Abstract
In eukaryotes, DNA is packaged into a basic unit, the nucleosome which consists of 147 bp of DNA wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. Nucleosome structures are diverse not only by histone variants, histone modifications, histone composition but also through accommodating different conformational states such as DNA breathing and dimer splitting. Variation in nucleosome structures allows it to perform a variety of cellular functions. Here, we identified a novel spontaneous conformational switching of nucleosomes under physiological conditions using single-molecule FRET. Using FRET probes placed at various positions on the nucleosomal DNA to monitor conformation of the nucleosome over a long period of time (30–60 min) at various ionic conditions, we identified conformational changes we refer to as nucleosome gaping. Gaping transitions are distinct from nucleosome breathing, sliding or tightening. Gaping modes switch along the direction normal to the DNA plane through about 5–10 angstroms and at minutes (1–10 min) time scale. This conformational transition, which has not been observed previously, may be potentially important for enzymatic reactions/transactions on nucleosomal substrate and the formation of multiple compression forms of chromatin fibers.
Highlights
In eukaryotes, DNA is packaged into a basic unit, the nucleosome [1]
Reconstituted nucleosomes display a single broad high FRET peak in the single molecule FRET (smFRET) efficiency histogram plus a low FRET peak attributed to the free DNA (Supplementary Figure S1). smFRET traces of ED2.8 in 50 mM NaCl solution revealed spontaneous transitions between three FRET states (Figure 1B and C): the high FRET state at 0.545 ± 0.003, the middle FRET state at 0.440 ± 0.001 and the low FRET state at 0.339 ± 0.003 (Figure 1C and Table 1, the errors represent standard errors of measurement)
Because the nucleosome stability is dependent on salt concentrations [13], we examined the conformational switching kinetics at different ionic conditions. smFRET histograms of the ED1.7 construct were recorded over a wide range of NaCl concentrations from 50 mM to 1 M
Summary
DNA is packaged into a basic unit, the nucleosome [1]. Nucleosomes are regularly arranged at approximately every 200 base pairs along the DNA like ‘beads on a string’, separated by short DNA linker [2]. Even intact nucleosomes are highly dynamic and may deviate from the canonical crystal structure by DNA breathing [8,9,10,11,12], H2A/H2B dimer splitting [2,11,13,14] and nucleosome gaping [15,16]. In DNA breathing and opening excursions, nucleosomal DNA ends unwrap from the histone core partially and reversibly on a rapid time scale (10–250 ms) [9,17]. Spontaneous unwrapping of nucleosomal DNA ends makes chromatin more accessible to DNA binding factors [8,18]. Nucleosome gaping was proposed theoretically in order to accommodate tightly package of chromatin higher order structure, the 30 nm fiber [15], but there has not been experimental demonstration
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