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 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.
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Etheridge
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Rémi L
authors.initials: RL
authors.lastname: Boulineau
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Herbert
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Adam T
authors.initials: AT
authors.lastname: Watson
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Daigaku
authors.affiliation: Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Jem
authors.initials: J
authors.lastname: Tucker
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Sophie
authors.initials: S
authors.lastname: George
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Jönsson
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Palayret
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: David
authors.initials: D
authors.lastname: Lando
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.
authors.forename: Ernest
authors.identifier: http://orcid.org/0000-0002-7476-4148
authors.initials: E
authors.lastname: Laue
authors.affiliation: Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Osborne
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.
authors.forename: David
authors.initials: D
authors.lastname: Klenerman
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge, UK.
authors.forename: Steven F
authors.initials: SF
authors.lastname: Lee
authors.affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK a.m.carr@sussex.ac.uk.
authors.forename: Antony M
authors.initials: AM
authors.lastname: Carr
mesh_terms: D002453:Cell Cycle; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000032:analysis*; D004058:Diffusion; D008856:Microscopy, Fluorescence / Q000379:methods*; D064148:Minichromosome Maintenance Complex Component 4 / Q000032:analysis; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D012568:Schizosaccharomyces / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis
chemical_list: D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D029702:Schizosaccharomyces pombe Proteins; D064148:Minichromosome Maintenance Complex Component 4; C078490:mcm4 protein, S pombe
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