Optical Tweezers Analysis of DNA–Protein Complexes

Heller, Iddo; Hoekstra, Tjalle P.; King, Graeme A.; Peterman, Erwin J. G.; Wuite, Gijs J. L.

doi:10.1021/cr4003006

Cited by 175 publications

(153 citation statements)

References 164 publications

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“…[1][2][3][4] The direct visualization of fluorescently labeled DNA undergoing Brownian motion using DNA-specific fluorescent dyes has allowed for a quantitative understanding of its thermal fluctuations. [5][6][7] Many dyes specific to double-stranded DNA (dsDNA) have been employed in fluorescence applications both in bulk and at the microscopic level.…”

Section: Introductionmentioning

confidence: 99%

A polarized view on DNA under tension

Mameren

Vermeulen

Wuite

2017

The Journal of Chemical Physics

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In the past decades, sensitive fluorescence microscopy techniques have contributed significantly to our understanding of the dynamics of DNA. The specific labeling of DNA using intercalating dyes has allowed for quantitative measurement of the thermal fluctuations the polymers undergo. On the other hand, recent advances in single-molecule manipulation techniques have unraveled the mechanical and elastic properties of this intricate polymer. Here, we have combined these two approaches to study the conformational dynamics of DNA under a wide range of tensions. Using polarized fluorescence microscopy in conjunction with optical-tweezers-based manipulation of YOYO-intercalated DNA, we controllably align the YOYO dyes using DNA tension, enabling us to disentangle the rapid dynamics of the dyes from that of the DNA itself. With unprecedented control of the DNA alignment, we resolve an inconsistency in reports about the tilted orientation of intercalated dyes. We find that intercalated dyes are on average oriented perpendicular to the long axis of the DNA, yet undergo fast dynamics on the time scale of absorption and fluorescence emission. In the overstretching transition of doublestranded DNA, we do not observe changes in orientation or orientational dynamics of the dyes. Only beyond the overstretching transition, a considerable depolarization is observed, presumably caused by an average tilting of the DNA base pairs. Our combined approach thus contributes to the elucidation of unique features of the molecular dynamics of DNA.

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Section: Introductionmentioning

confidence: 99%

A polarized view on DNA under tension

Mameren

Vermeulen

Wuite

2017

The Journal of Chemical Physics

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“…The stiffness constant, k, can be evaluated from the Stokes drag calibration method by monitoring the Brownian motion of an optically trapped polystyrene latex sphere. This kind of micro-spheres can be used as an optical handle to grab and manipulate other tiny objects, such as DNA [26,27], viruses [28] and molecules [29] by attaching them to the surface of spherical bead. This could be useful for biological studies and for optically assisted self-assembly processes.…”

Section: Resultsmentioning

confidence: 99%

Quantitative optical trapping and optical manipulation of micro-sized objects

Sayed¹

2017

Semicond. Phys. Quantum Electron. Optoelectron.

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Abstract. An optical tweezers technique is used for ultraprecise micromanipulation to measure positions of micrometer scale objects with a precision down to the nanometer scale. It consists of a high performance research microscope with motorized scanning stage and sensitive position detection system. Up to 10 traps can be used quasisimultaneously. Non photodamage optical trapping of Escherichia coli (E. coli) bacteria cells of 2 µm in length, as an example of motile bacteria, has been shown in this paper. Also, efficient optical trapping and rotation of polystyrene latex particles of 3 µm in diameter have been studied, as an optical handle for the pick and place of other tiny objects. A fast galvoscanner is used to produce multiple optical traps for manipulation of micro-sized objects and optical forces of these trapped objects quantified and measured according to explanation of ray optics regime. The diameter of trapped particle is bigger than the wavelength of the trapping laser light. The force constant (k) has been determined in real time from the positional time series recorded from the trapped object that is monitored by a CCD camera through a personal computer.

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“…To gain a comprehensive understanding of this process, our laboratory is using interdisciplinary approaches to reconstitute mitotic DNA decatenation in vitro. To achieve this, we combine ensemble biochemistry on model DNA substrates, with single-molecule optical tweezers coupled to fluorescence microscopy (Heller et al 2014). …”

Section: Modeling Ufbs In a Test Tubementioning

confidence: 99%

“…be tethered between streptavidin-coated polystyrene microspheres and manipulated in the flow cell of a fluorescence microscope (Heller et al 2014). The effect of tension applied to a model UFB can thus be addressed, and fluorescently tagged proteins can be directly visualized on the stretched DNA.…”

Section: Modeling Of Ufbs In Single-molecule Experimentsmentioning

confidence: 99%

Knotty Problems during Mitosis: Mechanistic Insight into the Processing of Ultrafine DNA Bridges in Anaphase

Sarlós

Biebricher

Petermann

et al. 2017

Cold Spring Harb Symp Quant Biol

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To survive and proliferate, cells have to faithfully segregate their newly replicated genomic DNA to the two daughter cells. However, the sister chromatids of mitotic chromosomes are frequently interlinked by so-called ultrafine DNA bridges (UFBs) that are visible in the anaphase of mitosis. UFBs can only be detected by the proteins bound to them and not by staining with conventional DNA dyes. These DNA bridges are presumed to represent entangled sister chromatids and hence pose a threat to faithful segregation. A failure to accurately unlink UFB DNA results in chromosome segregation errors and binucleation. This, in turn, compromises genome integrity, which is a hallmark of cancer. UFBs are actively removed during anaphase, and most known UFB-associated proteins are enzymes involved in DNA repair in interphase. However, little is known about the mitotic activities of these enzymes or the exact DNA structures present on UFBs. We focus on the biology of UFBs, with special emphasis on their underlying DNA structure and the decatenation machineries that process UFBs.

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Optical Tweezers Analysis of DNA–Protein Complexes

Cited by 175 publications

References 164 publications

A polarized view on DNA under tension

A polarized view on DNA under tension

Quantitative optical trapping and optical manipulation of micro-sized objects

Knotty Problems during Mitosis: Mechanistic Insight into the Processing of Ultrafine DNA Bridges in Anaphase

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