Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy |
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| Authors: | Thomas J. Etheridge Rémi L. Boulineau Alex Herbert Adam T. Watson Yasukazu Daigaku Jem Tucker Sophie George Peter J?nsson Matthieu Palayret David Lando Ernest Laue Mark A. Osborne David Klenerman Steven F. Lee Antony M. Carr |
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| Affiliation: | 1.Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK;2.Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Sussex, UK;3.Department of Chemistry, University of Cambridge, Cambridge, UK;4.Department of Biochemistry, University of Cambridge, Cambridge, UK |
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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. |
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