Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.

The conformation of DNA bound in nucleosomes depends on the DNA sequence. Questions such as how nucleosomes are positioned and how they potentially bind sequence-dependent nuclear factors require near-atomic resolution structures of the nucleosome core containing different DNA sequences; despite this, only the DNA for two similar α-satellite sequences and a sequence (601) selected in vitro have been visualized bound in the nucleosome core. Here we report the 2.6-Å resolution X-ray structure of a nucleosome core particle containing the DNA sequence of nucleosome A of the 3′-LTR of the mouse mammary tumor virus (147 bp MMTV-A). To our knowledge, this is the first nucleosome core particle structure containing a promoter sequence and crystallized from Mg 2+ ions. It reveals sequence-dependent DNA conformations not seen previously, including kinking into the DNA major groove.chromatin | nucleosome | DNA | X-ray structure | MMTV D NA in eukaryotic cells is wrapped repeatedly in nucleosomes to form chromatin, the substrate engaged by the nuclear machinery to carry out repair, replication, recombination, and transcription of genomes. Nucleosome mapping in situ combined with biochemical studies has revealed that nucleosome positions determine access to DNA regulatory sequences essential to these processes (1, 2). Nucleosome position is determined chiefly by DNA sequence and ATP-dependent chromatin remodeling factors (3-5). The nucleosome includes a linker DNA of variable length and a nucleosome core containing a histone octamer and 147 bp of DNA (6). Many high-resolution structures of nucleosome cores with differing DNA sequences are required to see how the details of DNA conformation could affect nucleosome positioning and dynamics, as well as nuclear factor binding. The DNA studied would most interestingly represent natural sequences of transcription promoters and enhancer elements.Our knowledge of sequence-dependent structure of DNA bound in the nucleosome core relative to the amount of DNA bound in genomes is extremely limited. A resolution of at least 2.6 Å is necessary to evaluate differences in DNA conformations and assess solvent interactions adequately. To date, this highly reliable "library" of DNA structural information pertinent to the nucleosome core consists primarily of two similar sequences of half α-satellite repeats and half the artificially "evolved" sequence 601 (7-10). Further investigations have been limited to substitution of short sequence elements in one of the α-satellite sequences (11, 12). These high-resolution structures have hinged on using palindromic sequences to avoid twofold averaging imposed by crystal packing. The lack of twofold symmetry in the full 601 sequence, for example, resulted in superposition of the electron density of the two different half-sequences (9).We describe here the X-ray structure of a nucleosome core particle (NCP) containing a DNA sequence from mouse mammary tumor virus (MMTV) determined at 2.6 Å resolution. To our knowledge, this is the first NCP structure conta...

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.

A major question in chromatin involves the exact organization of nucleosomes within the 30-nm chromatin fiber and its structural determinants of assembly. Here we investigate the structure of histone octamer helical tubes via the method of iterative helical real-space reconstruction. Accurate placement of the x-ray structure of the histone octamer within the reconstructed density yields a pseudoatomic model for the entire helix, and allows precise identification of molecular interactions between neighboring octamers. One such interaction that would not be obscured by DNA in the nucleosome consists of a twofold symmetric four-helix bundle formed between pairs of H2B-alpha3 and H2B-alphaC helices of neighboring octamers. We believe that this interface can act as an internucleosomal four-helix bundle within the context of the chromatin fiber. The potential relevance of this interface in the folding of the 30-nm chromatin fiber is discussed.

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