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

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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 Carr
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.

History

Publication status

  • Published

File Version

  • Published version

Journal

Nucleic Acids Research

ISSN

0305-1048

Publisher

Oxford University Press

Issue

19

Volume

42

Article number

e146

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2014-09-02

First Open Access (FOA) Date

2014-09-02

First Compliant Deposit (FCD) Date

2014-08-10

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