Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Mol Biol
2018 Jan 05;4301:45-57. doi: 10.1016/j.jmb.2017.10.029.
Show Gene links
Show Anatomy links
Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability.
Frouws TD
,
Barth PD
,
Richmond TJ
.
???displayArticle.abstract???
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.
Fig. 1. Location of site-specific crosslinks designed based on the 1.9-Å NCP structure (PDB ID 1KX5). (a) The NCP viewed up its dyad axis showing the two copies of H2A (yellow) and the modeled H2A-N38C point mutations (magenta). (b) The NCP viewed down its dyad axis showing the two copies of H3 (blue), and the modeled H3-R40C point mutations (magenta) and convertible guanosines (cyan) at position 70 on the I- and J-strand.
Fig. 2. Characterization of histone octamer and a nucleosome containing H2A-N38C. (a) Size exclusion on Superdex 200. The left panel shows chromatography in 2 M NaCl for oxidized H2A-N38C octamer (red) and w.t. octamer (black) at pH 7.5, and for oxidized H2A-N38C at pH 5.0 (green). The right panel shows chromatography in 0.8 M NaCl for w.t. octamer (black), oxidized H2A-N38C octamer (red) and reduced H2A-N38C octamer (cyan) at pH 7.5. (b) SDS PAGE of peak fractions from panel a. Each upper panel corresponds to a series of fractions from a peak underlined in panel a for H2A-N38C-containing or w.t. histone octamer. The bottom panels show the amount of each core histone relative to the amounts in the H2A-N38C 2 M NaCl panel as estimated by gel densitometry. The H2A-N38C disulfide of the H2A:H2A dimer is reduced on the addition of DTT (panel N38C 0.8 M NaCl versus N38C 0.8 M NaCl + DTT). Marker proteins are shown in the left lanes. (c) Native PAGE analysis of nucleosome 45N29 containing w.t. and N38C octamers. The octamer to DNA ratio used for each assembly reaction is indicated. Marker DNAs are shown in the left lane (M).
Fig. 3. The disulfide crosslink in the H2A-N38C NCP. (a) 2FO–FC electron density for the disulfide bond (black mesh, contour 1.4 × sigma) with the NCP structure superimposed. The two N38C substitutions are highlighted (magenta). (b) Structure cartoon of the H2A-N38C crosslink and associated H2A-L1 loops (yellow).
Fig. 4. Purification and characterization of H3-DNA NCP. (a) Purification of H3-DNA NCP by anion-exchange chromatography. The linear salt-gradient (gray line) maximizes the yield of NCP containing H3-DNA disulfide crosslinks. Three elution volumes were analyzed by native PAGE (inset) and correspond to crosslinked NCP (red), partially crosslinked and uncrosslinked products (green), and mainly free DNA (blue). The ethidium bromide dye used to visualize products is relatively ineffective on the crosslinked DNA (red). (b) SDS PAGE of purified H3-DNA and H3-DNA/H2A-N38C NCPs. H3-R40C migrates as a 40-kDa species when crosslinked to NCP DNA and as free H3 in the presence of DTT. Crosslinked H2A-N38C migrates as a dimer. Marker proteins are shown in the left lane (M). (c) Effect of dilution on H3-DNA NCP. Native PAGE of crosslinked (− DTT) and reduced (+ DTT) NCP upon dilution from 2.5 μM to 50 nM NCP. (d) Effect of lyophilization on H2A-N38C/H3-DNA NCP. Native PAGE of crosslinked (− DTT) and reduced (+ DTT) NCP before (pre-lyo) and after lyophilization and resuspension in solution (post-lyo). (e) Effect on multiple DNA positions for MET16 H3-DNA NCP. Native PAGE of crosslinked (− DTT) and reduced (+ DTT) NCP containing a DNA sequence from the Saccharomycescerevisiae MET16 promoter.
Fig. 5. The disulfide crosslink in the H3-DNA NCP. (a) 2F0–FC electron density for the disulfide bond (black mesh, contour 0.7 × sigma) with the NCP structure superimposed. An H3-R40C substitution (red) bound to a convertible guanosine at DNA position 70 NCP (yellow) is highlighted. (b) Structure cartoon of the H3-DNA disulfide crosslink. The two DNA backbones (gray) show the location of the minor groove.
Adams,
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
2010, Pubmed
Adams,
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
2010,
Pubmed
Andrews,
Nucleosome structure(s) and stability: variations on a theme.
2011,
Pubmed
Andrews,
A coupled equilibrium approach to study nucleosome thermodynamics.
2011,
Pubmed
Arents,
The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left-handed superhelix.
1991,
Pubmed
Ausio,
Nucleosome core particle stability and conformational change. Effect of temperature, particle and NaCl concentrations, and crosslinking of histone H3 sulfhydryl groups.
1984,
Pubmed
Banerjee,
Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA.
2005,
Pubmed
Brogaard,
A map of nucleosome positions in yeast at base-pair resolution.
2012,
Pubmed
Brünger,
Crystallography & NMR system: A new software suite for macromolecular structure determination.
1998,
Pubmed
Burton,
The interaction of core histones with DNA: equilibrium binding studies.
1978,
Pubmed
Cairns,
Chromatin remodeling: insights and intrigue from single-molecule studies.
2007,
Pubmed
Chiang,
A semisynthetic strategy to generate phosphorylated and acetylated histone H2B.
2009,
Pubmed
Chua,
3.9 Å structure of the nucleosome core particle determined by phase-plate cryo-EM.
2016,
Pubmed
,
Xenbase
Davey,
Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution.
2002,
Pubmed
,
Xenbase
De la Mora,
Effective scavenging at cryotemperatures: further increasing the dose tolerance of protein crystals.
2011,
Pubmed
Dorigo,
Chromatin fiber folding: requirement for the histone H4 N-terminal tail.
2003,
Pubmed
,
Xenbase
Dyer,
Reconstitution of nucleosome core particles from recombinant histones and DNA.
2004,
Pubmed
Eickbush,
The histone core complex: an octamer assembled by two sets of protein-protein interactions.
1978,
Pubmed
Emsley,
Features and development of Coot.
2010,
Pubmed
Fei,
The prenucleosome, a stable conformational isomer of the nucleosome.
2015,
Pubmed
Flaus,
Positioning and stability of nucleosomes on MMTV 3'LTR sequences.
1998,
Pubmed
Flaus,
Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.
1996,
Pubmed
Frouws,
X-ray structure of the MMTV-A nucleosome core.
2016,
Pubmed
Hall,
High-resolution dynamic mapping of histone-DNA interactions in a nucleosome.
2009,
Pubmed
Huang,
Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance.
1998,
Pubmed
Kabsch,
XDS.
2010,
Pubmed
Kornberg,
Structure of chromatin.
1977,
Pubmed
Lavelle,
Right-handed nucleosome: myth or reality?
2009,
Pubmed
Levendosky,
The Chd1 chromatin remodeler shifts hexasomes unidirectionally.
2016,
Pubmed
,
Xenbase
Lowary,
New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.
1998,
Pubmed
Luger,
Crystal structure of the nucleosome core particle at 2.8 A resolution.
1997,
Pubmed
Luger,
Preparation of nucleosome core particle from recombinant histones.
1999,
Pubmed
,
Xenbase
Luger,
Characterization of nucleosome core particles containing histone proteins made in bacteria.
1997,
Pubmed
,
Xenbase
Malecka,
Crystal structure of a p53 core tetramer bound to DNA.
2009,
Pubmed
McCoy,
Phaser crystallographic software.
2007,
Pubmed
Nilsson,
Integrated production of human insulin and its C-peptide.
1996,
Pubmed
Ong,
DNA stretching and extreme kinking in the nucleosome core.
2007,
Pubmed
,
Xenbase
Oohara,
Spectroscopic studies on histone-DNA interactions. II. Three transitions in nucleosomes resolved by salt-titration.
1987,
Pubmed
Pettersen,
UCSF Chimera--a visualization system for exploratory research and analysis.
2004,
Pubmed
Rhodes,
Assembly of nucleosomes and chromatin in vitro.
1989,
Pubmed
,
Xenbase
Richardson,
The anatomy and taxonomy of protein structure.
1981,
Pubmed
Richmond,
Structure of the nucleosome core particle at 7 A resolution.
,
Pubmed
Richmond,
Crystals of a nucleosome core particle containing defined sequence DNA.
1988,
Pubmed
Ruiz-Carrillo,
An octamer of core histones in solution: central role of the H3-H4 tetramer in the self-assembly.
1979,
Pubmed
Satchwell,
Sequence periodicities in chicken nucleosome core DNA.
1986,
Pubmed
Schmidt,
Allosteric disulfide bonds.
2006,
Pubmed
Shrader,
Artificial nucleosome positioning sequences.
1989,
Pubmed
Shrader,
Effects of DNA sequence and histone-histone interactions on nucleosome placement.
1990,
Pubmed
Thåström,
Histone-DNA binding free energy cannot be measured in dilution-driven dissociation experiments.
2004,
Pubmed
,
Xenbase
Thåström,
Nucleosomal locations of dominant DNA sequence motifs for histone-DNA interactions and nucleosome positioning.
2004,
Pubmed
Vasudevan,
Crystal structures of nucleosome core particles containing the '601' strong positioning sequence.
2010,
Pubmed
,
Xenbase
Wilhelm,
Reconstitution of chromatin: assembly of the nucleosome.
1978,
Pubmed
Yamada,
Structure and mechanism of the chromatin remodelling factor ISW1a.
2011,
Pubmed
,
Xenbase
van Aalten,
A structural snapshot of base-pair opening in DNA.
1999,
Pubmed
von Holt,
Isolation and characterization of histones.
1989,
Pubmed
