DNA polymerase plays a critical role in passing the genetic information of any living organism to its offspring. DNA polymerase from enterobacteria phage RB69 (RB69pol) has both polymerization and ...exonuclease activities and has been extensively studied as a model system for B-family DNA polymerases. Many binary and ternary complex structures of RB69pol are known, and they all contain a single polymerase-primer/template (P/T) DNA complex. Here, we report a crystal structure of the exonuclease-deficient RB69pol with the P/T duplex in a dimeric form at a resolution of 2.2 Å. The structure includes one new closed ternary complex with a single divalent metal ion bound and one new open binary complex in the pre-insertion state with a vacant dNTP-binding pocket. These complexes suggest that initial binding of the correct dNTP in the open state is much weaker than expected and that initial binding of the second divalent metal ion in the closed state is also much weaker than measured. Additional conformational changes are required to convert these complexes to high-affinity states. Thus, the measured affinities for the correct incoming dNTP and divalent metal ions are average values from many conformationally distinctive states. Our structure provides new insights into the order of the complex assembly involving two divalent metal ions. The biological relevance of specific interactions observed between one RB69pol and the P/T duplex bound to the second RB69pol observed within this dimeric complex is discussed.
PSD-95 is an abundant postsynaptic density (PSD) protein involved in the formation and regulation of excitatory synapses and dendritic spines, but the underlying mechanisms are not comprehensively ...understood. Here we report a novel PSD-95-interacting protein Preso that regulates spine morphogenesis. Preso is mainly expressed in the brain and contains WW (domain with two conserved Trp residues), PDZ (PSD-95/Dlg/ZO-1), FERM (4.1, ezrin, radixin, and moesin), and C-terminal PDZ-binding domains. These domains associate with actin filaments, the Rac1/Cdc42 guanine nucleotide exchange factor betaPix, phosphatidylinositol-4,5-bisphosphate, and the postsynaptic scaffolding protein PSD-95, respectively. Preso overexpression increases the density of dendritic spines in a manner requiring WW, PDZ, FERM, and PDZ-binding domains. Conversely, knockdown or dominant-negative inhibition of Preso decreases spine density, excitatory synaptic transmission, and the spine level of filamentous actin. These results suggest that Preso positively regulates spine density through its interaction with the synaptic plasma membrane, actin filaments, PSD-95, and the betaPix-based Rac1 signaling pathway.
Transcriptional regulation of ergosterol biosynthesis in fungi is crucial for sterol homeostasis and for resistance to azole drugs. In Saccharomyces cerevisiae, the Upc2 transcription factor ...activates the expression of related genes in response to sterol depletion by poorly understood mechanisms. We have determined the structure of the C-terminal domain (CTD) of Upc2, which displays a novel α-helical fold with a deep hydrophobic pocket. We discovered that the conserved CTD is a ligand-binding domain and senses the ergosterol level in the cell. Ergosterol binding represses its transcription activity, while dissociation of the ligand leads to relocalization of Upc2 from cytosol to nucleus for transcriptional activation. The C-terminal activation loop is essential for ligand binding and for transcriptional regulation. Our findings highlight that Upc2 represents a novel class of fungal zinc cluster transcription factors, which can serve as a target for the developments of antifungal therapeutics.
Microtubules are one of the major targets for anticancer drugs because of their role in cell proliferation and migration. However, as anticancer drugs targeting microtubules have side effects, ...including the death of normal cells, it is necessary to develop anticancer agents that can target microtubules by specifically acting on cancer cells only. In this study, we identified chemicals that can act as anticancer agents by specifically binding to acetylated microtubules, which are predominant in triple-negative breast cancer (TNBC). The chemical compounds disrupted acetylated microtubule lattices by interfering with microtubule access to alpha-tubulin acetyltransferase 1 (αTAT1), a major acetyltransferase of microtubules, resulting in the increased apoptotic cell death of MDA-MB-231 cells (a TNBC cell line) compared with other cells, such as MCF-10A and MCF-7, which lack microtubule acetylation. Moreover, mouse xenograft experiments showed that treatment with the chemical compounds markedly reduced tumor growth progression. Taken together, the newly identified chemical compounds can be selective for acetylated microtubules and act as potential therapeutic agents against microtubule acetylation enrichment in TNBC.
During DNA synthesis, base stacking and Watson–Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base ...stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB’s interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge–charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB’s surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.
RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes . The human Rad52 protein is a functional counterpart of ...RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two‐domain structure: the N‐terminal domain with a helix–hairpin–helix (HhH) motif and the C‐terminal domain with a Cys4 zinc‐finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring‐shaped tetramer of 222 symmetry with a central hole of 30−35 Å diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding.
Reliability assessment of lithium secondary batteries was mainly considered. Shape parameter (
β) and scale parameter (
η) were calculated from experimental data based on cycle life test. We also ...examined safety characteristics of lithium secondary batteries. As proposed by IEC 62133 (2002), we had performed all of the safety/abuse tests such as ‘mechanical abuse tests’, ‘environmental abuse tests’, ‘electrical abuse tests’.
This paper describes the cycle life of lithium secondary batteries, FMEA (failure modes and effects analysis) and the safety/abuse tests we had performed.
l-Arabinose isomerase (AI) catalyzes the isomerization of
l-arabinose to
l-ribulose. It can also convert
d-galactose to
d-tagatose at elevated temperatures in the presence of divalent metal ions. The
...araA genes, encoding AI, from the mesophilic bacterium
Bacillus halodurans and the thermophilic
Geobacillus stearothermophilus were cloned and overexpressed in
Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232
kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements.
B. halodurans AI has maximal activity at 50
°C under the assay conditions used and is not dependent on divalent metal ions. Its apparent
K
m values are 36
mM for
l-arabinose and 167
mM for
d-galactose, and the catalytic efficiencies (
k
cat/
K
m) of the enzyme were 51.4
mM
−1
min
−1 (
l-arabinose) and 0.4
mM
−1
min
−1 (
d-galactose). Unlike
B. halodurans AI,
G. stearothermophilus AI has maximal activity at 65–70
°C, and is strongly activated by Mn
2+. It also has a much higher catalytic efficiency of 4.3
mM
−1
min
−1 for
d-galactose and 32.5
mM
−1
min
−1for
l-arabinose, with apparent
K
m values of 117 and 63
mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of
B. halodurans AI (
T
m
=
65–67
°C) was unaffected by the presence of metal ions, whereas EDTA-treated
G. stearothermophilus AI had a lower
T
m (72
°C) than the holoenzyme (78
°C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo
G. stearothermophilus AI, and there is an active tertiary structure for
G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic
B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of
G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic
G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.
Starting at 512Mb 6Gb/s/pin 1, GDDR5's speed and density have been steadily developing for about 10 years; recently achieving 8Gb 9Gb/s/pin 2 with per-pin timing training. Although 8Gb GDDR5X can ...operate at 12Gb/s 3 by increasing the burst length (BL) from 8 to 16, a degradation in system performance at a data granularity of 64B is seen. The I/O specification, using PLL clocking that additionally causes PLL jitter, has not changed much compared with GDDR5. To overcome these issues, GDDR6 introduced a dual channel for a data granularity of 32B with a BL16, per-bit training of l/ REF , and an equalizer with PLL-less clocking. This paper presents a 16Gb 18Gb/s/pin GDDR6 DRAM with a die architecture and high-speed circuit techniques on 1.35V DRAM process.
Ca
regulates several cellular functions, including signaling events, energy production, and cell survival. These cellular processes are mediated by Ca
-binding proteins, such as EF-hand superfamily ...proteins. Among the EF-hand superfamily proteins, allograft inflammatory factor-1 (AIF-1) and swiprosin-1/EF-hand domain-containing protein 2 (EFhd2) are cytosolic actin-binding proteins. AIF-1 modulates the cytoskeleton and increases the migration of immune cells. EFhd2 is also a cytoskeletal protein implicated in immune cell activation and brain cell functions. EFhd1, a mitochondrial fraternal twin of EFhd2, mediates neuronal and pro-/pre-B cell differentiation and mitoflash activation. Although EFhd1 is important for maintaining mitochondrial morphology and energy synthesis, its mechanism of action remains unclear. Here, we report the crystal structure of the EFhd1 core domain comprising a C-terminus of a proline-rich region, two EF-hand domains, and a ligand mimic helix. Structural comparisons of EFhd1, EFhd2, and AIF-1 revealed similarities in their overall structures. In the structure of the EFhd1 core domain, two Zn
ions were observed at the interface of the crystal contact, suggesting the possibility of Zn
-mediated multimerization. In addition, we found that EFhd1 has Ca
-independent β-actin-binding and Ca
-dependent β-actin-bundling activities. These findings suggest that EFhd1, an actin-binding and -bundling protein in the mitochondria, may contribute to the Ca
-dependent regulation of mitochondrial morphology and energy synthesis.
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