DNA–DNA Interactions in Tight Supercoils Are Described by a Small Effective Charge Density

Maffeo, Christopher; Schöpflin, Robert; Brutzer, Hergen; Stehr, Rene; Aksimentiev, Aleksei; Wedemann, Gero; Seidel, Ralf

doi:10.1103/physrevlett.105.158101

Cited by 98 publications

(165 citation statements)

References 39 publications

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“…After DNA stretching and initial characterization of the DNA, proteins were added, and changes in DNA length were observed as a function of applied force and DNA turns. Torque values were calculated based on previous theoretical work (24,38) (SI Appendix). In-house-written software used for these calculations is available for download at the Web site of R.S.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes

Szczelkun¹,

Tikhomirova

Šinkūnas

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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435

499

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Clustered, regularly interspaced, short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) systems protect bacteria and archaea from infection by viruses and plasmids. Central to this defense is a ribonucleoprotein complex that produces RNA-guided cleavage of foreign nucleic acids. In DNA-targeting CRISPR-Cas systems, the RNA component of the complex encodes target recognition by forming a sitespecific hybrid (R-loop) with its complement (protospacer) on an invading DNA while displacing the noncomplementary strand. Subsequently, the R-loop structure triggers DNA degradation. Although these reactions have been reconstituted, the exact mechanism of Rloop formation has not been fully resolved. Here, we use singlemolecule DNA supercoiling to directly observe and quantify the dynamics of torque-dependent R-loop formation and dissociation for both Cascade-and Cas9-based CRISPR-Cas systems. We find that the protospacer adjacent motif (PAM) affects primarily the Rloop association rates, whereas protospacer elements distal to the PAM affect primarily R-loop stability. Furthermore, Cascade has higher torque stability than Cas9 by using a conformational locking step. Our data provide direct evidence for directional R-loop formation, starting from PAM recognition and expanding toward the distal protospacer end. Moreover, we introduce DNA supercoiling as a quantitative tool to explore the sequence requirements and promiscuities of orthogonal CRISPR-Cas systems in rapidly emerging gene-targeting applications.magnetic tweezers | genome engineering | crRNA C lustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems constitute an adaptable immune system that protects bacteria and archaea against foreign nucleic acids. The defense is initiated by a ribonucleoprotein (RNP) complex that mediates cleavage of dsDNA (1) or RNA (2, 3). The RNA component (crRNA) of the complex is derived by transcription and posttranscriptional processing from a locus containing CRISPRs (2, 4, 5) in which short spacer fragments were integrated from foreign nucleic acids (6-8). Each transcribed crRNA spacer sequence encodes the recognition of the targets. In DNA-targeting CRISPR-Cas systems, the crRNAs form a hybrid with a matching complement (protospacer) on an invading DNA, which leads to the displacement of the noncomplementary strand. The resulting structure is called an R-loop and constitutes the signal for subsequent DNA degradation. R-loop formation is additionally dependent on a short protospacer adjacent motif (PAM) (Fig. 1A), which provides discrimination between self and nonself DNA in CRISPR systems; it is absolutely required for recognition of the invading DNA but is absent from the host CRISPR array (9).On the basis of sequence homology, different CRISPR-Cas families have been identified (10). We investigate here a type IE and a type II system from Streptococcus thermophilus St-CRISPR4 and St-CRISPR3, respectively. The Cas proteins of type IE systems (4, 11, 12) associate with a crRNA into a mult...

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Section: Methodsmentioning

confidence: 99%

“…To clearly distinguish between the two models, we use here single-molecule DNA supercoiling to directly reveal the kinetics and the extent of R-loop formation and dissociation (22)(23)(24). Moreover, our assay provides quantitative insight into the dependence of these processes on DNA supercoiling.…”

mentioning

confidence: 99%

Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes

Szczelkun¹,

Tikhomirova

Šinkūnas

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

435

499

View full text Add to dashboard Cite

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“…The side-by-side interaction between DNA fragments has been studied in some detail previously [50][51][52]. Seidel et al [50] demonstrated that in the presence of monovalent ions, the effective repulsion between DNA supercoils corresponds to 40 % of the DNA charge. Thus, it is important to examine whether ultra-short DNA fragments show electrostatic repulsion sufficient to give rise to considerably anisotropic interaction that would lead to the formation of columns.…”

Section: Resultsmentioning

confidence: 99%

Understanding the origin of liquid crystal ordering of ultrashort double-stranded DNA

et al. 2017

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“…Assuming that, in physiological conditions, half of the charges from the phosphate groups are screened by counter ions (42), one can think that each bead (σ = 2.5 nm ' 8 bp) contains a total charge of q b = 16q e =2, where q e is the electron charge. Within this assumption, we can map the external force applied onto each bead to an effective electric field E = f =q b .…”

Section: Methodsmentioning

confidence: 99%

Topological patterns in two-dimensional gel electrophoresis of DNA knots

Michieletto

Marenduzzo

Orlandini

2015

Proc. Natl. Acad. Sci. U.S.A.

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Gel electrophoresis is a powerful experimental method to probe the topology of DNA and other biopolymers. Although there is a large body of experimental work that allows us to accurately separate different topoisomers of a molecule, a full theoretical understanding of these experiments has not yet been achieved. Here we show that the mobility of DNA knots depends crucially and subtly on the physical properties of the gel and, in particular, on the presence of dangling ends. The topological interactions between these and DNA molecules can be described in terms of an "entanglement number" and yield a nonmonotonic mobility at moderate fields. Consequently, in 2D electrophoresis, gel bands display a characteristic arc pattern; this turns into a straight line when the density of dangling ends vanishes. We also provide a novel framework to accurately predict the shape of such arcs as a function of molecule length and topological complexity, which may be used to inform future experiments.DNA knots | topology | gel electrophoresis T opology plays a key role in the biophysics of DNA and is intimately related to its functioning. For instance, transcription of a gene redistributes twist locally to create what is known as supercoiling, whereas catenanes or knots can prevent cell division; hence they need to be quickly and accurately removed by specialized enzymes known as topoisomerases. How can one establish experimentally the topological state of a given DNA molecule? By far the most successful and widely used technique to do so is gel electrophoresis (1, 2). This method exploits the empirical observation that the mobility of a charged DNA molecule under an electric field depends on its size, shape, and topology (2). Gel electrophoresis is so reliable that it can be used, for instance, to map replication origins and stalled replication forks (3), to separate plasmids with different amount of supercoiling (3, 4), and to identify DNA knots (5, 6). The most widely used variant of this technique nowadays is 2D gel electrophoresis, where a DNA molecule is subjected to a sequence of two fields, applied along orthogonal directions (2). The two runs are characterized by different field strengths and sometimes also gel concentrations (4); with suitable choices, the joint responses lead to increased sensitivity.Although gel electrophoresis is used very often, and is extremely well characterized empirically, there is still no comprehensive theory to quantitatively understand, or predict, what results will be observed in a particular experiment. Some aspects are reasonably well established. For instance, it is now widely accepted that the physics of the size-dependent migration of linear polymers can be explained by the theory of biased polymer reptation (7-12). Likewise, the behavior of, for example, nicked, torsionally relaxed, DNA knots in a sparse gel and under a weak field is analogous to that of molecules sedimenting under gravity (13-15). The terminal velocity can be estimated via a balance between the applied force and the fr...

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DNA–DNA Interactions in Tight Supercoils Are Described by a Small Effective Charge Density

Cited by 98 publications

References 39 publications

Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes

Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes

Understanding the origin of liquid crystal ordering of ultrashort double-stranded DNA

Topological patterns in two-dimensional gel electrophoresis of DNA knots

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