Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn(2+) ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment

Cleavage of a DNA–RNA–DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA–RNA junction with prokaryotic RNases HII

AbstractWe have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA–RNA–DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5′-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA–RNA junction (5′-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA

Care-givers’ perspectives of occupational engagement of persons with dementia

Author name used in this publication: Jenny C. C. ChungVersion of RecordPublishe

Structure specific recognition protein-1 (SSRP1) is an elongated homodimer that binds histones

The histone chaperone complex facilitates chromatin transcription (FACT) plays important roles in DNA repair, replication, and transcription. In the formation of this complex, structure-specific recognition protein-1 (SSRP1) heterodimerizes with suppressor of Ty 16 (SPT16). SSRP1 also has SPT16-independent functions, but how SSRP1 functions alone remains elusive. Here, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS) techniques, we characterized human SSRP1 and that from the amoeba Dictyostelium discoideum and show that both orthologs form an elongated homodimer in solution. We found that substitutions in the SSRP1 pleckstrin homology domain known to bind SPT16 also disrupt SSRP1 homodimerization. Moreover, AUC and SAXS analyses revealed that SSRP1 homodimerization and heterodimerization with SPT16 (resulting in FACT) involve the same SSRP1 surface, namely, the PH2 region, and that the FACT complex contains only one molecule of SSRP1. These observations suggest that SSRP1 homo- and heterodimerization might be mutually exclusive. Moreover, isothermal titration calorimetry analyses disclosed that SSRP1 binds both histones H2A-H2B and H3-H4 and that disruption of SSRP1 homodimerization decreases its histone-binding affinity. Together, our results provide evidence for regulation of SSRP1 by homodimerization and suggest a potential role for homodimerization in facilitating SPT16-independent functions of SSRP1

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement.

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes

Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

We determined the 2.45 Å crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human α-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein–DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged

Structural visualization of key steps in nucleosome reorganization by human FACT

Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA

Specific DNA structural attributes modulate platinum anticancer drug site selection and cross-link generation

Heavy metal compounds have toxic and medicinal potential through capacity to form strong specific bonds with macromolecules, and the interaction of platinum drugs at the major groove nitrogen atom of guanine bases primarily underlies their therapeutic activity. By crystallographic analysis of transition metal–and in particular platinum compound–DNA site selectivity in the nucleosome core, we establish that steric accessibility, which is controlled by specific structural parameters of the double helix, modulates initial guanine–metal bond formation. Moreover, DNA conformational features can be linked to both similarities and distinctions in platinum drug adduct formation between the naked and nucleosomal DNA states. Notably, structures that facilitate initial platinum–guanine bond formation can oppose cross-link generation, rationalizing the occurrence of long-lived therapeutically ineffective monofunctional adducts. These findings illuminate DNA structure-dependent reactivity and provide a novel framework for understanding metal–double helix interactions, which should facilitate the development of improved chromatin-targeting medicinal agents

DNA stretching in the nucleosome facilitates alkylation by an intercalating antitumour agent

DNA stretching in the nucleosome core can cause dramatic structural distortions, which may influence compaction and factor recognition in chromatin. We find that the base pair unstacking arising from stretching-induced extreme minor groove kinking near the nucleosome centre creates a hot spot for intercalation and alkylation by a novel anticancer compound. This may have far reaching implications for how chromatin structure can influence binding of intercalator species and indicates potential for the development of site selective DNA-binding agents that target unique conformational features of the nucleosome

Perturbations in nucleosome structure from heavy metal association

Heavy metals have the potential to engage in strong bonding interactions and can thus function in essential as well as toxic or therapeutic capacities. We conducted crystallographic analyses of heavy cation binding to the nucleosome core particle and found that Co2+ and Ni2+ preferentially associate with the DNA major groove, in a sequence- and conformation-dependent manner. Conversely, Rb+ and Cs+ are found to bind only opportunistically to minor groove elements of the DNA, in particular at narrow AT dinucleotide sites. Furthermore, relative to Mn2+ the aggressive coordination of Co2+ and Ni2+ to guanine bases is observed to induce a shift in histone–DNA register around the nucleosome center by stabilizing DNA stretching over one region accompanied by expulsion of two bases at an opposing location. These ‘softer’ transition metals also associate with multiple histone protein sites, including inter-nucleosomal cross-linking, and display a proclivity for coordination to histidine. Sustained binding and the ability to induce structural perturbations at specific locations in the nucleosome may contribute to genetic and epigenetic mechanisms of carcinogenesis mediated by Co2+ and Ni2+