Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Etheridge, Thomas J, Boulineau, Rémi L, Herbert, Alex, Watson, Adam T, Daigaku, Yasukazu, Tucker, Jem, George, Sophie, Jönsson, Peter, Palayret, Matthieu, Lando, David, Laue, Ernest, Osborne, Mark A, Klenerman, David, Lee, Steven F and Carr, Antony M (2014) Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy. Nucleic Acids Research, 42 (19). e146-e146. ISSN 0305-1048

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Abstract

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.

Item Type: Article
Schools and Departments: School of Life Sciences > Chemistry
Subjects: Q Science > QD Chemistry > QD0071 Analytical chemistry
Q Science > QD Chemistry > QD0450 Physical and theoretical chemistry
Q Science > QD Chemistry > QD0450 Physical and theoretical chemistry > QD0701 Photochemistry
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Depositing User: Mark Osborne
Date Deposited: 13 Apr 2016 11:11
Last Modified: 07 Mar 2017 05:08
URI: http://sro.sussex.ac.uk/id/eprint/60453

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