Visualizing single DNA-bound proteins using DNA as a scanning probe

Noom, Maarten C.; Broek, Bram van den; Mameren, Joost van; Wuite, Gijs J. L.

doi:10.1038/nmeth1126

Cited by 82 publications

(93 citation statements)

References 33 publications

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“…To determine the bimolecular association rate k on to a recognition site on linear DNA, for different polymer conformations, individual DNA molecules were tethered between two optically trapped beads in a multichannel flow chamber as described (25,26). The degree of DNA coiling was set by changing the distance between the two beads (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…EcoRV was recovered from ammonium sulfate precipitates as described (26). For the DNA cleavage experiments pCco5 plasmid (6.5 kbp in length) was linearized by SpeI digestion (25).…”

Section: Methodsmentioning

confidence: 99%

“…26. Because of nonmixing laminar buffer flow (39), assembled DNA constructs can be transported into a channel containing 1 nM EcoRV with an estimated timing error of 0.5 s. Before each experiment session a 10 mM Tris⅐HCl buffer (pH 7.5) containing 1 mg/ml BSA was flowed through the chamber for Ϸ30 min to coat the walls of the chamber and tubing to prevent sticking of EcoRV enzymes.…”

Section: Methodsmentioning

confidence: 99%

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How DNA coiling enhances target localization by proteins

Broek

Lomholt²,

Kalisch

et al. 2008

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

188

197

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Many genetic processes depend on proteins interacting with specific sequences on DNA. Despite the large excess of nonspecific DNA in the cell, proteins can locate their targets rapidly. After initial nonspecific binding, they are believed to find the target site by 1D diffusion (''sliding'') interspersed by 3D dissociation/reassociation, a process usually referred to as facilitated diffusion. The 3D events combine short intrasegmental ''hops'' along the DNA contour, intersegmental ''jumps'' between nearby DNA segments, and longer volume ''excursions.'' The impact of DNA conformation on the search pathway is, however, still unknown. Here, we show direct evidence that DNA coiling influences the specific association rate of EcoRV restriction enzymes. Using optical tweezers together with a fast buffer exchange system, we obtained association times of EcoRV on single DNA molecules as a function of DNA extension, separating intersegmental jumping from other search pathways. Depending on salt concentration, targeting rates almost double when the DNA conformation is changed from fully extended to a coiled configuration. Quantitative analysis by an extended facilitated diffusion model reveals that only a fraction of enzymes are ready to bind to DNA. Generalizing our results to the crowded environment of the cell we predict a major impact of intersegmental jumps on target localization speed on DNA.A n essential feature in biological processes on DNA is the ability of proteins to quickly locate specific DNA sequences in a vast surplus of nonspecific DNA (1, 2). A protein's search for the target site is thought to be accelerated by facilitated diffusion along nonspecific DNA (3-6). Recent work has yielded considerable insight in the possible search strategies of sitespecific proteins (7-15). Assisted by DNA looping some proteins can, for instance, intermittently bind to two DNA segments simultaneously. This way they can directly move from one to another chemically remote segment (16). This intersegmental transfer accelerates target finding on DNA because it assumes a constantly changing random configuration (11). Here, we demonstrate and quantify a similar mechanism, intersegmental jumping, for proteins with only one DNA-binding site.Little experimental work on facilitated diffusion is available. To date, most studies have been investigating DNA cleavage by restriction enzymes in bulk assays, measuring association times as a function of DNA length, or monitoring processivity on DNA constructs with two sites (7,(17)(18)(19)(20)(21). Although in these biochemical assays valuable information can be obtained, association rates of proteins to specific sites are difficult to measure, and the underlying kinetics of the target search mechanism are often obscured. Furthermore, it remains experimentally challenging to distinguish 1D and 3D search pathways. In previous singlemolecule assays only pure 1D protein search has been addressed (21-24). Here, we present single-molecule measurements of DNA cleavage by EcoRV on individual plasm...

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Section: Resultsmentioning

confidence: 99%

“…EcoRV was recovered from ammonium sulfate precipitates as described (26). For the DNA cleavage experiments pCco5 plasmid (6.5 kbp in length) was linearized by SpeI digestion (25).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

How DNA coiling enhances target localization by proteins

Broek

Lomholt²,

Kalisch

et al. 2008

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

188

197

View full text Add to dashboard Cite

show abstract

“…To generate the DNA construct with biotin labels on one of the strands on either side of the DNA (3Ј-3Ј attached DNA), we used a protocol to terminally label lambda DNA, as previously described (30,31). The DNA construct that possesses biotin-labels at both strands on either end (3Ј5Ј-5Ј3Ј attached DNA) was generated as follows.…”

Section: Methodsmentioning

confidence: 99%

Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

Mameren

Groß

Farge

et al. 2009

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

Self Cite

266

299

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Single-molecule manipulation studies have revealed that doublestranded DNA undergoes a structural transition when subjected to tension. At forces that depend on the attachment geometry of the DNA (65 pN or 110 pN), it elongates Ϸ1.7-fold and its elastic properties change dramatically. The nature of this overstretched DNA has been under debate. In one model, the DNA cooperatively unwinds, while base pairing remains intact. In a competing model, the hydrogen bonds between base pairs break and two single DNA strands are formed, comparable to thermal DNA melting. Here, we resolve the structural basis of DNA overstretching using a combination of fluorescence microscopy, optical tweezers, and microfluidics. In DNA molecules undergoing the transition, we visualize double-and single-stranded segments using specific fluorescent labels. Our data directly demonstrate that overstretching comprises a gradual conversion from double-stranded to singlestranded DNA, irrespective of the attachment geometry. We found that these conversions favorably initiate from nicks or free DNA ends. These discontinuities in the phosphodiester backbone serve as energetically favorable nucleation points for melting. When both DNA strands are intact and no nicks or free ends are present, the overstretching force increases from 65 to 110 pN and melting initiates throughout the molecule, comparable to thermal melting. These results provide unique insights in the thermodynamics of DNA and DNA-protein interactions.DNA melting ͉ fluorescence microscopy ͉ optical trapping ͉ single-molecule techniques ͉ single-stranded DNA

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“…Recently, state of the art braiding experiments have been developed [48] using four optically trapped beads, which potentially offer much greater control over the geometry of the two molecules than those of Ref. [16], most notably b .…”

Section: Discussion and Outlookmentioning

confidence: 99%

Self-consistent treatment of electrostatics in molecular DNA braiding through external forces

Lee

2014

Phys. Rev. E

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In this paper we consider a physical system in which two DNA molecules braid about each other. The distance between the two molecular ends, on either side of the braid, is held at a distance much larger than supercoiling radius of the braid. The system is subjected to an external pulling force, and a moment that induces the braiding. In a model, developed for understanding such a system, we assume that each molecule can be divided into a braided and unbraided section. We also suppose that the DNA is nicked so that there is no constraint of the individual linking numbers of the molecules. Included in the model are steric and electrostatic interactions, thermal fluctuations of the braided and unbraided sections of the molecule, as well as the constraint on the braid linking (catenation) number. We compare two approximations used in estimating the free energy of the braided section. One is where the amplitude of undulations of one molecule with respect to the other is determined only by steric interactions. The other is a self-consistent determination of the mean squared amplitude of these undulations. In this second approximation electrostatics should play an important role in determining this quantity, as suggested by physical arguments. We see that if the electrostatic interaction is sufficiently large there are indeed notable differences between the two approximations. We go on to test the self-consistent approximation, included in the full model, against experimental data, for such a system, and we find good agreement. However, there seems to by a slight left/right handed braid asymmetry in some of the experimental results. We discuss what might be the origin of this small asymmetry

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Visualizing single DNA-bound proteins using DNA as a scanning probe

Cited by 82 publications

References 33 publications

How DNA coiling enhances target localization by proteins

How DNA coiling enhances target localization by proteins

Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

Self-consistent treatment of electrostatics in molecular DNA braiding through external forces

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