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

Thomas J. Etheridge, Rémi L. Boulineau, Alex Herbert, Adam T. Watson, Yasukazu Daigaku, Jem Tucker, Sophie George, Peter Jönsson, Matthieu Palayret, David Lando, Ernest Laue, Mark A. Osborne, David Klenerman, Steven F. Lee, Antony M. Carr

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

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 microscopybased 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.

Original languageEnglish
Article numbere146
JournalNucleic acids research
Volume42
Issue number19
DOIs
Publication statusPublished - 2014 Oct 29
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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