Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

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The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses

Molecules ◽

10.3390/molecules26247532 ◽

2021 ◽

Vol 26 (24) ◽

pp. 7532

Author(s):

Clive R. Bagshaw ◽

Jendrik Hentschel ◽

Michael D. Stone

Keyword(s):

Time Course ◽

Systematic Errors ◽

Reverse Transcriptases ◽

Rigorous Approach ◽

Translocation Event ◽

Nucleotide Incorporation ◽

Standard Procedures ◽

Nucleotide Addition ◽

Basic Reaction ◽

Linear Chromosomes

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3′ end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase.

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Base pairing, structural and functional insights into N4-methylcytidine (m4C) and N4,N4-dimethylcytidine (m42C) modified RNA

Nucleic Acids Research ◽

10.1093/nar/gkaa737 ◽

2020 ◽

Vol 48 (18) ◽

pp. 10087-10100

Author(s):

Song Mao ◽

Bartosz Sekula ◽

Milosz Ruszkowski ◽

Srivathsan V Ranganathan ◽

Phensinee Haruehanroengra ◽

...

Keyword(s):

Base Pairing ◽

Reverse Transcriptases ◽

X Ray Crystallography ◽

Coding Efficiency ◽

Gene Replication ◽

Nucleotide Incorporation ◽

Duplex Stability ◽

Rna Duplexes ◽

Fine Tune ◽

Hiv 1

Abstract The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

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tiRNAs & tRFs Biogenesis and Regulation of Diseases: A Review

Current Medicinal Chemistry ◽

10.2174/0929867326666190124123831 ◽

2019 ◽

Vol 26 (31) ◽

pp. 5849-5861 ◽

Cited By ~ 3

Author(s):

Pan Jiang ◽

Feng Yan

Keyword(s):

Gene Expression ◽

Protein Synthesis ◽

Metabolic Diseases ◽

Viral Infections ◽

Regulatory Mechanisms ◽

Biological Functions ◽

Reverse Transcriptases ◽

Dna Damage Responses ◽

Potential Applications ◽

Viral Reverse Transcriptases

tiRNAs & tRFs are a class of small molecular noncoding tRNA derived from precise processing of mature or precursor tRNAs. Most tiRNAs & tRFs described originate from nucleus-encoded tRNAs, and only a few tiRNAs and tRFs have been reported. They have been suggested to play important roles in inhibiting protein synthesis, regulating gene expression, priming viral reverse transcriptases, and the modulation of DNA damage responses. However, the regulatory mechanisms and potential function of tiRNAs & tRFs remain poorly understood. This review aims to describe tiRNAs & tRFs, including their structure, biological functions and subcellular localization. The regulatory roles of tiRNAs & tRFs in translation, neurodegeneration, metabolic diseases, viral infections, and carcinogenesis are also discussed in detail. Finally, the potential applications of these noncoding tRNAs as biomarkers and gene regulators in different diseases is also highlighted.

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TCF19 Impacts a Network of Inflammatory and DNA Damage Response Genes in the Pancreatic β-Cell

Metabolites ◽

10.3390/metabo11080513 ◽

2021 ◽

Vol 11 (8) ◽

pp. 513

Author(s):

Grace H. Yang ◽

Danielle A. Fontaine ◽

Sukanya Lodh ◽

Joseph T. Blumer ◽

Avtar Roopra ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Damage Response ◽

Association Studies ◽

Cell Cycle Gene ◽

Genome Wide Association Studies ◽

Β Cell ◽

Nucleotide Incorporation ◽

Damage Response ◽

The Impact

Transcription factor 19 (TCF19) is a gene associated with type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that Tcf19 knockdown impairs β-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 β-cells and human islets on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNA-seq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in viral responses, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the β-cell and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways can provide potential targets for improving β-cell survival.

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Snapshots of a Non-Canonical RdRP in Action

Viruses ◽

10.3390/v13071260 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1260

Author(s):

Diego S. Ferrero ◽

Michela Falqui ◽

Nuria Verdaguer

Keyword(s):

Metal Ion ◽

Rna Viruses ◽

Ion Binding ◽

Genome Replication ◽

Metal Ion Binding ◽

Insect Virus ◽

X Ray ◽

Template Strand ◽

Nucleotide Incorporation ◽

Elongation Process

RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication and transcription. The closed “right hand” architecture of RdRPs encircles seven conserved structural motifs (A to G) that regulate the polymerization activity. The four palm motifs, arranged in the sequential order A to D, are common to all known template dependent polynucleotide polymerases, with motifs A and C containing the catalytic aspartic acid residues. Exceptions to this design have been reported in members of the Permutotetraviridae and Birnaviridae families of positive single stranded (+ss) and double-stranded (ds) RNA viruses, respectively. In these enzymes, motif C is located upstream of motif A, displaying a permuted C–A–B–D connectivity. Here we study the details of the replication elongation process in the non-canonical RdRP of the Thosea asigna virus (TaV), an insect virus from the Permutatetraviridae family. We report the X-ray structures of three replicative complexes of the TaV polymerase obtained with an RNA template-primer in the absence and in the presence of incoming rNTPs. The structures captured different replication events and allowed to define the critical interactions involved in: (i) the positioning of the acceptor base of the template strand, (ii) the positioning of the 3’-OH group of the primer nucleotide during RNA replication and (iii) the recognition and positioning of the incoming nucleotide. Structural comparisons unveiled a closure of the active site on the RNA template-primer binding, before rNTP entry. This conformational rearrangement that also includes the repositioning of the motif A aspartate for the catalytic reaction to take place is maintained on rNTP and metal ion binding and after nucleotide incorporation, before translocation.

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