer time scales, we show that continuous re-randomization due to nucleosome turnover results in chromatin tracing out an effective WLC with a dramatically smaller Kuhn length than bare DNA. Together, these observations demonstrate that heterogeneity in nucleosome spacing acts as the dominant source of chromatin elasticity and governs both local and global chromatin organization.

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 Diversity and Functions of Type II Topoisomerases

Acta Naturae ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 59-75
Author(s):  
Dmitry A. Sutormin ◽  
Alina Kh. Galivondzhyan ◽  
Alexander V. Polkhovskiy ◽  
Sofia O. Kamalyan ◽  
Konstantin V. Severinov ◽  
...  
Keyword(s):  
Genetic Information ◽  
Double Helix ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Specific Group ◽  
Functional Roles ◽  
Domains Of Life ◽  
Dna Double Helix ◽  
Catalytic Cycles ◽  
Transcription And Replication

The DNA double helix provides a simple and elegant way to store and copy genetic information. However, the processes requiring the DNA helix strands separation, such as transcription and replication, induce a topological side-effect supercoiling of the molecule. Topoisomerases comprise a specific group of enzymes that disentangle the topological challenges associated with DNA supercoiling. They relax DNA supercoils and resolve catenanes and knots. Here, we review the catalytic cycles, evolution, diversity, and functional roles of type II topoisomerases in organisms from all domains of life, as well as viruses and other mobile genetic elements.

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.

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