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. ISSN 0305-1048

[img]
Preview
PDF - Published Version
Available under License Creative Commons Attribution.

Download (1MB) | Preview

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 > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > Q Science (General)
Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Q Science > QH Natural history > QH0301 Biology
Depositing User: Thomas Etheridge
Date Deposited: 02 Sep 2014 14:15
Last Modified: 06 Aug 2019 07:43
URI: http://sro.sussex.ac.uk/id/eprint/49544

View download statistics for this item

📧 Request an update