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Rnase h

An Li, Shanzhong Gong, Kenneth A Johnson
Previous measurements of the rates of polymerization and pyrophosphate release with DNA templates showed that pyrophosphate (PPi) dissociation was fast following nucleotide incorporation so that it did not contribute to enzyme specificity (kcat/Km). Here, kinetic parameters governing nucleotide incorporation and PPi release were determined using an RNA template. Compared with a DNA template of the same sequence, the rate of chemistry increased by up to 10-fold (250 vs 24 s-1) while the rate of PPi release decreased to approximate 58 s-1, so that PPi release became the rate-limiting step...
October 24, 2016: Journal of Biological Chemistry
An Li, Jiawen Li, Kenneth A Johnson
HIV Reverse Transcriptase (RT) plays a central role in viral replication and requires coordination of both polymerase and RNase H activities. Although this coordination is crucial in viral replication, whether a DNA/RNA hybrid can simultaneously engage both active sites has yet to be determined since structural and kinetic analyses have provided contradictory results. Single nucleotide incorporation and RNase H cleavage were examined using pre-steady-state kinetics with global data analysis. The results revealed three interconverting RT-DNA/RNA species: 43% were active for both sites, 27% showed only polymerase activity, and the remaining 30% were nonproductive...
October 24, 2016: Journal of Biological Chemistry
Yan Guo, Jie Wu, Shilin Zhao, Fei Ye, Yinghao Su, Travis Clark, Quanhu Sheng, Brian Lehmann, Xiao-Ou Shu, Qiuyin Cai
Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N = 16). We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens. Results. Both rRNA depletion methods produced consistent data between the technical replicates...
2016: International Journal of Genomics
Dennis Walczyk, Markus Gößringer, Walter Rossmanith, Timofei S Zatsepin, Tatiana S Oretskaya, Roland K Hartmann
Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5'-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino or 2'-O-methyl substitutions...
October 18, 2016: Journal of Molecular Biology
Francesca Esposito, Ilaria Carli, Claudia Del Vecchio, Lijia Xu, Angela Corona, Nicole Grandi, Dario Piano, Elias Maccioni, Simona Distinto, Cristina Parolin, Enzo Tramontano
BACKGROUND: Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with new mode of action are still needed. An innovative approach is aimed to identify dual HIV-1 inhibitors, small molecules that can inhibit two viral functions at the same time. Rhubarb, originated from Rheum palmatum L. and Rheum officinale Baill., is one of the earliest and most commonly used medicinal plants in Traditional Chinese Medicine (TCM) practice. We wanted to explore TCM for the identification of new chemical scaffolds with dual action abilities against HIV-1...
November 15, 2016: Phytomedicine: International Journal of Phytotherapy and Phytopharmacology
Sijia Wu, Xinman Zhang, Jiuqiang Han
[This corrects the article DOI: 10.1371/journal.pone.0161913.].
2016: PloS One
Magnolia Bostick, Nathalie Bolduc, Alisa Lehman, Andrew Farmer
Next-generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA-seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non-coding RNAs. Commonly used methods for generating strand-specific RNA-seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean-up steps, making them time-intensive, insensitive, and unsuitable for processing several samples simultaneously...
October 10, 2016: Current Protocols in Molecular Biology
David J Boerema, Valentina A Tereshko, Junliang Zhang, Stephen B H Kent
In this paper, we have used total chemical synthesis of RNase A analogues in order to probe the molecular basis of enzyme catalysis. Our goal was to obligately fill the adenine-binding pocket on the enzyme molecule, and to thus pre-orient the imidazole side chain of His(119) in its catalytically productive orientation. Two designed analogues of the RNase A protein molecule that contained an adenine moiety covalently bound to distinct amino acid side chains adjacent to the adenine binding pocket were prepared...
September 21, 2016: Organic & Biomolecular Chemistry
Baofeng Zhang, Michael P D'Erasmo, Ryan P Murelli, Emilio Gallicchio
We report the results of a binding free energy-based virtual screening campaign of a library of 77 α-hydroxytropolone derivatives against the challenging RNase H active site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus-1. Multiple protonation states, rotamer states, and binding modalities of each compound were individually evaluated. The work involved more than 300 individual absolute alchemical binding free energy parallel molecular dynamics calculations and over 1 million CPU hours on national computing clusters and a local campus computational grid...
September 30, 2016: ACS Omega
Huning Jia, Ying Bu, Bingjie Zou, Jianping Wang, Shalen Kumar, Janet L Pitman, Guohua Zhou, Qinxin Song
Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA...
September 30, 2016: Analyst
Xiao Wang, Belete Teferedegne, Kenneth Shatzkes, Wei Tu, Haruhiko Murata
Using purified reaction components, a commercial monoclonal antibody (Ab) specific to RNase inhibitor (RI) was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNase A, RI, or a combination of the above. Following incubation for 1 h at 22 °C or 37 °C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation...
December 2016: Data in Brief
Agnieszka Ruszkowska, Elzbieta Lenartowicz, Walter N Moss, Ryszard Kierzek, Elzbieta Kierzek
The influenza A virus genome is comprised of eight negative-sense viral (v)RNA segments. The seventh segment of the genome encodes two essential viral proteins and is specifically packaged alongside the other seven vRNAs. To gain insight into the possible roles of RNA structure both within and without virions, a secondary structure model of a naked (protein-free) segment 7 vRNA has been determined using chemical mapping and thermodynamic energy minimization. The proposed structure model was validated using microarray mapping, RNase H cleavage and comparative sequence analysis...
September 30, 2016: Biochemical Journal
Alexandra East-Seletsky, Mitchell R O'Connell, Spencer C Knight, David Burstein, Jamie H D Cate, Robert Tjian, Jennifer A Doudna
Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear...
September 26, 2016: Nature
Keith W Vance
Capture Hybridization Analysis of RNA Targets (CHART) has recently been developed to map the genome-wide binding profile of chromatin-associated RNAs. This protocol uses a small number of 22-28 nucleotide biotinylated antisense oligonucleotides, complementary to regions of the target RNA that are accessible for hybridization, to purify RNAs from a cross-linked chromatin extract. RNA-chromatin complexes are next immobilized on beads, washed, and specifically eluted using RNase H. Associated genomic DNA is then sequenced using high-throughput sequencing technologies and mapped to the genome to identify RNA-chromatin associations on a large scale...
2017: Methods in Molecular Biology
Akira Sassa, Melike Ҫağlayan, Yesenia Rodriguez, William A Beard, Samuel H Wilson, Takehiko Nohmi, Masamitsu Honma, Manabu Yasui
Numerous ribonucleotides are incorporated into the genome during DNA replication. Oxidized ribonucleotides can also be erroneously incorporated into DNA. Embedded ribonucleotides destabilize the structure of DNA and retard DNA synthesis by DNA polymerases (pols), leading to genomic instability. Mammalian cells possess translesion DNA synthesis (TLS) pols that bypass DNA damage. The mechanism of TLS and repair of oxidized ribonucleotides remains to be elucidated. To address this, we analyzed the miscoding properties of the ribonucleotides riboguanosine (rG) and 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG) during TLS catalyzed by the human TLS pols κ and η in vitro...
September 22, 2016: Journal of Biological Chemistry
Angela Corona, Francesco Saverio di Leva, Giuseppe Rigogliuso, Luca Pescatori, Valentina Noemi Madia, Frederic Subra, Olivier Delelis, Francesca Esposito, Marta Cadeddu, Roberta Costi, Sandro Cosconati, Ettore Novellino, Roberto di Santo, Enzo Tramontano
HIV-1 integrase (IN) inhibitors are one of the most recent innovations in the treatment of HIV infection. The selection of drug resistance viral strains is however a still open issue requiring constant efforts to identify new anti-HIV-1 drugs. Pyrrolyl diketo acid (DKA) derivatives inhibit HIV-1 replication by interacting with the Mg(2+) cofactors within the HIV-1 IN active site or within the HIV-1 reverse-transcriptase associated ribonuclease H (RNase H) active site. While the interaction mode of pyrrolyl DKAs with the RNase H active site has been recently reported and substantiated by mutagenesis experiments, their interaction within the IN active site still lacks a detailed understanding...
October 2016: Antiviral Research
Pradeep S Pallan, Thazha P Prakash, Arnie R de Leon, Martin Egli
RNase H1 cleaves the RNA strand of RNA:DNA hybrids. Replacement of RNA 2'-hydroxyls by fluorine (FRNA) is commonly used to stabilize aptamers and siRNAs. However, FRNA:DNA hybrids fail to elicit RNase H activity. The underlying reasons are unclear, as 2'-OH groups are not directly involved in cleavage. We determined the crystal structure of Bacillus halodurans RNase H bound to a FRNA:DNA hybrid. The structure points to dynamic (slippage of the FRNA:DNA hybrid relative to the enzyme), geometric (different curvatures of FRNA:DNA and RNA:DNA hybrids), and electronic reasons (Mg(2+) absent from the active site of the FRNA:DNA complex) for the loss of RNaseH activity...
September 27, 2016: Biochemistry
Anirban Kar, Smaranda Willcox, Jack D Griffith
The formation of DNA loops at chromosome ends (t-loops) and the transcription of telomeres producing G-rich RNA (TERRA) represent two central features of telomeres. To explore a possible link between them we employed artificial human telomeres containing long arrays of TTAGGG repeats flanked by the T7 or T3 promoters. Transcription of these DNAs generates a high frequency of t-loops within individual molecules and homologous recombination events between different DNAs at their telomeric sequences. T-loop formation does not require a single strand overhang, arguing that both terminal strands insert into the preceding duplex...
September 7, 2016: Nucleic Acids Research
Xianbin Yang
The oligodeoxynucleotide phosphorodithioate modification (PS2-ODN) uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphordiester backbone linkage. Like a natural phosphodiester ODN backbone linkage, a PS2-modified backbone linkage is achiral at phosphorus. PS2-ODNs are highly stable to nucleases and numerous in vitro assays have demonstrated their biological activity. For example, PS2-ODNs activated RNase H in vitro, strongly inhibited human immunodeficiency virus (HIV) reverse transcriptase, induced B-cell proliferation and differentiation, and bound to protein targets in the form of PS2-aptamers (thioaptamers)...
2016: Current Protocols in Nucleic Acid Chemistry
Sijia Wu, Xinman Zhang, Jiuqiang Han
RNase H (RNH) is a pivotal domain in retrovirus to cleave the DNA-RNA hybrid for continuing retroviral replication. The crucial role indicates that RNH is a promising drug target for therapeutic intervention. However, annotated RNHs in UniProtKB database have still been insufficient for a good understanding of their statistical characteristics so far. In this work, a computational RNH model was proposed to annotate new putative RNHs (np-RNHs) in the retroviruses. It basically predicts RNH domains through recognizing their start and end sites separately with SVM method...
2016: PloS One
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