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The nucleosomal subunit organization of chromatin provides a multitude of functions. Nucleosomes elicit an initial ∼7-fold linear compaction of genomic DNA. They provide a critical mechanism for stable repression of genes and other DNA-dependent activities by restricting binding of trans-acting factors to cognate DNA sequences. Conversely they are engineered to be nearly meta-stable and disassembled (and reassembled) in a facile manner to allow rapid access to the underlying DNA during processes such as transcription, replication and DNA repair. Nucleosomes protect the genome from DNA damaging agents and provide a lattice onto which a myriad of epigenetic signals are deposited. Moreover, vast strings of nucleosomes provide a framework for assembly of the chromatin fiber and higher-order chromatin structures. Thus, in order to provide a foundation for understanding these functions, we present a review of the basic elements of nucleosome structure and stability, including the association of linker histones.

The core histone tail domains are "master control switches" that help define the structural and functional characteristics of chromatin at many levels. The tails modulate DNA accessibility within the nucleosome, are essential for stable folding of oligonucleosome arrays into condensed chromatin fibers, and are important for fiber-fiber interactions involved in higher order structures. Many nuclear signaling pathways impinge upon the tail domains, resulting in posttranslational modifications that are likely to alter the charge, structure, and/or interactions of the core histone tails or to serve as targets for the binding of ancillary proteins or other enzymatic functions. However, currently we have only a marginal understanding of the molecular details of core histone tail conformations and contacts. Here we review data related to the structures and interactions of the core histone tail domains and how these domains and posttranslational modifications therein may define the structure and function of chromatin.

R2 retrotransposons insert into the 28S rRNA genes of insects. Integration occurs by specific cleavage of the target site and utilization of the released DNA end to prime reverse transcription of the RNA transcript. Specificity of the protein to the target site is dependent upon nucleotide sequence recognition extending from 35 bp upstream to 15 bp downstream of the cleavage site. In this report, we show that sequence recognition and cleavage by the R2 protein can occur while the target site is assembled into nucleosomes. Reconstitution of DNA fragments containing the 28S gene sequence into a set of nucleosomes with different translational frames revealed that the R2 site adopted the same rotational orientation with respect to the histone octamer. Binding and cleavage by the R2 protein were most efficient when the upstream binding site for the R2 protein was near a nucleosome end. Interaction of the R2 protein with the nucleosome disrupted the histone:DNA contacts in the 50 bp region directly bound by R2, but did not modify the remainder of the nucleosome structure.

The organization of DNA within eukaryotic cell nuclei poses special problems and opportunities for the cell. For example, assembly of DNA into chromatin is thought to be a principle mechanism by which adventitious general transcription is repressed. However, access to genomic DNA for events such as DNA repair must be facilitated by energy-intensive processes that either directly alter chromatin structure or impart post-translational modifications, leading to increased DNA accessibility. The assembly of DNA into chromatin affects both the incidence of damage to DNA and repair of that damage. Correction of most damage to DNA caused by UV irradiation occurs via the nucleotide excision repair (NER) process. NER requires extensive involvement of large multiprotein complexes with relatively large stretches of DNA. Here, we review recent evidence suggesting that at least some steps of NER require ATP-dependent chromatin remodeling activities while perhaps others do not.

Exposure of Escherichia coli to a variety of DNA-damaging agents results in the induction of the global ‘SOS response’. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA-regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5′-TACTG(TA)5CAGTA-3′. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA-regulated genes. These searches identified a total of 69 potential LexA-regulated genes/operons with a heterology index of

Nucleosome mobility facilitates the transcription of chromatin templates containing only histone octamers. Inclusion of linker histones in chromatin inhibits nucleosome mobility, directs nucleosome positioning and represses transcription. Transcriptional repression by linker histone occurs preferentially on templates associated with histone octamers relative to naked DNA. Mobile nucleosomes and the restriction of mobility by linker histones might be expected to exert a major influence on the accessibility of chromatin to regulatory molecules.

Impediments to DNA access due to assembly of the eukaryotic genome into chromatin are in part overcome by the activity of ATP-dependent chromatin-remodeling complexes. These complexes employ energy derived from ATP hydrolysis to destabilize histone—DNA interactions and alter nucleosome positions, thereby increasing the accessibility of DNA-binding factors to their targets. However, the mechanism by which theses complexes accomplish this task remains unresolved. We review aspects of nucleosome alteration by the SWI/SNF complex, the archetypal remodeling enzyme. We focus on experiments that provide insights into how SWI/SNF induces nucleosome movement along DNA. Numerous biochemical activities have been characterized for this complex, all likely providing clues as to the molecular mechanism of translocation.

Nucleosome positioning is proposed to have an essential role in facilitating the regulated transcription of eukaryotic genes. Some transcription factors can bind to DNA when it is appropriately wrapped around the histone core, others cannot bind due to the severe deformation of DNA structure. The staged assembly of nucleosomes and positioning of histone-DNA contacts away from promoter elements can facilitate the access of transcription factors to DNA. Positioned nucleosomes can also facilitate transcription through providing the appropriate scaffolding to bring regulatory factors bound at dispersed sites into juxtaposition.

The native chromatin complex within most eukaryotic nuclei is very difficult to study by biochemical means, so researchers have developed methods for studying smaller portions of the complex. This unit details the use of DNase I and hydroxyl radicals to characterize histone-DNA interactions within such portions of the complex. DNase I digestion can be used to determine what regions of a DNA segment are intimately associated with the core histone proteins and what regions are more like naked DNA (i.e., linker DNA within the nucleosomal repeat). The finer details of histone-DNA interactions and DNA structure within these complexes is best characterized by digestion with the hydroxyl radical. Both reagents may be used to assess the degree and homogeneity of rotational and translational positioning within isolated chromatin complexes.