New recognition mode for a TG mismatch: the atomic structure of a very short patch repair endonuclease-DNA complex.
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Base Pair Mismatch↗
Biomedical subjects
K Morikawa
Publications and source records attributed to K Morikawa.
Purification, crystallization and preliminary X-ray analysis of a complex between granulocyte colony-stimulating factor and its soluble receptor.
Crystals of the complex between granulocyte colony-stimulating factor and its soluble receptor were obtained by a vapour-diffusion method using ammonium sulfate as a precipitant. Addition of 1, 4-dioxane was critical in order to grow the crystals to sufficient sizes. Cryoprotection was essential in order to collect diffraction data at atomic resolution. Two kinds of crystal forms were obtained depending on the cryoprotectants. In a cryosolvent with the same salt concentration as in the crystallization conditions, the crystal belonged to the space group I4(1)22. At higher salt concentrations, the crystal was converted to a different space group P4(1)2(1)2 (P4(3)2(1)2) with the same unit-cell parameters.
Animals↗
Calcium polycarbophil, a water absorbing polymer, increases bowel movement and prevents sennoside-induced diarrhea in dogs.
The effects of calcium polycarbophil (CP), a water-absorbing polymer, on bowel movement were examined in comparison with known laxatives and anti-diarrheal agents in dogs, a species that resembles humans for stool output. CP increased stool frequency, fecal water content and fecal weight in a dose-dependent manner, but did not induce diarrhea. Sennoside and carboxymethylcellulose sodium (CMC-Na) increased fecal water content and induced diarrhea at lower doses than that which enhanced stool frequency. Trimebutine decreased stool frequency, fecal weight and fecal water content, resulting in inhibition rather than stimulation of defecation. In sennoside-induced diarrhea, loperamide and CP improved stool consistency and this was accompanied by reduced fecal moisture and frequency of diarrhea. In contrast, CMC-Na aggravated stool consistency with increased fecal water content and frequency of diarrhea, and trimebutine had little noticeable effect apart from reducing fecal weight. Our results show that CP has both laxative and anti-diarrheal effects in dogs and differed from conventional laxatives and anti-diarrheal agents. CP may be a suitable agent for treatment of idiopathic constipation, secretory diarrhea and irritable bowel syndrome with alternating constipation and diarrhea and with either predominating in terms of less side effects such as diarrhea or constipation.
Acrylic Resins↗
Spontaneous intracranial hypotension associated with bilateral chronic subdural hematomas--case report.
A 34-year-old female presented with spontaneous intracranial hypotension (SIH) manifesting as severe postural headache and meningism. Magnetic resonance (MR) imaging with gadolinium showed diffuse pachymeningeal enhancement. She developed bilateral chronic subdural hematomas 4 weeks after the onset of the symptoms. MR imaging showed descent of the midline structures of the brain. The bilateral chronic subdural hematomas were surgically drained, with no remarkable pressure. Postoperative MR imaging showed complete resolution of the pachymeningeal enhancement and relevation of the midline structures of the brain. SIH is an uncommon and probably unrecognized condition because of the usually benign course. However, this case emphasizes that SIH is not entirely benign. SIH should be considered if there is no identifiable risk for intracranial hemorrhage, particularly in young patients. Neurosurgical intervention for the treatment of the underlying cerebrospinal fluid leak may be required if SIH persists.
Adult↗
[A new cytokine-receptor recognition scheme revealed by the complex structure of G-CSF and its receptor].
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Crystallography, X-Ray↗
Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.
Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions.
Animals↗
Recognition of a TG mismatch: the crystal structure of very short patch repair endonuclease in complex with a DNA duplex.
The crystal structure of very short patch repair (Vsr) endonuclease, in complex with Mg2+ and with duplex DNA containing a TG mismatch, has been determined at 2.3 A resolution. In E. coli, the enzyme recognizes a TG mismatched base pair, generated after spontaneous deamination of methylated cytosines, and cleaves the phosphate backbone on the 5' side of the thymine. Extensive interactions between the DNA and the protein characterize a novel recognition mechanism, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking. With the presence of a cleaved DNA intermediate in the active center, the structure of the Vsr/DNA complex provides detailed insights into the catalytic mechanism for endonuclease activity.
Base Pair Mismatch↗
PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. I. Purification and identification of the homing-type endonuclease activities.
We screened for proteins with specific binding activity to Holliday junction DNA from the hyperthermophilic archaeon Pyrococcus furiosus and found a protein that has specific affinity for DNA with a branched structure, like a three-way or four-way junction. The protein was identified as one of the two inteins encoded in the gene for ribonucleotide reductase (RNR) by gene cloning. These two inteins were spliced out from the precursor protein as polypeptides with molecular weights of 53.078 and 43.976 kDa, respectively. The amino acid sequences of these inteins have two copies of the LAGLIDADG motif, which is found in the site-specific DNA endonucleases. The purified proteins actually cleaved double-stranded DNA with the sequence of the intein(-)allele, and, therefore, they were designated PI- Pfu I and PI- Pfu II. They generate a 4 bp 3'-OH overhang with a 5'-phosphate, like other known homing endonucleases originating from inteins. The optimal conditions of the DNA cleavage reaction, including temperature, pH, and concentrations of KCl and MgCl(2), have been determined. The high affinity for junction DNA of PI- Pfu I was confirmed using the purified protein.
Amino Acid Motifs↗
PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.
PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing endonucleases and provides some of the catalytic residues necessary for DNA cleavage activity. Site-specific mutagenesis studies showed that two acidic residues in the motifs, Asp149 and Glu250 in PI- Pfu I, and Asp156 and Asp249 in PI- Pfu II, were critical for catalysis. The third residues of the active site triads, as predicted from the structure of PI- Sce I, were Asn225 in PI- Pfu I and Lys224 in PI- Pfu II. Substitution of Asn225 in PI- Pfu I by Ala did not affect catalysis. The cleavage activity of PI- Pfu II was 50-fold decreased by the substitution of Ala for Lys224. The binding affinity of the mutant protein for the substrate DNA also decreased 6-fold. The Lys in PI- Pfu II may play a direct or indirect role in catalysis of the endonuclease activity.
Amino Acid Motifs↗
Atomic structure of the GCSF-receptor complex showing a new cytokine-receptor recognition scheme.
Granulocyte colony-stimulating factor (GCSF) is the principal growth factor regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes and is used to treat neutropenia. GCSF is a member of the long-chain subtype of the class 1 cytokine superfamily, which includes growth hormone, erythropoietin, interleukin 6 and oncostatin M. Here we have determined the crystal structure of GCSF complexed to the BN-BC domains, the principal ligand-binding region of the GCSF receptor (GCSFR). The two receptor domains form a complex in a 2:2 ratio with the ligand, with a non-crystallographic pseudo-twofold axis through primarily the interdomain region and secondarily the BC domain. This structural view of a gp130-type receptor-ligand complex presents a new molecular basis for cytokine-receptor recognition.
Amino Acid Sequence↗
A Holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea.
The Holliday junction is an essential intermediate of homologous recombination. RecA of Bacteria, Rad51 of Eukarya, and RadA of Archaea are structural and functional homologs. These proteins play a pivotal role in the formation of Holliday junctions from two homologous DNA duplexes. RuvC is a specific endonuclease that resolves Holliday junctions in Bacteria. A Holliday junction-resolving activity has been found in both yeast and mammalian cells. To examine whether the paradigm of homologous recombination apply to Archaea, we assayed and found the activity to resolve a synthetic Holliday junction in crude extract of Pyrococcus furiosus cells. The gene, hjc (Holliday junction cleavage), encodes a protein composed of 123 amino acids, whose sequence is not similar to that of any proteins with known function. However, all four archaea, whose total genome sequences have been published, have the homologous genes. The purified Hjc protein cleaved the recombination intermediates formed by RecA in vitro. These results support the notion that the formation and resolution of Holliday junction is the common mechanism of homologous recombination in the three domains of life.
Amino Acid Sequence↗
Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana. Evidence for cold denaturation in regions outside of the DNA binding domain.
A transcriptional activator, CBF1, from Arabidopsis thaliana, which has the AP2 domain for DNA binding and regulates the cold acclimation response, was overexpressed in Escherichia coli, purified, and characterized. Analyses of the interaction between CBF1 and the C-repeat/dehydration-responsive element by fluorescence measurement showed that CBF1 binds to C-repeat/dehydration-responsive element as a monomer irrespective of the temperature. CD spectra of the intact and truncated CBF1 proteins (1-213, 41-213, 41-157, and 41-146) were measured to examine the temperature-dependent changes of the secondary structure of CBF1. The results suggested that the CBF1 protein has regions exhibiting reversible cold denaturation in the range between 30 and -5 degrees C and also has a region exhibiting thermal denaturation between 40 and 60 degrees C. This cold denaturation occurred in both the N-terminal and acidic regions. The thermal denaturation occurred in the region encompassing the AP2 domain. The difference between the retention time of CBF1 at 4 degrees C and that at 25 degrees C in gel filtration, and the decrease of the sedimentation coefficient, s20,w, caused by the temperature change from 25 to 3 degrees C, strongly suggested that the cold denaturation was accompanied by the extension of the molecule. The possible cold denaturation observed here might be a physiologically important structural response of CBF1 to cold stress.
Arabidopsis↗
Circadian-regulated expression of a nuclear-encoded plastid sigma factor gene (sigA) in wheat seedlings.
The activity of a light-responsive psbD promoter in plastids is known to be regulated by a circadian clock. However, the mechanism of the circadian regulation of the psbD light-responsive promotor, which is recognized by an Escherichia coli-type RNA polymerase, is not yet known. We examined the time course of mRNA accumulation of two E. coli-type RNA polymerase subunit genes, sigA and rpoA, under a continuous light condition after 12 h light/12 h dark entrainment. Accumulation of the sigA mRNA was found to be regulated by a circadian clock, while rpoA mRNA did not show any significant oscillation throughout the experiment.
Amino Acid Sequence↗
Binding and functional characterization of alpha1-adrenoceptor subtypes in the rat prostate.
The alpha1-adrenoceptor subtypes of rat prostate were characterized in binding and functional experiments. In binding experiments, [3H]tamsulosin bound to a single class of binding sites with an affinity (pKD) of 10.79+/-0.04 and Bmax of 87+/-2 fmol mg(-1) protein. This binding was inhibited by prazosin, 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil, alpha-ethyl-3,4,5,-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)-propyl)benzeneacetonitrile fumarate (HV723) and oxymetazoline with high efficacy, resulting in a good correlation with the binding characteristics of cloned alpha1a but not alpha1b and alpha1d-adrenoceptor subtypes. In functional studies, noradrenaline and oxymetazoline produced concentration-dependent contractions. These contractions were antagonized by tamsulosin, prazosin, WB4101 and 5-methylurapidil with an efficacy lower than that exhibited by these agents for inhibition of [3H]tamsulosin binding. The relationship between receptor occupancy and contractile amplitude revealed the presence of receptor reserve for noradrenaline, but the contraction induced by oxymetazoline was not in parallel with receptor occupation and developed after predicted receptor saturation. From these results, it is suggested that alpha1A-adrenoceptors are the dominant subtype in the rat prostate which can be detected with [3H]tamsulosin, but that the functional subtype mediating adrenergic contractions has the characteristics of the alpha1L-adrenoceptor subtype, having a lower affinity for prazosin and some other drugs than the alpha1A-adrenoceptor subtype.
Acetonitriles↗
Crystallographic and functional studies of very short patch repair endonuclease.
Vsr endonuclease plays a crucial role in the repair of TG mismatched base pairs, which are generated by the spontaneous degradation of methylated cytidines; Vsr recognizes the mismatched base pair and cleaves the phosphate backbone 5' to the thymidine. We have determined the crystal structure of a truncated form of this endonuclease at 1.8 A resolution. The protein contains one structural zinc-binding module. Unexpectedly, its overall topology resembles members of the type II restriction endonuclease family. Subsequent mutational and biochemical analyses showed that certain elements in the catalytic site are also conserved. However, the identification of a critical histidine and evidence of an active site metal-binding coordination that is novel to endonucleases indicate a distinct catalytic mechanism.
Alanine↗
Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region. Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride.
Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region. In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.
Bacterial Proteins↗
Comparative studies on sleep disturbance in the elderly based on questionnaire assessments in 1983 and 1996.
We made an attempt to compare the complaints about sleep disturbance in the elderly based on the questionnaires using self-rating scales carried out in 1983 and 1996. It was noted that the score of awakening frequency from the course of sleep was the highest of all the items in men and women in both the 1983 and 1996 inquiries. The scores of insomnia nights per week and the difficulty in falling asleep were slightly higher in women than in men in both inquiries.
Aged↗
Resonance assignments, solution structure, and backbone dynamics of the DNA- and RPA-binding domain of human repair factor XPA.
XPA is involved in the damage recognition step of nucleotide excision repair (NER). XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA). The domain consists of a zinc-containing subdomain with a compact globular structure and a C-terminal subdomain with a positively charged cleft in a novel alpha/beta structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods. 15N relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion. In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (Dparallel/Dperpendicular) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-terminal subdomain. This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA.
Binding Sites↗