Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain

A thermophilic strain producing an extracellular esterase/lipase was isolated from a hot spring in Tǎşnad, Romania, and was identified phenotypically and by 16S rDNA sequencing as Anoxybacillus flavithermus (GenBank ID: JQ267733). The gene encoding the putative carboxyl esterase (GenBank ID: JX494348) was cloned by direct PCR amplification from genomic DNA. The protein, consisting of 246 amino acids and having a predicted molecular weight of 28.03 kDa, is encoded by an ORF of 741 bps. Expression was achieved in Escherichia coli and a recombinant protein with esterolytic activity and estimated molecular weight of 25 kDa was recovered and purified from the periplasmic fraction by IMAC. The purified enzyme, most active at 60-65°C and in the near-neutral range (pH 6.5-8), displayed a half-life at 60°C of about 5 h. Est/Lip displayed a relative tolerance to methanol, DMSO, acetonitrile, and low detergent concentrations (SDS, Triton) increased its thermostability. Highest activity was attained with p-nitrophenyl butyrate, but the enzyme was also able to hydrolyze long chain fatty acid esters, as well as triolein. The primary sequence and predicted tridimensional structure of the enzyme are very similar to those of other Anoxybacillus and Geobacillus carboxyl esterases in a distinct, recently described lipase family. Est/Lip was highly enantioselective, with preference for the (S)-enantiomer of substrates

Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell

CRISPR/Cas9-Mediated Knock-Out of dUTPase in Mice Leads to Early Embryonic Lethality

Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2′-deoxyuridine 5′-triphosphate (dUTP) into 2′-deoxyuridine 5′-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/– animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut −/− embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements

Structural biology and regulation of protein import into the nucleus

Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g. importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g. importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals (cNLSs), recognized by the adaptor importin-α, and the PY-NLSs, recognized by transportin-1. Post-translational modifications, in particular phosphorylation, constitute key regulatory mechanisms operating in these pathways