Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer

Tomschik, Miroslav; Zheng, Haocheng; Holde, Kensal van; Zlatanova, Jordanka; Leuba, Sanford H.

doi:10.1073/pnas.0500189102

Cited by 127 publications

(115 citation statements)

References 42 publications

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“…Biological reactions that unravel nucleosomes should be readily discernable with such single spectroscopic measurements. We have recently observed long-range conformational changes in individual nucleosomes by spFRET with an EFFM [28]. As the SCFM has a much higher SNR (20:1) than the EFFM (≈5:1) [49], it should be possible to achieve much higher resolution time trajectories with the SCFM, especially if the instrument is connected to a high precision flow injection system, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Prior to an experiment, the DNA is reconstituted with core histones into a nucleosome, e.g. [13,15,27,28,101].…”

Section: Nucleosome Preparationmentioning

confidence: 99%

“…We know of no other method other than spFRET with which one can follow the dynamics of reversible processes of single molecules in real time, e.g. [28]. Concept of single pair fluorescence resonance energy transfer (spFRET) and example time trajectory.…”

Section: Future Directionsmentioning

confidence: 99%

“…Harp, was prepared with PyMOL [106] and Xfit [107] using pdb 1kx5 [108]. For details of preparation see [28]. (B) Example SCFM 10 μm × 10 μm image of individual nucleosomes labeled with a Cy3 dye and a Cy5 dye (for details on labeling see [15,28] …”

Section: Future Directionsmentioning

confidence: 99%

“…Single-molecule approaches have already led to significant new information about chromatin dynamics, reviewed in [7][8][9][10][11][12][13][14][15][16][17]. Examples are the determination of the piconewton (pN) forces to unravel individual nucleosomes [18][19][20][21][22][23][24], the demonstration that stretching forces applied to DNA prevent nucleosome assembly [18,[25][26][27], and the observation of reversible long-range opening and closing in individual nucleosomes in real time [28]. Single-molecule approaches can be used to achieve a fundamental understanding of biological processes occurring at the nanometer (nm) scale, in the millisecond (ms) time domain and at the pN force level.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

“…Prior to an experiment, the DNA is reconstituted with core histones into a nucleosome, e.g. [13,15,27,28,101].…”

Section: Nucleosome Preparationmentioning

confidence: 99%

Section: Future Directionsmentioning

confidence: 99%

Section: Future Directionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Zheng¹,

Goldner²,

Leuba³

2007

Methods

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Opening and Closing DNA: Theories on the Nucleosome

Kulić

Schiessel

2007

DNA Interactions With Polymers and Surfactants

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INTRODUCTIONDNA-the carrier of the genetic information-is at the base of many central life processes [1]: replication, transcription, and repair of genetic material depend on the unique properties of DNA, especially the base pairing. One has, however, to appreciate the fact that the molecular machinery of eucaryotes (plants and animals) does not deal with naked DNA but with chromatin, a DNA-protein complex in which DNA is wrapped and folded in a hierarchical fashion [2]. On the lowest level DNA is wrapped nearly twice around an octamer of histone proteins. A short stretch of the "linker DNA" connects to the next such protein spool. The resulting string of so-called nucleosomes folds into higher order structures, the details of which are still under debate (see Figure 7.1).The structure of the nucleosome core particle (NCP), the particle that is left when the linker DNA is digested away, is known in exquisite detail from X-ray crystallography at 2.8 A resolution [3] and more recently at 1.9 A[4]. The octamer is composed of two molecules each of the four core histone proteins H2A, H2B, H3, and H4. At physiological conditions the stable oligomeric aggregates of the core histones are the H3-H4 tetramer (an aggregate of two H3 and two H4 proteins) and the H2A-H2B dimer; the octamer is then only stable if it is associated with DNA [5]. The two dimers and the tetramer are put together in such a way that the resulting octamer forms a cylinder with about a 65 A diameter and about a 60 A height. With grooves, ridges, and binding sites the octamer defines the wrapping path of the DNA, a left-handed helical ramp of 1 and 3/4 turns, a 147 base-pairs (bp) length, and a roughly 28 A pitch. This aggregate has a twofold axis of symmetry (the dyad axis) that is perpendicular to the superhelix axis. A schematic view of the NCP is given in Figure 7.2. DNA Interactions with Polymers and Surfactants Edited by Rita Dias and Bj€ orn Lindman

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The Folding of the Nucleosome Chain

Rippe¹

2011

Genome Organization and Function in the Cell Nucleus

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Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer

Cited by 127 publications

References 42 publications

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

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

Opening and Closing DNA: Theories on the Nucleosome

The Folding of the Nucleosome Chain

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