Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.     

Determination of Residues Responsible for Substrate and Product Specificity of Solanum habrochaites Short-Chain cis-Prenyltransferases

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.Prenylation is an important derivatization of plant aromatics, contributing to the chemical diversification of phenolic secondary metabolites in plants due to differences in prenylation positions, prenyl chain lengths, and further modifications of prenyl chains. To date, about 1,000 prenylated aromatic compounds have been isolated as biologically active substances from various plant species, including many medicinal plants.Coumarins (α-benzopyrones) are a large group of plant secondary metabolites. Many biologically active coumarins are prenylated, with the prenyl residue enhancing the biological activities of the aromatic core compound. For example, imperatorin (dimethylallylated xanthotoxol), a strong inhibitor of a Manduca sexta midgut cytochrome P450, has 100-fold greater activity than the nonprenylated coumarin compound, suggesting that prenylation is involved in chemoprevention against biotic stress in plants (Neal and Wu, 1994). Prenylated compounds are also beneficial for human health. For example, geranylation of umbelliferone at the OH position to form auraptene results in a 25-fold enhancement of the inhibition of Epstein Barr virus activity, a test used to screen antitumor compounds (Murakami et al., 1997). Moreover, in tuberculosis, 8-geranyloxypsoralen was reported to decrease the growth rate of Mycobacterium smegmatis (Adams et al., 2006).There are many reports on the detection of prenyltransferase (PT) activities for coumarins in various plant species. For example, umbelliferone-dimethylallyltransferase activities were reported in cultured parsley (Petroselinum crispum) cells, Ruta graveolens, and Ammi majus, and plastidial localization of the enzyme activity is also reported (Ellis and Brown, 1974; Dhillon and Brown, 1976; Tietjen and Matern, 1983; Hamerski and Matern, 1988; Hamerski et al., 1990). In addition, bergaptol 5-O-geranyltransferase activity, which yields bergamottin, a major coumarin derivative, was characterized using the microsomal fraction of lemon (Citrus limon) peel flavedo, the outer part of the lemon fruit (Frérot and Decorzant, 2004; Munakata et al., 2012). In the lemon flavedo, 8-geranyltransferase activity for umbelliferone was also detected (Munakata et al., 2012). To date, only one gene encoding these enzymes has been described; this gene, which encodes a parsley PT (PcPT), was very recently isolated (Karamat et al., 2014).The first flavonoid-specific PT identified was naringenin 8-dimethylallyltransferase (SfN8DT1) from a leguminous medicinal plant, Sophora flavescens (Sasaki et al., 2008). Since then, genes encoding various flavonoid PTs have been identified in Leguminosae (Akashi et al., 2009; Sasaki et al., 2011; Shen et al., 2012). Although other prenylated aromatic compounds, including coumarins, xanthons, phenylpropanoids, and phloroglucinols, have been isolated from many plant species, no gene encoding a PT for those aromatics has been isolated, except for the gene encoding a phloroglucinol-specific enzyme (HlPT1) from hops (Humulus lupulus) and a the recently isolated coumarin dimethylallyltransferase from parsley (Tsurumaru et al., 2010, 2012; Karamat et al., 2014). These isolated plant aromatic PTs show strong preference for dimethylallyl diphosphate (DMAPP) as the prenyl donor substrate, although in nature, many geranylated phenolics and less farnesylated phenolics have been described. This raises questions about the enzymes and reaction mechanisms involved in the synthesis of these phenolic compounds, such as substrate specificity and prenylation sites. Better understanding of these reactions requires the identification of PTs with other enzymatic activities. It is also necessary to identify PTs producing prenylated phenolics in nonleguminosaeous plants. Four different tracks should be explored to identify enzymes that (1) recognize nonflavonoid substrates, e.g. coumarins, phenylpropanoids, and xanthons, (2) are specific for longer chain prenyl diphosphates such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), (3) are from nonlegume origins, and (4) catalyze O-prenylation.Citrus species, including lemons, contain large quantities of geranylated coumarins. We therefore isolated a complementary DNA (cDNA) encoding a PT from lemon peel, identifying the novel PT-encoding gene ClPT1. Phylogenetic analysis showed that this enzyme shares homologies with homogentisate PTs involved in vitamin E and plastoquinone biosynthesis but is located in a new clade. We provide evidence showing that this unique enzyme is highly specific for GPP as a prenyl donor and coumarin as a prenyl acceptor. We also show that the gene product is targeted to plastid in plant cells.     

Targeted exome sequencing of unselected heavy‐ion beam‐irradiated populations reveals less‐biased mutation characteristics in the rice genome

Heavy‐ion beams have been widely utilized as a novel and effective mutagen for mutation breeding in diverse plant species, but the induced mutation spectrum is not fully understood at the genome scale. We describe the development of a multiplexed and cost‐efficient whole‐exome sequencing procedure in rice, and its application to characterize an unselected population of heavy‐ion beam‐induced mutations. The bioinformatics pipeline identified single‐nucleotide mutations as well as small and large (>63 kb) insertions and deletions, and showed good agreement with the results obtained with conventional polymerase chain reaction (PCR) and sequencing analyses. We applied the procedure to analyze the mutation spectrum induced by heavy‐ion beams at the population level. In total, 165 individual M₂ lines derived from six irradiation conditions as well as eight pools from non‐irradiated ‘Nipponbare’ controls were sequenced using the newly established target exome sequencing procedure. The characteristics and distribution of carbon‐ion beam‐induced mutations were analyzed in the absence of bias introduced by visual mutant selections. The average (±SE) number of mutations within the target exon regions was 9.06 ± 0.37 induced by 150 Gy irradiation of dry seeds. The mutation frequency changed in parallel to the irradiation dose when dry seeds were irradiated. The total number of mutations detected by sequencing unselected M₂ lines was correlated with the conventional mutation frequency determined by the occurrence of morphological mutants. Therefore, mutation frequency may be a good indicator for sequencing‐based determination of the optimal irradiation condition for induction of mutations.     

Importance of methionine residues in the enzymatic carboxylation of biotin-containing peptides representing the local biotinyl site of E. coli acetyl-CoA carboxylase

A biotin-containing hexapeptide Ac-Glu-Ala-Met-Bct-Met-Met (1) that represents the local biotin-containing site of Escherichia coli acetyl-CoA carboxylase has been prepared by the solid phase method. Peptide 1 is carboxylated by the biotin carboxylase subunit dimer of E. coli acetyl-CoA carboxylase with the following kinetic parameters; Km 12 mM, Vmax 2.8 microM X min-1. These compare with the parameters for biotin of Km 214 mM and Vmax 28 microM X min -1. Hence, the overall reactivity (Vmax/Km) of 1 is 1.8 times greater than that of free biotin. When all methionines in 1 are replaced by alanine, the resulting peptide (2) retains a similar binding ability but with a much decreased Vmax. It was also found that peptide 3, which carries an N epsilon-benzyloxycarbonyllysine in place of biocytin in 1, decreases the Km of biotin threefold.     

Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways

Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.     

Theste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.     

Glutamate levels control HT22 murine hippocampal cell death by regulating biphasic patterns of Erk1/2 activation: role of metabolic glutamate receptor 5

Extracellular glutamate concentration is a critical determinant of neuronal cell fate. We recently demonstrated that HT22 murine hippocampal cell viability was reduced by exposure to high concentrations of glutamate, whereas low concentrations promoted cell survival. Extracellular signal-regulated kinase (Erk)1/2 activation by glutamate is important for both glutamate-induced cell death and survival. In this study, we investigated the role of glutamate-induced or hydrogen peroxide (H₂O₂)-induced Erk1/2 activation in HT22 cell fate determination. Glutamate and H₂O₂ treatment similarly induced early (<1 h) Erk1/2 phosphorylation regardless of concentration. On the other hand, persistent Erk1/2 phosphorylation (16–24 h) was observed only in the presence of excess glutamate. Only the latter contributed to glutamate-induced cell death, which involved metabolic glutamate receptor 5. Our findings suggest that glutamate concentration modulates two distinct phases of Erk1/2 activation, which can explain the glutamate concentration-dependent determination of HT22 cell fate.     

Triphala,a formulation of traditional Ayurvedic medicine,shows protective effect against X-radiation in HeLa cells

Ayurveda is a holistic medical system of traditional medicine, and Triphala is one of the most popular formulations in Ayurveda. Triphala is composed of three kinds of herb, Terminalia chebula, Terminalia bellirica, and Emblica officinalis. Since Triphala is shown to exhibit a protective activity against ionizing radiation in mice, we investigated its activity in HeLa cells. We found that Triphala showed the protective effects against X-radiation and bleomycin, both of which generate DNA strand breaks, in HeLa cells. Further, Triphala efficiently eliminated reactive oxygen species (ROS) in HeLa cells. Thus, the antioxidant activity of Triphala would likely play a role in its protective actions against X-radiation and bleomycin because both agents damage DNA through the generation of ROS. These observations suggested that the radioprotective activity of Triphala can be, at least partly, studied with the cells cultured in vitro. The simple bioassay system with human cultured cells would facilitate the understanding of the molecular basis for the beneficial effects of Triphala.     

Novel families of vacuolar amino acid transporters

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed.     

Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9-O-acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography-immunostaining, two-dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9-O- acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O-acetyl ganglioside synthase were not up-regulated. Tis21 which had been reported to be a 9-O-acetylation inducer was partially down-regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9-O-acetyl-GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9-O-acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild-type brain might be able to synthesize very low levels of 9-O-acetyl GD3. Increased 9-O-acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.