Structural dynamics of individual Holliday junctions

McKinney, Sean A.; Déclais, Anne-Cécile; Lilley, David M.J.; Ha, Taekjip

doi:10.1038/nsb883

Cited by 338 publications

(482 citation statements)

References 28 publications

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“…[43][44][45] In the presence of Na + and Mg 2+ , a HJ folds into two stacked X-shaped conformers by coaxial pairwise stacking of its four helical arms (conf-I and conf-II, Figure 1A, B). [42][43][44]46 In one conformer, two of the DNA strands run through a pair of stacked helical arms similarly as in a B-form DNA while the other two strands exchange between stacked helical pairs ( Figure 1B). 42,46 The four strands switch positions in the other conformer.…”

Section: Methodsmentioning

confidence: 99%

Engineered Holliday Junctions as Single-Molecule Reporters for Protein−DNA Interactions with Application to a MerR-Family Regulator

et al. 2007

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Protein-DNA interactions are essential for gene maintenance, replication and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Since the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo-and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.

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Section: Methodsmentioning

confidence: 99%

Engineered Holliday Junctions as Single-Molecule Reporters for Protein−DNA Interactions with Application to a MerR-Family Regulator

et al. 2007

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“…Oligonucleotides of the following sequences were synthesized using phosphoramidite chemistry implemented on DNA synthesizers (IDTDNA.com): Cy5-J3b, 5′-Cy5-CCCTAGCAAGGGGCTGCTACGG; Cy3-J3h, 5′-Cy3-CCGTAGCAGCCTGAGCGGTGGG; Biot-J3r, 5′-Biotin-CCCACCGCTCAACTCAACTGGG; and J3x, 5′-CCCAGTTGAGTCCTTGCTAGGG [36]. We mixed all the oligonucleotides together at 65 °C and then allowed them to come slowly to room temperature to hybridize completely.…”

Section: Holliday Junction Preparationmentioning

confidence: 99%

“…A scanning confocal fluorescence microscope (SCFM) that is sensitive enough to detect single fluorescent molecules can be used to observe single-pair fluorescence resonance energy transfer (spFRET) [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45]. Fluorescence resonance energy transfer (FRET) is a spectroscopic process in which non-radiative energy transfer occurs between an excited dipole (the donor) and another dipole (the acceptor) that has an absorption spectrum that overlaps the emission spectrum of the donor [46].…”

Section: Introductionmentioning

confidence: 99%

“…Changes in the conformation of the molecule are detected as changes in the energy transfer between the donor and acceptor molecule. In this fashion, single molecular-pair FRET has been used to study, e.g., nanometer conformational motions in individual RNA molecules [29][30][31][32][33][34][35], individual DNA molecules [36][37][38][39][40][41] and individual helicases and other motor proteins on DNA [42][43][44][45]. By following changes in FRET efficiency over time, it is possible to observe nanometer distance changes due to macromolecular rearrangements.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Zheng¹,

Goldner²,

Leuba³

2007

Methods

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Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/ acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.

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“…[3][4][5][6] In particular, intramolecular distances can now be probed by attaching dye molecules to sites of interest and measuring the efficiency of fluorescence resonance energy transfer (FRET). 3,5 This approach has been applied to monitor folding of biopolymers (proteins and nucleic acids), [7][8][9] enzyme-substrate complex formation, [10][11][12][13][14] and the operation of molecular motors. [15][16][17][18][19][20] A persistent issue in such studies is that they explicitly follow the dynamics of one, or at most a few, of the many degrees of freedom of a macromolecular system.…”

Section: Introductionmentioning

confidence: 99%

Models of Single-Molecule Experiments with Periodic Perturbations Reveal Hidden Dynamics in RNA Folding

Liu

et al. 2009

J. Phys. Chem. B

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Traditionally, microscopic fluctuations of molecules have been probed by measuring responses of an ensemble to perturbations. Now, single-molecule experiments are capable of following fluctuations without introducing perturbations. However, dynamics not readily sampled at equilibrium should be accessible to nonequilibrium single-molecule measurements. In a recent study [Qu, X. et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6602-6607], the efficiency of fluorescence resonance energy transfer (FRET) between probes on the L18 loop and 3′ terminus of the 260 nucleotide RNase P RNA from Bacillus stearothermophilus was found to exhibit complex kinetics that depended on the (periodically alternating) concentration of magnesium ions ([Mg 2+ ]) in solution. Specifically, this time series was found to exhibit a quasi-periodic response to a squarewave pattern of [Mg 2+ ] changes. Because these experiments directly probe only one of the many degrees of freedom in the macromolecule, models are needed to interpret these data. We find that Hidden Markov Models are inadequate for describing the nonequilibrium dynamics, but they serve as starting points for the construction of models in which a discrete observable degree of freedom is coupled to a continuously evolving (hidden) variable. Consideration of several models of this general form indicates that the quasi-periodic response in the nonequilibrium experiments results from the switching (back and forth) in positions of the minima of the effective potential for the hidden variable. This switching drives oscillation of that variable and synchronizes the population to the changing [Mg 2+ ]. We set the models in the context of earlier theoretical and experimental studies and conclude that single-molecule experiments with periodic peturbations can indeed yield qualitatively new information beyond that obtained at equilibrium.

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Structural dynamics of individual Holliday junctions

Cited by 338 publications

References 28 publications

Engineered Holliday Junctions as Single-Molecule Reporters for Protein−DNA Interactions with Application to a MerR-Family Regulator

Engineered Holliday Junctions as Single-Molecule Reporters for Protein−DNA Interactions with Application to a MerR-Family Regulator

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Models of Single-Molecule Experiments with Periodic Perturbations Reveal Hidden Dynamics in RNA Folding

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