Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm

<p>Abstract</p> <p>Background</p> <p><it>Pyrococcus furiosus </it>Hjm (<it>Pfu</it>Hjm) is a structure-specific DNA helicase that was originally identified by <it>in vitro </it>screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Θ (PolΘ), HEL308, and <it>Drosophila </it>Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that <it>Pfu</it>Hjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by <it>Pfu</it>Hjm will require its three-dimensional structure at atomic resolution.</p> <p>Results</p> <p>We determined the crystal structures of <it>Pfu</it>Hjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0–2.7 Å. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of <it>Archaeoglobus fulgidus </it>Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolΘ, indicating the strong conformational conservation between archaea and eukarya.</p> <p>Conclusion</p> <p>The detailed comparison between our DNA-free <it>Pfu</it>Hjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.</p

Architectures of archaeal GINS complexes, essential DNA replication initiation factors

<p>Abstract</p> <p>Background</p> <p>In the early stage of eukaryotic DNA replication, the template DNA is unwound by the MCM helicase, which is activated by forming a complex with the Cdc45 and GINS proteins. The eukaryotic GINS forms a heterotetramer, comprising four types of subunits. On the other hand, the archaeal GINS appears to be either a tetramer formed by two types of subunits in a 2:2 ratio (α₂β₂) or a homotetramer of a single subunit (α₄). Due to the low sequence similarity between the archaeal and eukaryotic GINS subunits, the atomic structures of the archaeal GINS complexes are attracting interest for comparisons of their subunit architectures and organization.</p> <p>Results</p> <p>We determined the crystal structure of the α₂β₂GINS tetramer from <it>Thermococcus kodakaraensis </it>(<it>Tko</it>GINS), comprising Gins51 and Gins23, and compared it with the reported human GINS structures. The backbone structure of each subunit and the tetrameric assembly are similar to those of human GINS. However, the location of the C-terminal small domain of Gins51 is remarkably different between the archaeal and human GINS structures. In addition, <it>Tko</it>GINS exhibits different subunit contacts from those in human GINS, as a consequence of the different relative locations and orientations between the domains. Based on the GINS crystal structures, we built a homology model of the putative homotetrameric GINS from <it>Thermoplasma acidophilum </it>(<it>Tac</it>GINS). Importantly, we propose that a long insertion loop allows the differential positioning of the C-terminal domains and, as a consequence, exclusively leads to the formation of an asymmetric homotetramer rather than a symmetrical one.</p> <p>Conclusions</p> <p>The DNA metabolizing proteins from archaea are similar to those from eukaryotes, and the archaeal multi-subunit complexes are occasionally simplified versions of the eukaryotic ones. The overall similarity in the architectures between the archaeal and eukaryotic GINS complexes suggests that the GINS function, directed through interactions with other protein components, is basically conserved. On the other hand, the different subunit contacts, including the locations and contributions of the C-terminal domains to the tetramer formation, imply the possibility that the archaeal and eukaryotic GINS complexes contribute to DNA unwinding reactions by significantly different mechanisms in terms of the atomic details.</p

Adaptability and selectivity of human peroxisome proliferator-activated receptor (PPAR) pan agonists revealed from crystal structures

The structures of the ligand-binding domains (LBDs) of human peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ) in complexes with a pan agonist, an α/δ dual agonist and a PPARδ-specific agonist were determined. The results explain how each ligand is recognized by the PPAR LBDs at an atomic level

A Western single-center experience with endoscopic submucosal dissection for early gastrointestinal cancers

Endoscopic submucosal dissection (ESD) has gained worldwide acceptance as a treatment for early gastrointestinal cancers (EGICs). However, the management of these tumors in the Western world is still mainly surgical. Our aim was to evaluate the safety and feasibility of ESD at a European center. Based on the knowledge transferred by one of the most experienced Japanese institutions, we conducted a pilot study on 25 consecutive patients with EGICs located in the esophagus (n = 3), stomach (n = 7), duodenum (n = 1), and colon (n = 14) at our tertiary center over a 2-year-period. The main outcome measurements were complete (R0) resection, as well as en-bloc resection and the management of complications. The R0 and en-bloc resection rates were 100% and 84%, respectively. There were three cases of bleeding and five cases of perforation. With a median follow up of 18 months, two recurrences were observed. We conclude that ESD for early esophageal and gastric cancers is feasible and effective, while colonic ESD requires more expertise

EISCAT_3D（次世代欧州非干渉散乱レーダー）計画の進捗状況（４）

第6回極域科学シンポジウム[OS] 宙空圏11月16日（月）　国立極地研究所 2階 大会議

T-REX: Bare electro-dynamic tape-tether technology experimetn on sounding rocket S520

The project to verify the performance of space tether technology was successfully demonstrated by the launch of the sounding rocket S520 the 25tu. The project is the space demonstration of science and engineering technologies of a bare tape electrodynamic tether (EDT) in the international campaign between Japan, USA, Europe and Australia. Method of "Inverse ORIGAMI (Tape tether folding)" was employed in order to deploy the bare tape EDT in a short period time of the suborbital flight. The deployment of tape tether was tested in a various experimental schemes on ground to show high reliability of tape tether deployment. The rocket was launched on the summer of 2010 and deployed a bare electro-dynamic tape tether with length 132.6 m, which is the world record of the length deployment of tape tether. The verification of tether technology has found a variety kind of science and technology results as the first in the humankind and will lead a large number of applications of space tether technologie

Propagation direction of the mesospheric gravity waves derived from OH airglow images at Tromsø, Norway

第3回極域科学シンポジウム　横断セッション「中層大気・熱圏」 11月26日（月）、27日（火） 国立極地研究所 ２階ラウン

Structural and functional studies of MinD ATPase: implications for the molecular recognition of the bacterial cell division apparatus

Proper placement of the bacterial cell division site requires the site-specific inactivation of other potential division sites. In Escherichia coli, selection of the correct mid-cell site is mediated by the MinC, MinD and MinE proteins. To clarify the functional role of the bacterial cell division inhibitor MinD, which is a membrane-associated ATPase that works as an activator of MinC, we determined the crystal structure of a Pyrococcus furiosus MinD homologue complexed with a substrate analogue, AMPPCP, and with the product ADP at resolutions of 2.7 and 2.0 Å, respectively. The structure reveals general similarities to the nitrogenase iron protein, the H-Ras p21 and the RecA-like ATPase domain. Alanine scanning mutational analyses of E.coli MinD were also performed in vivo. The results suggest that the residues around the ATP-binding site are required for the direct interaction with MinC, and that ATP binding and hydrolysis play a role as a molecular switch to control the mechanisms of MinCDE-dependent bacterial cell division