Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.
Nucleic Acids Res
; 42(19): e146, 2014 Oct 29.
Article
em En
| MEDLINE
| ID: mdl-25106872
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Ligação a DNA
/
Microscopia de Fluorescência
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article