Nucl._Acids_Res.-2014-Etheridge-nar-gku726.pdf (1.69 MB)
Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy
journal contribution
posted on 2023-06-08, 18:04 authored by Thomas Etheridge, Rémi L Boulineau, Alex Herbert, Adam WatsonAdam Watson, Yasukazu Daigaku, Jem Tucker, Sophie George, Peter Jönsson, Matthieu Palayret, David Lando, Ernest Laue, Mark Osborne, David Klenerman, Steven F Lee, Antony CarrAntony CarrDevelopment 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.
History
Publication status
- Published
File Version
- Published version
Journal
Nucleic Acids ResearchISSN
0305-1048Publisher
Oxford University PressExternal DOI
Issue
19Volume
42Article number
e146Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2014-09-02First Open Access (FOA) Date
2014-09-02First Compliant Deposit (FCD) Date
2014-08-10Usage metrics
Categories
No categories selectedKeywords
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC