@article{e8178f18efad4c43a18ff353569eda58,
title = "Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy",
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.",
author = "Etheridge, {Thomas J.} and Boulineau, {R{\'e}mi L.} and Alex Herbert and Watson, {Adam T.} and Yasukazu Daigaku and Jem Tucker and Sophie George and Peter J{\"o}nsson and Matthieu Palayret and David Lando and Ernest Laue and Osborne, {Mark A.} and David Klenerman and Lee, {Steven F.} and Carr, {Antony M.}",
note = "Funding Information: We thank the members of the Carr, Murray and Osborne labs (University of Sussex) and the Klenerman and Laue labs (University of Cambridge). We also thank Alessandro Bianchi for the gift of plasmid DNA. Authors Contributions: T.J.E. and S.F.L. conceived the approach. All authors contributed to the design of experiments. T.J.E. and R.L.B. performed microscope experiments. T.J.E. analysed localization numbers, reconstructed super-resolution images and performed simulations. R.L.B. performed single-particle tracking analysis. S.F.L., R.L.B. and S.G. designed and built the microscope. A.H. developed single-molecule fitting routines and simulation. T.J.E., A.W., Y.D. and S.G. created yeast strains. J.T., P.J. and M.A.O. designed the setup; R.L.B. and J.T. performed FCS experiments. T.J.E., R.L.B., S.F.L. and A.M.C. wrote the manuscript. European Research Council [268788-SMI-DDR to A.M.C.]. Funding for open access charge: European Research Council [268788-SMI-DDR]. Publisher Copyright: {\textcopyright} The Author(s) 2014.",
year = "2014",
month = oct,
day = "29",
doi = "10.1093/nar/gku726",
language = "English",
volume = "42",
journal = "Nucleic Acids Research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "19",
}