Synthesis of a universal 5-nitroindole ribonucleotide and incorporation into RNA by a viral RNA-dependent RNA polymerase.
Ontology highlight
ABSTRACT:
SUBMITTER: Harki DA
PROVIDER: S-EPMC2194811 | biostudies-literature | 2007 Aug
REPOSITORIES: biostudies-literature
Publications
Synthesis of a universal 5-nitroindole ribonucleotide and incorporation into RNA by a viral RNA-dependent RNA polymerase.
Harki Daniel A DA Graci Jason D JD Edathil Jocelyn P JP Castro Christian C Cameron Craig E CE Peterson Blake R BR
Chembiochem : a European journal of chemical biology 20070801 12
Similar Datasets
Project description:Fluorescent RNA is an important analytical tool in medical diagnostics, RNA cytochemistry, and RNA aptamer development. We have synthesized the fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP) and tested it as substrate for T7 RNA polymerase in transcription reactions, a convenient route for generating RNA in vitro. When transcribing a guanine, T7 RNA polymerase incorporates tCTP with 2-fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC. Remarkably, T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors <10, T7 RNA polymerase discriminates against tC-A base pair formation by factors of 40 and 300 when operating in the elongation and initiation mode, respectively. These catalytic properties make T7 RNA polymerase an ideal tool for synthesizing large fluorescent RNA, as we demonstrated by generating a approximately 800 nucleotide RNA in which every cytosine was replaced with tC.
| S-EPMC2819109 | biostudies-literature
Project description:Ribonucleotides (rNs) incorporated in the genome by DNA polymerases (Pols) are removed by RNase H2. Cytidine and guanosine preferentially accumulate over the other rNs. Here we show that human Pol η can incorporate cytidine monophosphate (rCMP) opposite guanine, 8-oxo-7,8-dihydroguanine, 8-methyl-2΄-deoxyguanosine and a cisplatin intrastrand guanine crosslink (cis-PtGG), while it cannot bypass a 3-methylcytidine or an abasic site with rNs as substrates. Pol η is also capable of synthesizing polyribonucleotide chains, and its activity is enhanced by its auxiliary factor DNA Pol δ interacting protein 2 (PolDIP2). Human RNase H2 removes cytidine and guanosine less efficiently than the other rNs and incorporation of rCMP opposite DNA lesions further reduces the efficiency of RNase H2. Experiments with XP-V cell extracts indicate Pol η as the major basis of rCMP incorporation opposite cis-PtGG. These results suggest that translesion synthesis by Pol η can contribute to the accumulation of rCMP in the genome, particularly opposite modified guanines.
| S-EPMC5389505 | biostudies-literature
Project description:The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has caused a global pandemic. The SARS-CoV-2 RNA genome is replicated by a conserved “core” replication-transcription complex (RTC) containing an error-prone RNA-dependent RNA polymerase holoenzyme (holo-RdRp, nsp12-nsp7-nsp8) and a RNA proofreading nuclease (nsp14-nsp10). Although structures and functions of SARS-CoV-2 holo-RdRp have been extensively studied and ribonucleotide-analog inhibitors, such as Remdesivir, have been treated for COVID-19 patients, the substrate and nucleotide specificity of SARS-CoV-2 holo-RdRp remain unknown. Here, our biochemical analysis of SARS-CoV-2 holo-RdRp reveals that it has a robust DNA-dependent RNA polymerase activity, in addition to its intrinsic RNA-dependent RNA polymerase activity. Strikingly, SARS-CoV-2 holo-RdRp fully extends RNAs with a low-fidelity even when only ATP and pyrimidine nucleotides, in particular CTP, are provided. This ATP-dependent error-prone ribonucleotide incorporation by SARS-CoV-2 holo-RdRp resists excision by the RNA proofreading nuclease in vitro. Our collective results suggest that a physiological concentration of ATP likely contributes to promoting the error-prone incorporation of ribonucleotides and ribonucleotide-analogs by SARS-CoV-2 holo-RdRp and provide a useful foundation to develop ribonucleotide analogs as an effective therapeutic strategy to combat coronavirus-mediated outbreak.
| S-EPMC9344795 | biostudies-literature
Project description:Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.
| S-EPMC4759156 | biostudies-literature
Project description:There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5'-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5'-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5'-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5'-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104 This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.
| S-EPMC5786792 | biostudies-literature
Project description:Fluorescent nucleobase analogues that respond to changes in their microenvironment are valuable for studying RNA structure, dynamics, and recognition. The most commonly used fluorescent ribonucleoside is 2-aminopurine, a highly responsive purine analogue. Responsive isosteric fluorescent pyrimidine analogues are, however, rare. Appending five-membered aromatic heterocycles at the 5-position on a pyrimidine core has recently been found to provide a family of responsive fluorescent nucleoside analogues with emission in the visible range. To explore the potential utility of this chromophore for studying RNA-ligand interactions, an efficient incorporation method is necessary. Here we describe the synthesis of the furan-containing ribonucleoside and its triphosphate, as well as their basic photophysical characteristics. We demonstrate that T7 RNA polymerase accepts this fluorescent ribonucleoside triphosphate as a substrate in in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides, generating fluorescent constructs. Furthermore, we utilize this triphosphate for the enzymatic preparation of a fluorescent bacterial A-site, an RNA construct of potential therapeutic utility. We show that the binding of this RNA target to aminoglycoside antibiotics, its cognate ligands, can be effectively monitored by fluorescence spectroscopy. These observations are significant since isosteric emissive U derivatives are scarce and the trivial synthesis and effective enzymatic incorporation of the furan-containing U triphosphate make it accessible to the biophysical community.
| S-EPMC2517582 | biostudies-literature
Project description:Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host-pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein--its binding partner within replication complexes--leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity.
| S-EPMC3273391 | biostudies-literature
Project description:Ribonucleoside triphosphates (rNTPs) are frequently incorporated during DNA synthesis by replicative DNA polymerases (DNAPs), and once incorporated are not efficiently edited by the DNAP exonucleolytic function. We examined the kinetic mechanisms that govern selection of complementary deoxyribonucleoside triphosphates (dNTPs) over complementary rNTPs and that govern the probability of a complementary ribonucleotide at the primer terminus escaping exonucleolytic editing and becoming stably incorporated. We studied the quantitative responses of individual Φ29 DNAP complexes to ribonucleotides using a kinetic framework, based on our prior work, in which transfer of the primer strand from the polymerase to exonuclease site occurs prior to translocation, and translocation precedes dNTP binding. We determined transition rates between the pre-translocation and post-translocation states, between the polymerase and exonuclease sites, and for dNTP or rNTP binding, with single-nucleotide spatial precision and submillisecond temporal resolution, from ionic current time traces recorded when individual DNAP complexes are held atop a nanopore in an electric field. The predominant response to the presence of a ribonucleotide in Φ29 DNAP complexes before and after covalent incorporation is significant destabilization, relative to the presence of a deoxyribonucleotide. This destabilization is manifested in the post-translocation state prior to incorporation as a substantially higher rNTP dissociation rate and manifested in the pre-translocation state after incorporation as rate increases for both primer strand transfer to the exonuclease site and the forward translocation, with the probability of editing not directly increased. In the post-translocation state, the primer terminal 2'-OH group also destabilizes dNTP binding.
| S-EPMC4283934 | biostudies-literature
Project description:While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase μ (Pol μ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol μ with a ribonucleotide bound at the active site. These structures reveal that Pol μ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol μ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol μ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations.
| S-EPMC5587726 | biostudies-literature
Project description:Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5'-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2'-C-methyl- and 2'-C-ethynyl-substituted analog 5'-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.
| S-EPMC5328514 | biostudies-literature