Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes pre-replication complex formation.

GRWD1 was previously identified as a novel Cdt1-binding protein that possesses histone-binding and nucleosome assembly activities and promotes MCM loading, probably by maintaining chromatin openness at replication origins. However, the molecular mechanisms underlying these activities remain unknown. We prepared reconstituted mononucleosomes from recombinant histones and a DNA fragment containing a nucleosome positioning sequence, and investigated the effects of GRWD1 on them. GRWD1 could disassemble these preformed mononucleosomes in vitro in an ATP-independent manner. Thus, our data suggest that GRWD1 facilitates removal of H2A-H2B dimers from nucleosomes, resulting in formation of hexasomes. The activity was compromised by deletion of the acidic domain, which is required for efficient histone binding. In contrast, nucleosome assembly activity of GRWD1 was not affected by deletion of the acidic domain. In HeLa cells, the acidic domain of GRWD1 was necessary to maintain chromatin openness and promote MCM loading at replication origins. Taken together, our results suggest that GRWD1 promotes chromatin fluidity by influencing nucleosome structures, e.g., by transient eviction of H2A-H2B, and thereby promotes efficient MCM loading at replication origins.

Speciation of two gobioid species, Pterogobius elapoides and Pterogobius zonoleucus revealed by multi-locus nuclear and mitochondrial DNA analyses.

To understand how geographical differentiation of gobioid fish species led to speciation, two populations of the Pacific Ocean and the Sea of Japan for each of the two gobioid species, Pterogobius elapoides and Pterogobius zonoleucus, were studied in both morphological and molecular features. Analyzing mitochondrial genes, Akihito et al. (2008) suggested that P. zonoleucus does not form a monophyletic clade relative to P. elapoides, indicating that "Sea of Japan P. zonoleucus" and P. elapoides form a clade excluding "Pacific P. zonoleucus" as an outgroup. Because morphological classification clearly distinguish these two species and a gene tree may differ from a population tree, we examined three nuclear genes, S7RP, RAG1, and TBR1, in this work, in order to determine whether nuclear and mitochondrial trees are concordant, thus shedding light on the evolutionary history of this group of fishes. Importantly, nuclear trees were based on exactly the same individuals that were used for the previously published mtDNA trees. The tree based on RAG1 exon sequences suggested a closer relationship of P. elapoides with "Sea of Japan P. zonoleucus", which was in agreement with the mitochondrial tree. In contrast, S7RP and TBR1 introns recovered a monophyletic P. zonoleucus. If the mitochondrial tree represents the population tree in which P. elapoides evolved from "Sea of Japan P. zonoleucus", the population size of P. elapoides is expected to be smaller than that of "Sea of Japan P. zonoleucus". This is because a smaller population of the new species is usually differentiated from a larger population of the ancestral species when the speciation occurred. However, we found no evidence of such a small population size during the evolution of P. elapoides. Therefore, we conclude that the monophyletic P. zonoleucus as suggested by S7RP and TBR1 most likely represents the population tree, which is consistent with the morphological classification. In this case, it is possible that the incongruent mitochondrial and RAG1 trees are either due to incomplete lineage sorting of ancestral polymorphisms or to introgression by hybridization. Because of a smaller effective population size of mitochondria compared with nuclear genes, the introgression might be a more likely scenario in explaining the incongruent mitochondrial tree than the incomplete lineage sorting. Because of smaller effective population size of "Sea of Japan P. zonoleucus" than that of P. elapoides, the direction of the introgression was likely to be from the latter to the former. This evolutionary work of the two gobioid species highlights the need of analyzing multiple gene trees for both nuclear and mitochondrial genes as well as scrutinization of morphological characteristics to obtain a population tree representing the organismal evolutionary history.

Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture.

Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins.

Evolution of Pacific Ocean and the Sea of Japan populations of the gobiid species, Pterogobius elapoides and Pterogobius zonoleucus, based on molecular and morphological analyses.

Pterogobius elapoides and Pterogobius zonoleucus are common free-swimming gobies found in rocky and weedy shores along the temperate coast of Japan. We collected individuals of both species from 23 locations around the coast of Japan and compared the mitochondrial nucleotide sequences of two gene regions, CytB and ND2. Phylogenetic trees constructed using the neighbor-joining, maximum parsimony, and maximum likelihood methods consistently indicated that all 125 samples of the two species, which are collected from a variety of locations in Japan, can be clearly divided into the following four clades: "Pacific P. elapoides" (Pa-ela), "Sea of Japan P. elapoides" (SJ-ela), "Pacific P. zonoleucus" (Pa-zon), and "Sea of Japan P. zonoleucus" (SJ-zon). These four monophyletic clades were supported with very high bootstrap values. Although Pa-ela and SJ-ela composed a monophyletic clade, it is noteworthy that the two clades of P. elapoides also formed a monophyletic group together with SJ-zon with a bootstrap value of 95% and 97% by the maximum likelihood and neighbor-joining methods, respectively. We observed several morphological differences between Pa-ela and SJ-ela, including; 1) six dark bands on the body in the former versus seven dark bands in the latter and 2) more pectoral-fin rays numbering 21-24 (mode 22) in the latter compared to the former (19-22, mode 21). Furthermore, the scatter plots of scores on principal components 1 and 2 based on the morphometric characters roughly separated the populations from each other. Moreover, we documented the following morphological differences between Pa-zon and SJ-zon for the first time; 1) six light bands on the body in the former versus five light bands in the latter and 2) the light bands from both eyes forming a complete U-shaped marking on the occipital region occurred in 55% of the specimens in the former versus 16% in the latter. However, no significant differences were found in the morphometric characters between the two populations of P. zonoleucus. The estimated divergence time of the two P. zonoleucus populations was 15.06+/-2.72 (mean+/-1 S.E.) times earlier than that of the two P. elapoides populations. However, the morphological differences between the two populations of the former were much smaller than those of the latter. An explanation for this obvious discrepancy between morphological and molecular features is proposed from an evolutionary point of view.

VISCANA: visualized cluster analysis of protein-ligand interaction based on the ab initio fragment molecular orbital method for virtual ligand screening.

We have developed a visualized cluster analysis of protein-ligand interaction (VISCANA) that analyzes the pattern of the interaction of the receptor and ligand on the basis of quantum theory for virtual ligand screening. Kitaura et al. (Chem. Phys. Lett. 1999, 312, 319-324.) have proposed an ab initio fragment molecular orbital (FMO) method by which large molecules such as proteins can be easily treated with chemical accuracy. In the FMO method, a total energy of the molecule is evaluated by summation of fragment energies and interfragment interaction energies (IFIEs). In this paper, we have proposed a cluster analysis using the dissimilarity that is defined as the squared Euclidean distance between IFIEs of two ligands. Although the result of an ordered table by clustering is still a massive collection of numbers, we combine a clustering method with a graphical representation of the IFIEs by representing each data point with colors that quantitatively and qualitatively reflect the IFIEs. We applied VISCANA to a docking study of pharmacophores of the human estrogen receptor alpha ligand-binding domain (57 amino acid residues). By using VISCANA, we could classify even structurally different ligands into functionally similar clusters according to the interaction pattern of a ligand and amino acid residues of the receptor protein. In addition, VISCANA could estimate the correct docking conformation by analyzing patterns of the receptor-ligand interactions of some conformations through the docking calculation.

Development of KiBank, a database supporting structure-based drug design.

KiBank is a database of inhibition constant (Ki) values with 3D structures of target proteins and chemicals. Ki values were accumulated from peer-reviewed literature searched via PubMed. The 3D structure files of target proteins were originally from Protein Data Bank (PDB), while the 2D structure files of the chemicals were collected together with the Ki values and then converted into 3D ones. In KiBank, the chemical and protein 3D structures with hydrogen atoms were optimized by energy minimization and stored in MDL MOL and PDB format, respectively. KiBank is designed to support structure-based drug design. It provides structure files of proteins and chemicals ready for use in virtual screening through automated docking methods, while the Ki values can be applied for tests of docking/scoring combinations, program parameter settings, and calibration of empirical scoring functions. Additionally, the chemical structures and corresponding Ki values in KiBank are useful for lead optimization based on quantitative structure-activity relationship (QSAR) techniques. KiBank is updated on a daily basis and is freely available at . As of August 2004, KiBank contains 8000 Ki values, over 6000 chemicals and 166 proteins covering the subtypes of receptors and enzymes.

[KiBank: a database for computer-aided drug design based on protein-chemical interaction analysis].

KiBank is a database for computer-aided drug design and consists of binding affinities and chemical and target protein structures. Each chemical or protein structure with hydrogen atoms added was optimized by energy minimization and stored in PDB or MDL MOL file format, so that the structural data can be directly used for in silico binding studies. To describe the extent of inhibition, the inhibition constant (K(i)) value is used to simplify comparisons of strengths among chemical-protein bindings. As of April 2004, KiBank contained 142 proteins, over 5000 chemicals, and over 6000 binding affinity values that were published in peer-reviewed journals. The binding affinity values are currently mostly for membrane and nuclear receptors but are soon being expanded to other drug targets. KiBank is updated daily and can be accessed on the Web at http://kibank.iis.u-tokyo.ac.jp/at no charge.