No abstract

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 < 15 and included all previously characterized LexA‐regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage‐inducible gene expression in a wild‐type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non‐inducible lexA(Ind−) cell. These experiments have allowed us to identify seven new LexA‐regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA‐regulated gene is discussed.

We describe the application of the hydroxyl radical footprinting technique to examine the histone-DNA interactions of a nucleosome that includes part of the 5S ribosomal RNA gene ofXenopus borealis. We establish that two distinct regions of DNA with different helical periodicities exist within the nucleosome and demonstrate a change in the helical periodicity of this DNA upon nucleosome formation. In particular, we find that on average the helical periodicity of DNA in this nucleosome is 10.18 ± 0.05 base pairs per turn. The same DNA, when bound to a calcium phosphate surface, has a periodicity of 10.49 ± 0.05 base pairs per turn, similar to that of random sequence DNA. Modulations in minor groove width within the naked DNA detected by the hydroxyl radical are maintained and exaggerated in nucleosomal DNA. These features correlate with regions in the DNA previously suggested to be important for nucleosome positioning.

Chromatin disruption and modification are associated with transcriptional regulation by diverse coactivators and corepressors. Here we discuss the possible structural basis and functional consequences of the observed alterations in chromatin associated with transcriptional activation and repression. Recent advances in defining the roles of individual histones and their domains in the assembly and maintenance of regulatory architectures provide a framework for understanding how chromatin remodelling machines, histone acetyltransferases and deacetylases function.

Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein.Single-base resolution •OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.nucleosome structure | chromatin higher order structure T he nucleosome is the fundamental repeating unit of chromatin in the nucleus of eukaryotic cells. The composition and the basic organization of the nucleosome is well established, and the structure of the nucleosomal core particle (NCP) has been described with nearly atomic precision by X-ray diffraction (1). However, similar information for the structure of a complete nucleosome, i.e., the NCP with associated linker DNA segments and a linker histone, is still lacking. Electron microscopy and electron cryomicroscopy (ECM) imaging have provided a relatively low-resolution picture of the complete nucleosome, both native (2) and reconstituted (3). However, important features of the structure remain obscure.Linker histones are typically ∼200 aa in length with a rather short nonstructured N terminus, followed by a ∼70-80 aa structured ("globular") domain, and a ∼100 aa long apparently unstructured C terminal domain, highly enriched in lysines. The globular domain of the linker histone appears to be internally located in the 30-nm chromatin fiber (4, 5), but its exact position within the nucleosome remains a subject of debate (for review, see ref. 6). A second question not yet resolved concerns the interactions and location of the linker histone C terminus. These issues have their origin in difficulties related to the preparation of well-defined nucleosomal samples. Indeed, direct binding of linker histone to nucleosomes in vitro is inefficient and complicated by the formation of large aggregates because of the nonspecific association of linker histones with DNA (7, 8).The situation can be considerably improved by using chaperones for linker histone deposition in vitro, a mechanism that is likely used in vivo (9). It was recently shown that NAP...

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.

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We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of -10.7 base pairs per turn, while flanking regions have a periodicity of -10.0 base pairs per turn. Removal ofthe histone tails does not change the hydroxyl radical cleavage pattern in either mixedor unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.Many studies have investigated the role of both DNA and the histone proteins in the architecture of the nucleosome (1-4). The structure of DNA in the nucleosome is altered in two ways from that found when the DNA is free in solution. First, DNA in the nucleosome is highly bent (1). This leads to the preferential association of the histone core with inherently curved DNA molecules, contributing to nucleosome positioning with respect to DNA sequence (5-9). Second, the average helical periodicity ofDNA is altered from 410.5 base pairs (bp) per turn when free in solution to an average of -10.2 bp per turn when in the nucleosome (5, 10-12). In addition, high-resolution analysis using the hydroxyl radical DNA cleavage reagent has revealed that two distinct regions of DNA with different helical periodicities exist in a nucleosome containing part of the 5S ribosomal RNA gene of Xenopus borealis (12). These changes in DNA structure upon incorporation into a nucleosome are expected to influence both the translational positioning of DNA with respect to the histone core and the way nucleosomal DNA interacts with other DNA-binding proteins.The central role of histones H3 and H4 in nucleosome structure and assembly, in vivo and in vitro, is supported by both physical (for a review, see ref. 13) and biochemical (14) evidence. Histones H3 and H4 form a tetramer, (H3/H4)2, which binds to DNA and directs the subsequent association of histones H2A and H2B (15-17). Alterations in the histone components within the nucleosome are associated with many biological processes. Deficiency of histones H2A and H2B within chromatin leads to an increase in accessibility to RNA polymerases (18)(19)(20). Modification of the highly conserved N-terminal basic domains of the histone proteins (tails) is also correlated with increased transcription (21,22). Acetylation ofthe histone tails influences the organization of nucleosomal DNA in some unknown way (23,24). In spite of these observations, the influence of the core histone tails on the actual structure of DNA within a nucleosome and the extent to whic...