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

被引：24

作者：

Etheridge, Thomas J. ^{[1
]}

Boulineau, Remi L. ^{[1
]}

Herbert, Alex ^{[1
]}

Watson, Adam T. ^{[1
]}

Daigaku, Yasukazu ^{[1
]}

Tucker, Jem ^{[2
]}

George, Sophie ^{[1
]}

Joensson, Peter ^{[3
]}

Palayret, Matthieu ^{[3
]}

Lando, David ^{[4
]}

Laue, Ernest ^{[4
]}

Osborne, Mark A. ^{[2
]}

Klenerman, David ^{[3
]}

Lee, Steven F. ^{[3
]}

Carr, Antony M. ^{[1
]}

机构：

[1] Univ Sussex, Sch Life Sci, Genome Damage & Stabil Ctr, Falmer, Sussex, England

[2] Univ Sussex, Sch Life Sci, Dept Chem, Falmer, Sussex, England

[3] Univ Cambridge, Dept Chem, Cambridge, England

[4] Univ Cambridge, Dept Biochem, Cambridge, England

来源：

NUCLEIC ACIDS RESEARCH | 2014年 / 42卷 / 19期

基金：

欧洲研究理事会; 英国工程与自然科学研究理事会;

关键词：

FLUORESCENCE CORRELATION SPECTROSCOPY; SCHIZOSACCHAROMYCES-POMBE; FISSION YEAST; SUPERRESOLUTION MICROSCOPY; REPLICATION; CHROMATIN; STABILITY; DYNAMICS; TRACKING; ELG1;

D O I：

10.1093/nar/gku726

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页数：11

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