A complex of Drosha with DGCR8 (or its homolog Pasha) cleaves primary microRNA (pri-miRNA) substrates into precursor miRNA and initiates the microRNA maturation process. Drosha provides the catalytic site for this cleavage, whereas DGCR8 or Pasha provides a frame for anchoring substrate pri-miRNAs. To clarify the molecular basis underlying recognition of pri-miRNA by DGCR8 and Pasha, we determined the crystal structure of the human DGCR8 core (DGCR8S, residues 493-720). In the structure, the two double-stranded RNA-binding domains (dsRBDs) are arranged with pseudo two-fold symmetry and are tightly packed against the C-terminal helix. The H2 helix in each dsRBD is important for recognition of pri-miRNA substrates. This structure, together with fluorescent resonance energy transfer and mutational analyses, suggests that the DGCR8 core recognizes pri-miRNA in two possible orientations. We propose a model for DGCR8's recognition of pri-miRNA.

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172-368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.

Werner syndrome is a human autosomal recessive disorder and is associated with premature aging and an increased incidence of cancer (1, 2). Werner syndrome patients exhibit an increased predisposition to arteriosclerosis, osteoporosis, type II diabetes mellitus, and a variety of tumors, which are normally observed during aging (2, 3). Fibroblast cultures from Werner syndrome patients show a reduced life span and a variety of chromosomal abnormalities including reciprocal translocations, deletions, and inversions (4), abnormalities in S phase initiation or transit (5), and attenuated replicative potential (6, 7). In addition, some Werner syndrome cell lines have shown aberrant mitotic recombination (8) and increased levels of homologous recombination (9). Werner syndrome cell lines are also hypersensitive to the DNA-damaging agent, 4-nitroquinoline-1-oxide (10). Together, these cellular phenotypes suggest that Werner syndrome is associated with one or more defects in DNA metabolism.Most Werner syndrome cases have been causally related to mutations in a single gene, WRN, which encodes a multifunctional protein, WRN, a member of the RecQ family of helicases (11). In humans, this family includes proteins such as Bloom syndrome and Rothmund-Thompson syndrome proteins, whose germline mutations are responsible for diseases associated with genomic instability (12, 13).The precise molecular function of WRN is unclear. However, WRN is known to interact with several key proteins that play critical roles in DNA replication, recombination, and repair, which suggests that WRN may maintain genomic integrity and life span by participating in DNA transactions in mammalian cells (13).WRN contains an exonuclease domain that is unique among RecQ family members (14 -16). Although the biological importance of the Werner exonuclease domain (WRNexo) 3 has not been precisely defined, the facts that WRNexo cleaves diverse substrates including the long fork form of DNA and Holliday junctions, and that WRN interacts with proliferating cell nuclear antigen (PCNA) suggest that WRNexo may participate in the DNA replication process (16). Recent studies show that inactivation of WRNexo alters DNA end-joining in human cells (17). Moreover, Ku70/80, an important regulator of genomic stability, stimulates the exonuclease activity of WRNexo, implying that WRNexo may play an important role in DNA repair (17). Interestingly, Ku70/80 inhibits the 3Ј-5Ј exonuclease of Klenow fragment (KFexo), a structurally conserved 3Ј-5Ј exonuclease, which suggests that the Ku-mediated activation mechanism could be unique to WRNexo. Thus, elucidation of structure and function of WRNexo may provide important insights to understand the molecular mechanism by which * This work was supported by funds from the National Creative Research Initiatives (Ministry of Science and Technology). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Sect...

HGF/c-Met pathway activation is associated with SLNM of the central neck in PTMC.

We report quantitative results on interactions between a tumor suppressor protein, p53, also known as a prognostic cancer marker, and its antibody. The p53 antibody molecules immobilized on an (R)-lipo-diaza-18-crown-6 self-assembled monolayer (SAM)-modified gold disk electrode were shown to effectively capture the p53 protein by Western blot, quartz crystal microbalance, and electrochemical impedance experiments. The p53 protein thus captured modulated the ability of the electrode for charge transfer to and from a redox probe in the solution in a p53 concentration range of approximately 0.1-30 microg/mL. The same interaction was also observed in the human embryonic kidney cell lysate, demonstrating that the SAM-modified electrode can serve as a selective platform for electrochemically monitoring the cellular p53 concentration.

The initiation of eukaryotic DNA replication requires the tightly controlled assembly of a set of replication factors. Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2-7 complex and DNA polymerases. The central domain, which contains the CCCH zinc-binding motif, is most conserved within Mcm10 and binds to DNA and several proteins, including proliferative cell nuclear antigen. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. An X-ray diffraction data set was collected to a resolution of 2.6 A on a synchrotron beamline. The crystals formed belonged to space group R3, with unit-cell parameters a = b = 99.5, c = 133.0 A. According to Matthews coefficient calculations, the crystals were predicted to contain six MCM10 central domain molecules in the asymmetric unit.

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acephala (kale) [2] and solved the crystal structure by molecular replacement method. The model structure, Lepidium WSCP (PDB code: 1WYA), shares 41% identity of primary sequence. Kale WSCP possesses a homo-tetrameric structure consisting of 19 kDa subunits, and each monomer contains one Chl but no carotenoid, as in the case of Lepidium WSCP. The remarkable structural feature is that all four Chls are packed in a hydrophobic core at the inter-subunit interface. Because the Chls are secluded from solvent, it is unlikely that the excitation energy of Chl transfers to oxygen and generates radical species.