ScienceWeek

GENOME BIOLOGY: STRUCTURE OF DNA IN THE NUCLEOSOME

The following points are made by T.J. Richmond and C.A. Davey (Nature 2003 423:145):

1) DNA in eukaryotic cells is packaged repetitively into nucleosomes by means of extensive association with histone proteins. The hierarchical chromatin structure formed is the genomic substrate relevant to the vital processes of DNA replication, recombination, transcription, repair, and chromosome segregation, and to the pathological progression of cancer and viral disease. Although nucleosomal organization of DNA is essentially ubiquitous throughout genomes and generally repressive to gene expression, it also contributes to gene transcription in a gene-specific manner, suggesting that nucleosome positioning in gene promoter regions is important for genuine gene regulation in vivo. The question therefore arises of how chromatin structure, in which DNA is normally highly compacted, permits site-specific access to regulatory factors and more extensive exposure to the transcription apparatus.

2) The answer is likely to require a knowledge of DNA conformation in the nucleosome core. The core comprises 147 base pairs (bp) of DNA and the histone octamer; compared with the nucleosome, it lacks only 10–90 bp of linker DNA envisaged to be naked or bound to histone H1. The histone-fold domains of the octamer organize the central 129 of 147 bp in 1.59 left-handed superhelical turns with a diameter only fourfold that of the double helix. The relatively straight 9-bp terminal segments contribute little to the curvature of the complete 1.67-turn superhelix. So far, the site-specific regulatory factors that have been discovered bind the linker or terminal regions of the intact nucleosome. The lack of binding to the central region of the superhelix might simply be a consequence of bending the double helix or, additionally, of unusual DNA conformations induced by histone binding. At least one protein, HIV-1 integrase, does prefer DNA bent around the nucleosome in contrast to naked DNA.

3) The initiation of DNA-dependent nuclear processes in the context of chromatin implies that nucleosome position is biased by the DNA sequence to facilitate access by initiation factors. Numerous examples of positioned nucleosomes in gene promoter regions have been described both in vivo and in vitro. Preferential positioning could place factor-binding sequences in nucleosome linker or terminal region DNA. Furthermore, nucleosomes are intrinsically mobile and yield access to their DNA in vitro, allowing even RNA polymerase to transcribe nucleosomal DNA without causing dissociation of the histone octamer. In vivo, energy-dependent chromatin remodeling factors, targeted by gene regulatory proteins and acting directly on the nucleosome core, augment nucleosome mobility. Their mechanism of action most probably derives from the innate ability of nucleosomes to "slide" along DNA without releasing it.

4) In summary: The 1.9-angstrom-resolution crystal structure of the nucleosome core particle containing 147 DNA base pairs reveals the conformation of nucleosomal DNA with unprecedented accuracy. The DNA structure is remarkably different from that in oligonucleotides and non-histone protein–DNA complexes. The DNA base-pair-step geometry has, overall, twice the curvature necessary to accommodate the DNA superhelical path in the nucleosome. DNA segments bent into the minor groove are either kinked or alternately shifted. The unusual DNA conformational parameters induced by the binding of histone protein have implications for sequence-dependent protein recognition and nucleosome positioning and mobility. Comparison of the 147-base-pair structure with two 146-base-pair structures reveals alterations in DNA twist that are evidently common in bulk chromatin, and which are of probable importance for chromatin fibre formation and chromatin remodeling.

Nature http://www.nature.com/nature

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ON NUCLEOSOMES AND DNA PACKING

In eukaryotic chromosomes, approximately every 200 nucleotides, the DNA double helix is coiled around a complex of 8 histone proteins, the entire assembly having the appearance of beads on a string. The beads (nucleosomes) are in turn supercoiled into a solenoid structure, and the entire complex of the eukaryotic chromosome is called "chromatin". The small histone proteins are basic (as opposed to acidic) proteins, and they are essential in forming nucleosomes. Chemically, histones are single polypeptide chains, molecular mass 11 to 21 kilodaltons, 25 percent lysine and arginine amino acids.

The following points are made by B.D. Brower-Toland et al (Proc. Nat. Acad. Sci. 2002 99:1960):

1) Nucleosomes are the fundamental organizational unit of the eukaryotic genome, occurring on average every 200 base pairs. The foundation of the nucleosome is the nucleosome core particle, consisting of 147 base pairs of DNA wrapped 1.65 times around an octamer of histone proteins. The nucleosome core particle must be a stable and yet dynamic structure, both maintaining eukaryotic DNA in a condensed state and also permitting regulated access to genetic information contained therein. Equilibrium accessibility of DNA in the nucleosome core particle has been demonstrated by using restriction enzyme accessibility assays. As visualized by cryoelectron microscopy, variability in the amount of DNA associated with the histone octamer and in the angle of exit and entry of DNA from the nucleosome core particle are also consistent with spontaneous peeling of DNA ends from the octamer surface. Spontaneous peeling presents a means by which the transcriptional apparatus might invade nucleosomal DNA, especially if this process were facilitated by force-generating molecular motors and destabilizing covalent histone modifications.

2) Single-molecule mechanical manipulation techniques offer a direct approach to the investigation of the forces and displacements required for enzymatic access to nucleosome-bound DNA. These techniques already have provided insights into the higher-order structure of chromatin fibers and the kinetics of fiber assembly. However, the resolution of these studies has not permitted observations of the interactions within individual nucleosomes.

3) The authors report that the dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 base pairs of DNA released from the histone core at low stretching force. Subsequently, 80 base pairs are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. Kinetic parameters for nucleosome disassembly were also determined.

References (abridged):

1. Kornberg, R. & Thomas, J. (1974) Science 184, 865-868

2. Luger, K. , Mader, A. , Richmond, R. , Sargent, D. & Richmond, T. (1997) Nature (London) 389, 251-260

3. Anderson, J. & Widom, J. (2000) J. Mol. Biol. 296, 79-987

4. Furrer, P. , Bednar, J. , Dubochet, J. , Hamiche, A. & Prunell, A. (1995) J. Struct. Biol. 114, 177-183

5. Cui, Y. & Bustamante, C. (2000) Proc. Natl. Acad. Sci. USA 97, 27-132

Proc. Nat. Acad. Sci. http://www.pnas.org

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