title: Methods for Investigating DNA Accessibility with Single Nucleosomes.
abstract: Nucleosomes are the fundamental organizing unit of all eukaryotic genomes. Understanding how proteins gain access to DNA-binding sites located within nucleosomes is important for understanding DNA processing including transcription, replication, and repair. Single-molecule total internal reflection fluorescence (smTIRF) microscopy measurements can provide key insight into how proteins gain and maintain access to DNA sites within nucleosomes. Here, we describe methods for smTIRF experiments including the preparation of fluorophore-labeled nucleosomes, the smTIRF system, data acquisition, analysis, and controls. These methods are presented for investigating transcription factor binding within nucleosomes. However, they are applicable for investigating the binding of any site-specific DNA-binding protein within nucleosomes.
authors: The Ohio State University, Columbus, OH, United States.
authors: M D
authors: MD
authors: Gibson
authors: The Ohio State University, Columbus, OH, United States.
authors: M
authors: M
authors: Brehove
authors: The Ohio State University, Columbus, OH, United States.
authors: Y
authors: Y
authors: Luo
authors: The Ohio State University, Columbus, OH, United States.
authors: J
authors: J
authors: North
authors: The Ohio State University, Columbus, OH, United States. Electronic address: poirier.18@osu.edu.
authors: M G
authors: MG
authors: Poirier
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D009707:Nucleosomes / Q000737:chemistry*; D000072760:Single Molecule Imaging / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D009707:Nucleosomes; D004247:DNA
keywords: Fluorophore-labeled nucleosomes; Nucleosome accessibility; Single-molecule fluorescence; Transcription factor-binding dynamics