Journal of Fluorescence

, Volume 19, Issue 1, pp 53–62

Nucleosome Dynamics as Studied by Single-pair Fluorescence Resonance Energy Transfer: A Reevaluation

Original Paper

DOI: 10.1007/s10895-008-0379-1

Cite this article as:
Tomschik, M., van Holde, K. & Zlatanova, J. J Fluoresc (2009) 19: 53. doi:10.1007/s10895-008-0379-1

Abstract

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.

Keywords

Evanescent field fluorescence microscopeNucleosome DNA unwrappingnucleosome openingphotoblinking

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Department of Molecular Biology, College of AgricultureUniversity of WyomingLaramieUSA
  2. 2.Department of Biochemistry and BiophysicsOregon State UniversityCorvallisUSA