Single-pair FRET microscopy reveals mononucleosome dynamics.
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.
Koopmans
W J A
WJ
Physics of Life Processes, Leiden University, Leiden Institute of Physics, Niels Bohrweg 2, Leiden, The Netherlands.
Brehm
A
A
Logie
C
C
Schmidt
T
T
van Noort
J
J
D001483:Base Sequence; D001710:Biotin; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D009707:Nucleosomes / Q000737:chemistry*; D013053:Spectrophotometry; D013816:Thermodynamics
D017931:DNA Primers; D005456:Fluorescent Dyes; D009707:Nucleosomes; D001710:Biotin; D004247:DNA