DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

Price, Allen C.; Pilkiewicz, Kevin R.; Graham, Thomas G.W.; Song, Dan; Eaves, Joel D.; Loparo, Joseph J.

doi:10.1016/j.bpj.2015.04.022

Cited by 18 publications

(27 citation statements)

References 39 publications

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“…Using our channel geometry and flow rate, we calculated Wi = 19. We previously showed that at Wi = 19 the extension of QD labeled  DNA is 66% and is relatively constant from the tether point out to the second QD (29). We next experimentally measured the extension using position measurements of QDs 1 and 2 and found the extension to be 65%, in agreement with our first method.…”

Section: Resultssupporting

confidence: 79%

“…The number of hopping cycles will not depend strongly on salt, since it only depends on the probability of recurrence once the protein has dissociated, a probability which is determined by the statistics of the three-dimensional diffusion. In addition, the mean time per hop is expected to be very small compared to the time in sliding, as it is controlled by the three-dimensional diffusion coefficient (15 µm 2 /s from ref (29)), which is significantly greater than that of the one-dimensional diffusion. Therefore, the scaling exponent we determined should reflect that of the intrinsic off rate from the sliding (non-specifically bound) state.…”

Section: The Interaction Of Ecori With Non-specific Dnamentioning

confidence: 99%

“…We can use our previous measurement of the hydrodynamic radius of the QD labeled EcoRI (13.70.4 nm , ref. (29)), and estimate ROC  13.7 1.0 nm. This leads to a reduction factor of 1500 (9%) due solely to the rotation coupling as calculated using Eq.…”

Section: Rotational Coupling and The Energy Landscape Of Slidingmentioning

confidence: 99%

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The sequence dependent search mechanism of EcoRI

Piatt

Loparo

Price

2018

Preprint

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One-dimensional search is an essential step in DNA target recognition. Theoretical studies have suggested that the sequence dependence of one-dimensional diffusion can help resolve the competing demands of fast search and high target affinity, a conflict known as the speedselectivity paradox. The resolution requires that the diffusion energy landscape is correlated with the underlying specific binding energies. In this work, we report observations of onedimensional search by QD labeled EcoRI. Our data supports the view that proteins search DNA via rotation coupled sliding over a corrugated energy landscape. We observed that while EcoRI primarily slides along DNA at low salt concentrations, at higher concentrations its diffusion is a combination of sliding and hopping. We also observed long-lived pauses at genomic star sites which differ by a single nucleotide from the target sequence. To reconcile these observations with prior biochemical and structural data, we propose a model of search in which the protein slides over a sequence independent energy landscape during fast search, but rapidly interconverts with a "hemi-specific" binding mode in which a half site is probed. This half site interaction stabilizes the transition to a fully specific mode of binding which can then lead to target recognition.

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Section: Resultssupporting

confidence: 79%

Section: The Interaction Of Ecori With Non-specific Dnamentioning

confidence: 99%

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The sequence dependent search mechanism of EcoRI

Piatt

Loparo

Price

2018

Preprint

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“…When compiling binding distributions from hundreds of such DNA molecules (spanning several flowcells), this approximation results in an apparent broadening of the resulting histogram. This broadening has also been observed for both site-and sequencespecific DNA-binding proteins in similar flow-stretches assays (e.g., EcoRI(E111Q) (40,41), FtsK (42,43), and DNA repair proteins (44,45)). Nonetheless, these results confirm that high-quality, recombinant λ-DNA can be rapidly modified at multiple sites and purified for single-molecule studies.…”

Section: A Molecular Toolkit For Modifying λ-Phage Dnasupporting

confidence: 57%

Multi-site internal modification of long DNA substrates for single-molecule studies

Kim

Leal

et al. 2016

Preprint

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Single-molecule studies of protein-nucleic acid interactions frequently require sitespecific modification of long DNA substrates. DNA isolated from bacteriophage λ (λ-DNA) is a convenient source of high quality long (48.5 kb) DNA. However, introducing specific DNA sequences, tertiary structures, and chemical modifications into λ-DNA remains technically challenging. Most current approaches rely on multistep ligations with low yields and incomplete products. Here, we describe a molecular toolkit for rapid preparation of modified λ-DNA. A set of PCR cassettes facilitates the introduction of recombinant DNA sequences into λ-DNA with 90-100% yield. Furthermore, various DNA structures and chemical modifications can be inserted at user-defined sites via an improved nicking enzyme-based strategy. As a proof-of-principle, we explore the interactions of Proliferating Cell Nuclear Antigen (PCNA) with modified DNA sequences and structures incorporated within λ-DNA. Our results demonstrate that PCNA can load on both 5'-ssDNA flaps and a 13xCAG triplet repeat. However, PCNA remains trapped on the 13xCAG structure, confirming a proposed mechanism for triplet repeat expansion. Although we focus on λ-DNA, this method is applicable to all long DNA substrates. We anticipate that this molecular toolbox will be broadly useful for both ensemble-and singlemolecule studies that require site-specific modification of long DNA substrates.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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“…Single molecule techniquesoffer the ultimate in sensitivity, but often make acquiring statistically significant data difficult. Extending our previous work on flow stretching of DNA [4,5,6], we have developed a multiplexing single molecule technique which measuresthe cleavage of hundreds of DNAs, providing statistically significant data in a single experiment.…”

mentioning

confidence: 99%