Single molecule fluorescence methodologies for investigating transcription factor binding kinetics to nucleosomes and DNA

Abstract

Site specific DNA binding complexes must bind their DNA target sites and then reside there for a sufficient amount of time for proper regulation of DNA processing including transcription, replication and DNA repair. In eukaryotes, the occupancy of DNA binding complexes at their target sites is regulated by chromatin structure and dynamics. Methodologies that probe both the binding and dissociation kinetics of DNA binding proteins with naked and nucleosomal DNA are essential for understanding the mechanisms by which these complexes function. Here, we describe single-molecule fluorescence methodologies for quantifying the binding and dissociation kinetics of transcription factors at a target site within DNA, nucleosomes and nucleosome arrays. This approach allowed for the unexpected observation that nucleosomes impact not only binding but also dissociation kinetics of transcription factors and is well-suited for the investigation of numerous DNA processing complexes that directly interact with DNA organized into chromatin.

Keywords: Chromatin; Fluorescence resonance energy transfer; Nucleosomes; Single molecule fluorescence; Transcription factors.