Characterization of specific nucleosomal states by use of selective substitution reagents in model octamer and tetramer structures.
Packaging of the DNA in nucleosomes restricts its access to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein-DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a topologically constrained DNA minicircle allows the visualization of different conformations of the nucleoprotein particle. The single cysteine located at position 110 of histone H3 can be titrated with thiol reagents, such as iodoacetamide (IAM), N-ethylmaleimide (NEM), and dithiobisnitrobenzoic acid (DTNB), in both histone octamers and histone (H3-H4)(2) tetramers. Treatment of histone H3 with IAM, NEM, and DTNB allows the trapping of different conformations of the (H3-H4)(2) tetramer within the nucleoprotein particle. When H3 cysteines are titrated within the histone octamer, IAM, NEM, and DTNB block the tetramer in the left-handed conformation, the conformation it adopts within the nucleosome. The left-handed conformation is initially dictated by H2A-H2B dimers and then frozen by the thiol reagents. When cysteines are titrated within the histone tetramer, the flexibility of the particle becomes apparent. NEM and IAM behave differently from DTNB. The first two reagents block the particle in its left-handed conformation while DTNB treatment favors the right-handed conformation. These thiol reagents that block the nucleoprotein particles in a given conformation should allow their structural analysis. They may also help the investigation of the role of the (H3-H4)(2) nucleoprotein particle structural transition in biological processes involving nucleosome dynamics, such as DNA transcription, replication, and repair.
Hamiche
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A
Institut de Biologie Moléculaire et de Génétique, Centre National de la Recherche Scientifique, 118 route de Narbonne, Toulouse Cedex, 31062, France.
Richard-Foy
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H
D008967:Molecular Biology / Q000379:methods*; D009707:Nucleosomes / Q000737:chemistry* / Q000187:drug effects*; D020836:Protein Structure, Quaternary; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D009707:Nucleosomes; D013438:Sulfhydryl Compounds