Nucleosome disassembly intermediates characterized by single-molecule FRET

Abstract

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated ''Widom 601'' positioning sequence labeled with donor and acceptor fluorophores. Reversible dissociation was induced by increasing NaCl concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less histones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.chromatin ͉ mononucleosomes ͉ structure T he nucleosome, the basic unit of genome compaction (1), consists of Ϸ2 turns of double-stranded DNA wound around a histone protein octamer. Its structure is known to a resolution of 1.9 Å (2). DNA sequence, chemical modifications and histone composition modulate gene activity through this structure.Central to nucleosomal function is its restructuring during processes that act on DNA, e.g., transcription or replication. Biochemical data indicate that nucleosome dissociation is temporary. Mechanisms for nucleosome unfolding, unwrapping or repositioning have been proposed (3), but so far none has been proven by direct physical evidence (e.g., detection of intermediate states). Here, we use single molecule Förster resonance energy transfer (FRET) to obtain quantitative structural information for elucidating such mechanisms.Bulk solution FRET is a proven tool for measuring average distances; nucleosome dynamics have been quantified by FRET between dyes attached to DNA and/or histone proteins (4-6). Analyzing FRET from single molecules (7, 8) can give detailed information on structural diversity; e.g., FRET on surfacetethered nucleosomes demonstrated spontaneous structure fluctuations (9, 10), whereas confocal spFRET experiments (11, 12) on freely diffusing single nucleosomes detected structural subpopulations under various conditions.The transit of a single molecule through the laser focus ...

Partially Assembled Nucleosome Structures at Atomic Detail

“…We mapped positions of the dyes utilized by Gansen et al (28) onto the 147-bp DNA fragment in the nucleosome structure of PDB: 1KX5 using the primer sequences from Gansen et al (28). Distances between the phosphorus atoms of these nucleotides were compared to the reported interdye distances.…”

“…APS-treated mica was used for immobilization of nucleosomes on the surface (59). The nucleosome samples were diluted to final concentration 2 nM with 10 mM Tris-HCl buffer, pH 7.5, and 140 mM NaCl, leading to accumulation of PANS, observed at low concentrations of the nucleosomes (28). Samples were deposited on a mica surface, rinsed with Milli-Q Ultrapure water (Millipore, Billerica, MA), and dried under argon flow (see section 2.1 in the Supporting Material).…”

“…Dilution of the nucleosome sample to nanomolar concentrations gives rise to the full repertoire of PANS in a single experiment (28,80,81). We used this well-characterized technique to visualize a representative set of naturally occurring PANS by AFM.…”

“…Specifically, it was shown that during the nucleosome assembly two copies of the H3$H4 dimer bind to the DNA first, forming a tetrasome, followed by sequential addition of each H2A$H2B dimers (27). The nucleosome can disassemble through the reverse reaction that starts with an opening of the (H3$H4) 2 tetramer-H2A$H2B dimers interface, followed by the release of H2A$H2B dimers from the DNA (14,28). It was also shown that H2A$H2B complexes with the DNA are effectively suppressed in vivo, even though the affinity of highly positively charged dimer to the DNA is substantial (29).…”

“…Further, because one of our goals here is to demonstrate utility of our atomistic models for interpretation of both in vivo and in vitro experiments (28,31,32), we focus on just three key PANS: the disome, the tetrasome, and the hexasome (Table 1). These subnucleosomal structures have been deemed important in vivo, and are already well characterized in vitro, both thermodynamically and by low-resolution methods such as Förster resonance energy transfer (FRET) or small-angle x-ray scattering (SAXS).…”