Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability

We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to st...

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Bibliographic Details
Published in:Journal of molecular biology 2018-01, Vol.430 (1), p.45-57
Main Authors: Frouws, Timothy D., Barth, Philip D., Richmond, Timothy J.
Format: Article
Language:English
Subjects:
Animals
chromatin
Crystallization - methods
Cysteine - genetics
Dimerization
Disulfides - metabolism
DNA
DNA - genetics
Escherichia coli - genetics
hexasome
histone
Histones - genetics
Nucleosomes - genetics
Point Mutation - genetics
X-ray crystallography
X-Rays
Xenopus laevis - genetics
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Summary:We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy. [Display omitted] •Crosslinked nucleosome core particles have increased stability against H2A/H2B dimer loss and DNA dissociation.•A site-specific disulfide crosslink was introduced between the two copies of H2A in the histone octamer to stabilize its quaternary structure.•Site-specific disulfide crosslinks were introduced between histone H3 and DNA within the nucleosome core particle.•Three X-ray crystal structures of crosslinked nucleosome core particles were determined at high resolution.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2017.10.029