Spakman, D;
King, GA;
Peterman, EJG;
Wuite, GJL;
(2020)
Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies.
Scientific Reports
, 10
, Article 9903. 10.1038/s41598-020-66259-4.
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Abstract
As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.
| Type: | Article |
|---|---|
| Title: | Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1038/s41598-020-66259-4 |
| Publisher version: | https://doi.org/10.1038/s41598-020-66259-4 |
| Language: | English |
| Additional information: | This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10102313 |
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