Advanced

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

Etheridge, Thomas J; Boulineau, Rémi L; Herbert, Alex; Watson, Adam T; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter LU ; Palayret, Matthieu and Lando, David, et al. (2014) In Nucleic Acids Research 42(19). p.146-146
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... (More)

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.

(Less)
Please use this url to cite or link to this publication:
author
, et al. (More)
(Less)
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Cell Cycle, DNA Replication, DNA-Binding Proteins, Diffusion, Microscopy, Fluorescence, Minichromosome Maintenance Complex Component 4, Proliferating Cell Nuclear Antigen, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Journal Article, Research Support, Non-U.S. Gov't
in
Nucleic Acids Research
volume
42
issue
19
pages
146 - 146
publisher
Oxford University Press
external identifiers
  • scopus:84925284247
ISSN
1362-4962
DOI
10.1093/nar/gku726
language
English
LU publication?
no
id
27921556-ee23-409c-9e94-d06d9af7b77f
date added to LUP
2018-01-26 10:24:54
date last changed
2018-07-15 04:47:15
@article{27921556-ee23-409c-9e94-d06d9af7b77f,
  abstract     = {<p>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.</p>},
  author       = {Etheridge, Thomas J and Boulineau, Rémi L and Herbert, Alex and Watson, Adam T and Daigaku, Yasukazu and Tucker, Jem and George, Sophie and Jönsson, Peter and Palayret, Matthieu and Lando, David and Laue, Ernest and Osborne, Mark A and Klenerman, David and Lee, Steven F and Carr, Antony M},
  issn         = {1362-4962},
  keyword      = {Cell Cycle,DNA Replication,DNA-Binding Proteins,Diffusion,Microscopy, Fluorescence,Minichromosome Maintenance Complex Component 4,Proliferating Cell Nuclear Antigen,Schizosaccharomyces,Schizosaccharomyces pombe Proteins,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  month        = {10},
  number       = {19},
  pages        = {146--146},
  publisher    = {Oxford University Press},
  series       = {Nucleic Acids Research},
  title        = {Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy},
  url          = {http://dx.doi.org/10.1093/nar/gku726},
  volume       = {42},
  year         = {2014},
}