Revealing transient structures of nucleosomes as DNA unwinds

Abstract

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.

Partially Assembled Nucleosome Structures at Atomic Detail

“…Scattering by DNA component of the sample was simulated by selective application of g_sans to trajectories of DNA atoms only. Experimental I(q) values obtained for 601NP nucleosome particles in varying salt conditions and with contrast variation in 50% sucrose (32) were fitted by a linear weighted combination of the computed intensity curves (see section 1.8 in the Supporting Material).…”

“…Recently, salt-induced nucleosome disassembly was investigated using SAXS with contrast variation (32). SAXS is a label-free technique that provides information about the average composition, size, and shape of macromolecules.…”

“…This concentration of sucrose has virtually no effect on the salt-dependent equilibrium of the nucleosome particle (32). The standard approaches of calculating SAXS curves based on static structural models (85) appear to have only a limited utility in this case due to the presence of relatively large and mobile fragments of free DNA.…”

“…We thank Dr. Suren Felekyan and Professor Claus Seidel for a fruitful discussion of the FRET data. We thank Professor Lois Pollack, Joshua Tokuda, and Yujie Chen for providing SAXS spectra from Chen et al (32). A.V.O.…”

“…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).…”