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J Fluoresc. 2007 Nov;17(6):785-95. Epub 2007 Jul 4.

Single-pair FRET microscopy reveals mononucleosome dynamics.

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  • 1Physics of Life Processes, Leiden University, Leiden Institute of Physics, Niels Bohrweg 2, Leiden, The Netherlands.

Abstract

We applied spFRET microscopy for direct observation of intranucleosomal DNA dynamics. Mononucleosomes, reconstituted with DNA containing a FRET pair at the dyad axis and exit of the nucleosome core particle, were immobilized through a 30 bp DNA tether on a polyethyleneglycol functionalized slide and visualized using Total Internal Reflection Fluorescence microscopy. FRET efficiency time-traces revealed two types of dynamics: acceptor blinking and intramolecular rearrangements. Both Cy5 and ATTO647N acceptor dyes showed severe blinking in a deoxygenated buffer in the presence of 2% betaME. Replacing the triplet quencher betaME with 1 mM Trolox eliminated most blinking effects. After suppression of blinking three subpopulations were observed: 90% appeared as dissociated complexes; the remaining 10% featured an average FRET efficiency in agreement with intact nucleosomes. In 97% of these intact nucleosomes no significant changes in FRET efficiency were observed in the experimentally accessible time window ranging from 10 ms to 10's of seconds. However, 3% of the intact nucleosomes showed intervals with reduced FRET efficiency, clearly distinct from blinking, with a lifetime of 120 ms. These fluctuations can unambiguously be attributed to DNA breathing. Our findings illustrate not only the merits but also typical caveats encountered in single-molecule FRET studies on complex biological systems.

PMID:
17609864
PMCID:
PMC2064943
DOI:
10.1007/s10895-007-0218-9
[PubMed - indexed for MEDLINE]
Free PMC Article
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