Nucleosome Dynamics as Studied by Single-pair Fluorescence Resonance Energy Transfer: A Reevaluation
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Accessibility of nucleosomal DNA to protein factor binding is ensured by at least three mechanisms: post-synthetic modifications to the histones, chromatin remodeling, and spontaneous unwrapping of the DNA from the histone core. We have previously used single-pair fluorescence resonance energy transfer (spFRET) experiments to investigate long-range conformational fluctuations in nucleosomal DNA (Tomschik M, Zheng H, van Holde K, Zlatanova J, Leuba SH in Proc Natl Acad Sci USA 102(9):3278–3283, 2005). Recent work has drawn attention to a major artifact in such studies due to photoblinking of the acceptor fluorophore. We have now used formaldehyde-crosslinked nucleosomes and imaging in the presence of Trolox, an efficient triplet-state quencher that suppresses photoblinking, to reevaluate our previous conclusions. Careful analysis of the data indicates that most of the events previously characterized as nucleosome ‘opening’ must have corresponded to photoblinking. There is, nevertheless, evidence for the existence of infrequent, rapid opening events.
KeywordsEvanescent field fluorescence microscope Nucleosome DNA unwrapping nucleosome opening photoblinking
We thank Dr. Ivan Rasnik (Emory University, Atlanta, GA) for help with the IDL and Matlab based data analysis, for valuable discussions and critical reading the manuscript. We thank Dr. Jean-Mark Victor (Université Pierre et Marie Curie, Paris) for providing the all-atom models for Fig. 1a, and for useful discussions on the gaping transition. This work was supported in part by NSF grant 0504239 to JZ, Eppley Foundation for Scientific Research to MT, and start-up funds from the University of Wyoming.
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