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https://www.readbyqxmd.com/read/28077560/mechanistic-insights-into-type-i-toxin-antitoxin-systems-in-helicobacter-pylori-the-importance-of-mrna-folding-in-controlling-toxin-expression
#1
Hélène Arnion, Dursun Nizam Korkut, Sara Masachis Gelo, Sandrine Chabas, Jérémy Reignier, Isabelle Iost, Fabien Darfeuille
Type I toxin-antitoxin (TA) systems have been identified in a wide range of bacterial genomes. Here, we report the characterization of a new type I TA system present on the chromosome of the major human gastric pathogen, Helicobacter pylori We show that the aapA1 gene encodes a 30 amino acid peptide whose artificial expression in H. pylori induces cell death. The synthesis of this toxin is prevented by the transcription of an antitoxin RNA, named IsoA1, expressed on the opposite strand of the toxin gene. We further reveal additional layers of post-transcriptional regulation that control toxin expression: (i) transcription of the aapA1 gene generates a full-length transcript whose folding impedes translation (ii) a 3' end processing of this message generates a shorter transcript that, after a structural rearrangement, becomes translatable (iii) but this rearrangement also leads to the formation of two stem-loop structures allowing formation of an extended duplex with IsoA1 via kissing-loop interactions...
January 10, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28076346/cryo-em-structure-of-a-human-spliceosome-activated-for-step-2-of-splicing
#2
Karl Bertram, Dmitry E Agafonov, Wen-Ti Liu, Olexandr Dybkov, Cindy L Will, Klaus Hartmuth, Henning Urlaub, Berthold Kastner, Holger Stark, Reinhard Lu Hrmann
Spliceosome rearrangements facilitated by RNA helicase Prp16 before catalytic step 2 of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after Prp16 action (C*). The architecture of the catalytic U2-U6 RNP core of the human C* spliceosome is highly similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated (by ~20 Å) from the catalytic centre, and its position close to the U6 snRNA ACAGA box is stabilised by interactions with the Prp8 RNase H-like and Prp17 WD40 domains...
January 11, 2017: Nature
https://www.readbyqxmd.com/read/28065739/r-loop-depletion-by-over-expressed-rnase-h1-in-mouse-b-cells-increases-activation-induced-deaminase-access-to-the-transcribed-strand-without-altering-frequency-of-isotype-switching
#3
Robert W Maul, Hyongi Chon, Kiran Sakhuja, Susana M Cerritelli, Lina A Gugliotti, Patricia J Gearhart, Robert J Crouch
R-loops, three-strand structures consisting of mRNA hybridized to the complementary DNA and a single-stranded DNA loop, are formed in switch regions on the heavy-chain immunoglobulin locus. To determine if R-loops have a direct effect on any of the steps involved in isotype switching, we generated a transgenic mouse that over-expressed RNase H1, an enzyme that cleaves the RNA of RNA/DNA hybrids in B cells. R-loops in the switch μ region were depleted by 70% in ex vivo activated splenic B cells. Frequencies of isotype switching to IgG1, IgG2b, IgG2c, and IgG3 were the same as C57BL/6 control cells...
January 6, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28038925/rapid-monitoring-of-rna-degradation-activity-in%C3%A2-vivo-for-mammalian-cells
#4
Hidenori Tani, Hiroaki Sato, Masaki Torimura
We have developed a rapid fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA degradation activity in mammalian cells. In this technique, double-stranded RNA (dsRNA) fluorescent probes are used. The dsRNA fluorescent probes consist of a 5' fluorophore-labeled strand hybridized to a 3' quencher-labeled strand, and the fluorescent dye is quenched by a quencher dye. When the dsRNA is degraded by nascent RNases in cells, the fluorescence emission of the fluorophore is induced following the degradation of the double strands...
December 23, 2016: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28026819/-nitrobenzofuroxane-derivatives-as-dual-action-hiv-1-inhibitors
#5
S P Korolev, M A Pustovarova, A M Starosotnikov, M A Bastrakov, Yu Yu Agapkina, S A Shevelev, M B Gottikh
Human immunodeficiency virus first type (HIV-1) is a main cause of one of the most dangerous diseases, AIDS. The search for new inhibitors of the virus still remains an urgent task. One approach to suppress the HIV infection is to use a double-acting inhibitors, i.e. inhibitors directed to two stages of the viral life cycle. The catalytic domain of HIV-1 integrase has a similar spatial organization with ribonuclease (RNase H) domain of HIV-1 reverse transcriptase, and approach aimed to create HIV-1 integrase and RNase H double-acting is very promising...
November 2016: Biomedit︠s︡inskai︠a︡ Khimii︠a︡
https://www.readbyqxmd.com/read/28003490/lineage-a-betacoronavirus-ns2-proteins-and-homologous-torovirus-berne-pp1a-carboxyterminal-domain-are-phosphodiesterases-that-antagonize-activation-of-rnase-l
#6
Stephen A Goldstein, Joshua M Thornbrough, Rong Zhang, Babal K Jha, Yize Li, Ruth Elliott, Katherine Quiroz-Figueroa, Annie I Chen, Robert H Silverman, Susan R Weiss
: Viruses in the family Coronaviridae, with the Nidovirus order, are etiologic agents of a range of human and animal diseases, including both mild and severe respiratory disease in humans. These viruses encode conserved replicase and structural proteins, and more diverse accessory proteins in the 3' end of their genomes that often act as host cell antagonists. We have previously shown that 2', 5' phosphodiesterases (PDE) encoded by the prototypical Betacoronavirus, mouse hepatitis virus (MHV), Middle East respiratory syndrome-associated coronavirus antagonize the oligoadenylate - ribonuclease L (OAS-RNase L) pathway...
December 21, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27959588/exosome-mediated-telomere-instability-in-human-breast-epithelial-cancer-cells-after-x-irradiation
#7
Ammar H J Al-Mayah, Scott J Bright, Debbie A Bowler, Predrag Slijepcevic, Edwin Goodwin, Munira A Kadhim
In directly irradiating cells, telomere metabolism is altered and similar effects have been observed in nontargeted cells. Exosomes and their cargo play dominant roles in communicating radiation-induced bystander effects with end points related to DNA damage. Here we report novel evidence that exosomes are also responsible for inducing telomere-related bystander effects. Breast epithelial cancer cells were exposed to either 2 Gy X rays, or exposed to irradiated cell conditioned media (ICCM), or exosomes purified from ICCM...
December 13, 2016: Radiation Research
https://www.readbyqxmd.com/read/27938663/rnase-h-enables-efficient-repair-of-r-loop-induced-dna-damage
#8
Jeremy D Amon, Douglas Koshland
R-loops, three-stranded structures that form when transcripts hybridize to chromosomal DNA, are potent agents of genome instability. This instability has been explained by the ability of R-loops to induce DNA damage. Here, we show that persistent R-loops also compromise DNA repair. Depleting endogenous RNase H activity impairs R-loop removal in Saccharomyces cerevisiae, causing DNA damage that occurs preferentially in the repetitive ribosomal DNA locus (rDNA). We analyzed the repair kinetics of this damage and identified mutants that modulate repair...
December 10, 2016: ELife
https://www.readbyqxmd.com/read/27936595/physiological-mg-2-conditions-significantly-alter-the-inhibition-of-hiv-1-and-hiv-2-reverse-transcriptases-by-nucleoside-and-non-nucleoside-inhibitors-in-vitro
#9
Vasudevan Achuthan, Kamlendra Singh, Jeffrey J DeStefano
Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg(2+), whereas the free Mg(2+) concentration in cells is much lower. Artificially high Mg(2+) concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg(2+). At low concentrations of Mg(2+), NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg(2+), whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg(2+) concentrations...
December 27, 2016: Biochemistry
https://www.readbyqxmd.com/read/27899605/poly-a-specific-ribonuclease-regulates-the-processing-of-small-subunit-rrnas-in-human-cells
#10
Hideaki Ishikawa, Harunori Yoshikawa, Keiichi Izumikawa, Yutaka Miura, Masato Taoka, Yuko Nobe, Yoshio Yamauchi, Hiroshi Nakayama, Richard J Simpson, Toshiaki Isobe
Ribosome biogenesis occurs successively in the nucleolus, nucleoplasm, and cytoplasm. Maturation of the ribosomal small subunit is completed in the cytoplasm by incorporation of a particular class of ribosomal proteins and final cleavage of 18S-E pre-rRNA (18S-E). Here, we show that poly(A)-specific ribonuclease (PARN) participates in steps leading to 18S-E maturation in human cells. We found PARN as a novel component of the pre-40S particle pulled down with the pre-ribosome factor LTV1 or Bystin. Reverse pull-down analysis revealed that PARN is a constitutive component of the Bystin-associated pre-40S particle...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27899559/division-of-labor-among-mycobacterium-smegmatis-rnase-h-enzymes-rnase-h1-activity-of-rnha-or-rnhc-is-essential-for-growth-whereas-rnhb-and-rnha-guard-against-killing-by-hydrogen-peroxide-in-stationary-phase
#11
Richa Gupta, Debashree Chatterjee, Michael S Glickman, Stewart Shuman
RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27890900/in-vitro-and-in-vivo-biophysical-properties-of-oligonucleotides-containing-5-thio-nucleosides
#12
Md Ariful Islam, Reiko Waki, Aki Fujisaka, Kosuke Ramon Ito, Satoshi Obika
Phosphorothioate modification is one of the most widely investigated and promising chemical modifications in oligonucleotide (ON) based therapeutics. Structurally similar 5'-thio or phosphorothiolate-modified nucleotides, in which a 5'-bridging oxygen is replaced with a sulfur atom, are gaining importance for ON-based research. Several reports have been published describing the synthesis of 5'-thio-modified ONs but no detailed in vitro and in vivo data are available. Here, we report the synthesis of 5'-thio-modified 2'-deoxy-5-methylcytidine...
2016: Drug Discoveries & Therapeutics
https://www.readbyqxmd.com/read/27886596/an-end-point-method-based-on-graphene-oxide-for-rnase-h-analysis-and-inhibitors-screening
#13
Chuan Zhao, Jialong Fan, Lan Peng, Lijian Zhao, Chunyi Tong, Wei Wang, Bin Liu
As a highly conserved damage repair protein, RNase H can hydrolysis DNA-RNA heteroduplex endonucleolytically and cleave RNA-DNA junctions as well. In this study, we have developed an accurate and sensitive RNase H assay based on fluorophore-labeled chimeric substrate hydrolysis and the differential affinity of graphene oxide on RNA strand with different length. This end-point measurement method can detect RNase H in a range of 0.01 to 1 units /mL with a detection limit of 5.0×10(-3) units/ mL under optimal conditions...
April 15, 2017: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/27881652/inhibition-of-human-cytomegalovirus-pul89-terminase-subunit-blocks-virus-replication-and-genome-cleavage
#14
Yan Wang, Lili Mao, Jayakanth Kankanala, Zhengqiang Wang, Robert J Geraghty
: Human cytomegalovirus terminase complex cleaves the concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage and the domain responsible is reported to have an RNase H-like fold...
November 23, 2016: Journal of Virology
https://www.readbyqxmd.com/read/27881299/transient-rna-dna-hybrids-are-required-for-efficient-double-strand-break-repair
#15
Corina Ohle, Rafael Tesorero, Géza Schermann, Nikolay Dobrev, Irmgard Sinning, Tamás Fischer
RNA-DNA hybrids are a major internal cause of DNA damage within cells, and their degradation by RNase H enzymes is important for maintaining genomic stability. Here, we identified an unexpected role for RNA-DNA hybrids and RNase H enzymes in DNA repair. Using a site-specific DNA double-strand break (DSB) system in Schizosaccharomyces pombe, we showed that RNA-DNA hybrids form as part of the homologous-recombination (HR)-mediated DSB repair process and that RNase H enzymes are essential for their degradation and efficient completion of DNA repair...
November 3, 2016: Cell
https://www.readbyqxmd.com/read/27875905/identification-of-novel-resistance-related-polymorphisms-in-hiv-1-subtype-c-rt-connection-and-rnase-h-domains-from-patients-under-virological-failure-in-brazil
#16
Maria F M Barral, Arielly K P Sousa, André F Santos, Celina M Abreu, Amilcar Tanuri, Marcelo A Soares
Mutations in the connection and RNase H C-terminal reverse transcriptase (RT) domains of HIV-1 have been shown to impact drug resistance to RT inhibitors. However, their impact in the context of non-B subtypes has been poorly assessed. This study aimed to characterize resistance-related mutations in the C-terminal portions of RT in treatment-failing patients from southern Brazil, a region with endemic HIV-1 subtype C (HIV-1C). Viral RNA was isolated and reverse transcribed from 280 infected subjects, and genomic regions were analyzed by polymerase chain reaction, DNA sequencing, and phylogenetic analysis...
December 27, 2016: AIDS Research and Human Retroviruses
https://www.readbyqxmd.com/read/27830012/human-mesenchymal-stromal-cell-derived-extracellular-vesicles-alleviate-renal-ischemic-reperfusion-injury-and-enhance-angiogenesis-in-rats
#17
Xiangyu Zou, Di Gu, Xiaoyu Xing, Zhongliang Cheng, Dongliang Gong, Guangyuan Zhang, Yingjian Zhu
BACKGROUND: Mesenchymal stromal cells (MSCs) derived extracellular vesicles (EVs) were regarded as a potent medium for kidney injury repair and angiogenesis were regarded as an important step in tissue regeneration. However, the pro-angiogenesis effect of MSC-EVs in ischemia-reperfusion induced kidney injury and its potential mechanisms have yet to be determined. METHODS: EVs were isolated from the medium of human umbilical cord-derived MSCs (huMSCs) were injected in rats intravenously after unilateral kidney ischemia...
2016: American Journal of Translational Research
https://www.readbyqxmd.com/read/27825885/endonuclease-controlled-aggregation-of-gold-nanoparticles-for-the-ultrasensitive-detection-of-pathogenic-bacterial-dna
#18
Claire McVey, Fumin Huang, Christopher Elliott, Cuong Cao
The development of an ultrasensitive biosensor for the low-cost and on-site detection of pathogenic DNA could transform detection capabilities within food safety, environmental monitoring and clinical diagnosis. Herein, we present an innovative approach exploiting endonuclease-controlled aggregation of plasmonic gold nanoparticles (AuNPs) for label-free and ultrasensitive detection of bacterial DNA. The method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA...
October 27, 2016: Biosensors & Bioelectronics
https://www.readbyqxmd.com/read/27812578/pcr-free-multiple-ligase-reactions-and-probe-cleavages-for-the-snp-detection-of-kras-mutation-with-attomole-sensitivity
#19
Joong Hyun Kim
A method to produce multiple ligated primers without PCR for a target DNA containing a single point mutation is presented. A strand displacing hairpin was introduced into the reaction, enabling separation of the ligated primer target DNA duplex without any thermal denaturing process. The ligated product was cycled to allow cleavage of fluorescence labeled substrates for RNase H on gold nanoparticles, leading to target specific fluorescence amplification. As a result, 10 attomoles of target DNA containing a point mutation in the KRAS gene were detected...
November 4, 2016: Analyst
https://www.readbyqxmd.com/read/27799545/evolutionary-trend-toward-kinetic-stability-in-the-folding-trajectory-of-rnases-h
#20
Shion A Lim, Kathryn M Hart, Michael J Harms, Susan Marqusee
Proper folding of proteins is critical to producing the biological machinery essential for cellular function. The rates and energetics of a protein's folding process, which is described by its energy landscape, are encoded in the amino acid sequence. Over the course of evolution, this landscape must be maintained such that the protein folds and remains folded over a biologically relevant time scale. How exactly a protein's energy landscape is maintained or altered throughout evolution is unclear. To study how a protein's energy landscape changed over time, we characterized the folding trajectories of ancestral proteins of the ribonuclease H (RNase H) family using ancestral sequence reconstruction to access the evolutionary history between RNases H from mesophilic and thermophilic bacteria...
November 15, 2016: Proceedings of the National Academy of Sciences of the United States of America
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