Preparation of DNA and nucleoprotein samples for AFM imaging

Lyubchenko, Yuri L.

doi:10.1016/j.micron.2010.08.011

Cited by 79 publications

(70 citation statements)

References 104 publications

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“…The vertical displacement of the tip during scanning is detected using the optical lever principle, in which the position of the light spot on the PSPD is measured. The figure was reproduced from paper (Lyubchenko 2011b) with Copyright © 2012 permission from Elsevier.…”

Section: Figmentioning

confidence: 99%

Nanoscale nucleosome dynamics assessed with time-lapse AFM

Lyubchenko

2013

Biophys Rev

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A fundamental challenge associated with chromosomal gene regulation is accessibility of DNA within nucleosomes. Recent studies performed by various techniques, including single-molecule approaches, led to the realization that nucleosomes are dynamic structures rather than static systems, as it was once believed. Direct data is required in order to understand the dynamics of nucleosomes more clearly and answer fundamental questions, including: What is the range of nucleosome dynamics? Does a non-ATP dependent unwrapping process of nucleosomes exist? What are the factors facilitating the large scale opening and unwrapping of nucleosomes? This review summarizes the results of nucleosome dynamics obtained with time-lapse AFM, including a high-speed version (HS-AFM) capable of visualizing molecular dynamics on the millisecond time scale. With HS-AFM, the dynamics of nucleosomes at a sub-second time scale was observed allowing one to visualize various pathways of nucleosome dynamics, such as sliding and unwrapping, including complete dissociation. Overall, these findings reveal new insights into the dynamics of nucleosomes and the novel mechanisms controlling spontaneous chromatin dynamics.

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Section: Figmentioning

confidence: 99%

Nanoscale nucleosome dynamics assessed with time-lapse AFM

Lyubchenko

2013

Biophys Rev

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“…AFM is a powerful technique that has provided high-resolution images for a variety of protein-DNA complexes (20)(21)(22)(23)(24)(25). Here we used AFM to visualize whether LacI divides a supercoiled DNA molecule into distinct topological domains.…”

Section: Atomic Force Microscope Images Revealed That Laci Divided a mentioning

confidence: 99%

Dividing a supercoiled DNA molecule into two independent topological domains

Leng

Chen

Dunlap

2011

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

102

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Both prokaryotic and eukaryotic chromosomes are organized into many independent topological domains. These topological domains may be formed through constraining each DNA end from rotating by interacting with nuclear proteins; i.e., DNA-binding proteins. However, so far, evidence to support this hypothesis is still elusive. Here we developed two biochemical methods; i.e., DNA-nicking and DNA-gyrase methods to examine whether certain sequence-specific DNA-binding proteins are capable of separating a supercoiled DNA molecule into distinct topological domains. Our approach is based on the successful construction of a series of plasmid DNA templates that contain many tandem copies of one or two DNA-binding sites in two different locations. With these approaches and atomic force microscopy, we discovered that several sequence-specific DNA-binding proteins; i.e., lac repressor, gal repressor, and λ O protein, are able to divide a supercoiled DNA molecule into two independent topological domains. These topological domains are stable under our experimental conditions. Our results can be explained by a topological barrier model in which nucleoprotein complexes confine DNA supercoils to localized regions. We propose that DNA topological barriers are certain nucleoprotein complexes that contain stable toroidal supercoils assembled from DNA-looping or tightly wrapping DNA around DNA-binding proteins. The DNA topological barrier model may be a general mechanism for certain DNA-binding proteins, such as histone or histone-like proteins, to modulate topology of chromosome DNA in vivo.

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“…Other problems with this method were identified and discussed in refs. [18, 44]. On the contrary, the AP-mica and APS-mica procedures are much more flexible.…”

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Visualization of DNA and Protein–DNA Complexes with Atomic Force Microscopy

Lyubchenko¹,

Gall²,

Shlyakhtenko³

2013

Methods in Molecular Biology

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This article describes sample preparation techniques for AFM imaging of DNA and protein–DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purifi cation of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein–DNA complexes.

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Preparation of DNA and nucleoprotein samples for AFM imaging

Cited by 79 publications

References 104 publications

Nanoscale nucleosome dynamics assessed with time-lapse AFM

Nanoscale nucleosome dynamics assessed with time-lapse AFM

Dividing a supercoiled DNA molecule into two independent topological domains

Visualization of DNA and Protein–DNA Complexes with Atomic Force Microscopy

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