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. 2020 Jan 28;11:540. doi: 10.1038/s41467-019-14217-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Pictorial representation of the challenges involved in tracking the target cognate site in the genomic DNA of a eukaryotic cell. b 3D Structure of the specific complex between EngHD and DNA (PDB: 1HDD). c Schematic diagram of how to determine binding of EngHD to DNA using FCS. (top) The miniscule illumination volume of a confocal microscope is used to monitor fluctuations in the emission of fluorescent-labeled EngHD molecules (purple) while in diffusive transit. When bound to the much larger DNA molecule (orange), EngHD experiences delayed diffusion, staying longer within the illumination volume. The fluorescence autocorrelation decay (bottom) represents the average diffusion, which depends on the fraction of free and bound EngHD molecules. FCS experiments at various concentrations of DNA permit to accurately determine the dissociation constant from the combined autocorrelation decays.