Structural Insight into Polymerase Mechanism via a Chiral Center Generated with a Single Selenium Atom.

DNA synthesis catalyzed by DNA polymerase is essential for all life forms, and phosphodiester bond formation with phosphorus center inversion is a key step in this process. Herein, by using a single-selenium-atom-modified dNTP probe, we report a novel strategy to visualize the reaction stereochemistry and catalysis. We capture the before- and after-reaction states and provide explicit evidence of the center inversion and in-line attacking SN2 mechanism of DNA polymerization, while solving the diastereomer absolute configurations. Further, our kinetic and thermodynamic studies demonstrate that in the presence of Mg2+ ions (or Mn2+), the binding affinity (Km) and reaction selectivity (kcat/Km) of dGTPαSe-Rp were 51.1-fold (or 19.5-fold) stronger and 21.8-fold (or 11.3-fold) higher than those of dGTPαSe-Sp, respectively, indicating that the diastereomeric Se-Sp atom was quite disruptive of the binding and catalysis. Our findings reveal that the third metal ion is much more critical than the other two metal ions in both substrate recognition and bond formation, providing insights into how to better design the polymerase inhibitors and discover the therapeutics.

Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.

One-Enzyme RTX-PCR for the Detection of RNA Viruses from Multiple Virus Genera and Crop Plants.

Reverse transcription PCR (RT-PCR) is a popular method for detecting RNA viruses in plants. RT-PCR is usually performed in a classical two-step procedure: in the first step, cDNA is synthesized by reverse transcriptase (RT), followed by PCR amplification by a thermostable polymerase in a separate tube in the second step. However, one-step kits containing multiple enzymes optimized for RT and PCR amplification in a single tube can also be used. Here, we describe an RT-PCR single-enzyme assay based on an RTX DNA polymerase that has both RT and polymerase activities. The expression plasmid pET_RTX_(exo-) was transferred to various E. coli genotypes that either compensated for codon bias (Rosetta-gami 2) or contained additional chaperones to promote solubility (BL21 (DE3) with plasmids pKJE8 or pTf2). The RTX enzyme was then purified and used for the RT-PCR assay. Several purified plant viruses (TMV, PVX, and PVY) were used to determine the efficiency of the assay compared to a commercial one-step RT-PCR kit. The RT-PCR assay with the RTX enzyme was validated for the detection of viruses from different genera using both total RNA and crude sap from infected plants. The detection endpoint of RTX-PCR for purified TMV was estimated to be approximately 0.01 pg of the whole virus per 25 µL reaction, corresponding to 6 virus particles/µL. Interestingly, the endpoint for detection of TMV from crude sap was also 0.01 pg per reaction in simulated crude plant extracts. The longest RNA fragment that could be amplified in a one-tube arrangement was 2379 bp long. The longest DNA fragment that could be amplified during a 10s extension was 6899 bp long. In total, we were able to detect 13 viruses from 11 genera using RTX-PCR. For each virus, two to three specific fragments were amplified. The RT-PCR assay using the RTX enzyme described here is a very robust, inexpensive, rapid, easy to perform, and sensitive single-enzyme assay for the detection of plant viruses.

Current Drugs to Treat Infections with Herpes Simplex Viruses-1 and -2.

Herpes simplex viruses-1 and -2 (HSV-1 and -2) are two of the three human alphaherpesviruses that cause infections worldwide. Since both viruses can be acquired in the absence of visible signs and symptoms, yet still result in lifelong infection, it is imperative that we provide interventions to keep them at bay, especially in immunocompromised patients. While numerous experimental vaccines are under consideration, current intervention consists solely of antiviral chemotherapeutic agents. This review explores all of the clinically approved drugs used to prevent the worst sequelae of recurrent outbreaks by these viruses.

Effects of Chemopreventive Natural Compounds on the Accuracy of 8-oxo-7,8-dihydro-2'-deoxyguanosine Translesion Synthesis.

Translesion synthesis is a DNA damage tolerance mechanism that relies on a series of specialized DNA polymerases able to bypass a lesion on a DNA template strand during replication or post-repair synthesis. Specialized translesion synthesis DNA polymerases pursue replication by inserting a base opposite to this lesion, correctly or incorrectly depending on the lesion nature, involved DNA polymerase(s), sequence context, and still unknown factors. To measure the correct or mutagenic outcome of 8-oxo-7,8-dihydro-2'-deoxyguanosine bypass by translesion synthesis, a primer-extension assay was performed in vitro on a template DNA bearing this lesion in the presence of nuclear proteins extracted from human intestinal epithelial cells (FHs 74 Int cell line); the reaction products were analyzed by both denaturing capillary electrophoresis (to measure the yield of translesion elongation) and pyrosequencing (to determine the identity of the nucleotide inserted in front of the lesion). The influence of 14 natural polyphenols on the correct or mutagenic outcome of translesion synthesis through 8-oxo-7,8-dihydro-2'-deoxyguanosine was then evaluated in 2 experimental conditions by adding the polyphenol either (i) to the reaction mix during the primer extension assay; or (ii) to the culture medium, 24 h before cell harvest and nuclear proteins extraction. Most of the tested polyphenols significantly influenced the outcome of translesion synthesis, either through an error-free (apigenin, baicalein, sakuranetin, and myricetin) or a mutagenic pathway (epicatechin, chalcone, genistein, magnolol, and honokiol).

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Thiophene-linked tetramethylbodipy-labeled nucleotide for viscosity-sensitive oligonucleotide probes of hybridization and protein-DNA interactions.

Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

Breaking Down the Barriers to a Natural Antiviral Agent: Antiviral Activity and Molecular Docking of Erythrina speciosa Extract, Fractions, and the Major Compound.

The in vitro cytotoxic activity in Vero cells and the antiviral activity of Erythrina speciosa methanol extract, fractions, and isolated vitexin were studied. The results revealed that E. speciosa leaves ethyl acetate soluble fraction of the methanol extract (ESLE) was the most active against herpes simplex virus type 1 (HSV-1). Bioactivity-guided fractionation was performed on ESLE to isolate the bioactive compounds responsible for this activity. One sub-fraction from ESLE (ESLE IV) showed the highest activity against HSV-1 and Hepatitis A HAV-H10 viruses. Vitexin isolated from ESLE VI exhibited a significant antiviral activity (EC50 =35±2.7 and 18±3.3 µg/mL against HAV-H10 and HSV-1 virus, respectively), which was notably greater than the activity of the extract and the fractions. Molecular docking studies were carried out to explore the molecular interactions of vitexin with different macromolecular targets. Analysis of the in silico data together with the in vitro studies validated the antiviral activity associated with vitexin. These outcomes indicated that vitexin is a potential candidate to be utilized commendably in lead optimization for the development of antiviral agents.

Extensive free-energy simulations identify water as the base in nucleotide addition by DNA polymerase.

Transphosphorylation of nucleotide triphosphates is the central reaction in DNA replication by DNA polymerase as well as many other biological processes. Despite its importance, the microscopic chemical mechanism of transphosphorylation of nucleotide triphosphates is, in most cases, unknown. Here we use extensive simulations of DNA polymerase η to test mechanistic hypotheses. We systematically survey the reactive space by calculating 2D free-energy surfaces for 10 different plausible mechanisms that have been proposed. We supplement these free-energy surfaces with calculations of pKa for a number of potentially acidic protons in different states relevant to the catalytic cycle. We find that among all of the conditions that we test, the smallest activation barrier occurs for a reaction where a Mg2+-coordinated water deprotonates the nucleophilic 3'-OH, and this deprotonation is concerted with the phosphoryl transfer. The presence of a third Mg2+ in the active site lowers the activation barrier for the water-as-base mechanism, as does protonation of the pyrophosphate leaving group, which is consistent with general acid catalysis. The results demonstrate the value of simulations, when used in conjunction with experimental data, to help establish a microscopic chemical mechanism in a complex environment.

Virtual Pharmacophore Screening Identifies Small-Molecule Inhibitors of the Rev1-CT/RIR Protein-Protein Interaction.

Translesion synthesis (TLS) has emerged as a mechanism through which several forms of cancer develop acquired resistance to first-line genotoxic chemotherapies by allowing replication to continue in the presence of damaged DNA. Small molecules that inhibit TLS hold promise as a novel class of anticancer agents that can serve to enhance the efficacy of these front-line therapies. We previously used a structure-based rational design approach to identify the phenazopyridine scaffold as an inhibitor of TLS that functions by disrupting the protein-protein interaction (PPI) between the C-terminal domain of the TLS DNA polymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) of other TLS DNA polymerases. To continue the identification of small molecules that disrupt the Rev1-CT/RIR PPI, we generated a pharmacophore model based on the phenazopyridine scaffold and used it in a structure-based virtual screen. In vitro analysis of promising hits identified several new chemotypes with the ability to disrupt this key TLS PPI. In addition, several of these compounds were found to enhance the efficacy of cisplatin in cultured cells, highlighting their anti-TLS potential.

Synthesis of Selenium-Triphosphates (dNTPαSe) for More Specific DNA Polymerization.

2'-Deoxynucleoside 5'-(alpha-P-seleno)-triphosphates (dNTPαSe) have been conveniently synthesized using a protection-free, one-pot strategy. One of two diastereomers of each dNTPαSe can be efficiently recognized by DNA polymerases, while the other is neither a substrate nor an inhibitor. Furthermore, this Se-atom modification can significantly inhibit non-specific DNA polymerization caused by mis-priming. Se-DNAs amplified with dNTPαSe via polymerase chain reaction have sequences identical to the corresponding native DNA. In conclusion, a simple strategy for more specific DNA polymerization has been established by replacing native dNTPs with dNTPαSe.

An aptamer biosensor for leukemia marker mRNA detection based on polymerase-assisted signal amplification and aggregation of illuminator.

A novel electrochemical luminescence (ECL) aptamer biosensor via polymerase amplification is constructed for label-free detection of leukemia marker mRNA (miR-16). In order to achieve the ultrasensitive detection of the target mRNA, the cyclic target chain displacement polymerization of leukemia marker mRNA assisted with Klenow fragment of DNA polymerase is employed. The determination is carried out by recording the ECL emission of pyridine ruthenium (Ru(bpy)32+) complexes embedded into the assistance DNA (ADNA) loaded on the nanogold surface, after the hybridization reaction between the probe DNA (PDNA) and the remaining sequence of the CP's stem part, and the formation of a core-shell sun-like structure. The mercapto-modified capture DNA (CP) is immobilized on the surface of a magneto-controlled glassy carbon electrode by Au-S bond. The CP is opened and hybridized with the target mRNA to form double-stranded DNA. In the presence of polymerase, primer DNA, and bases (dNTPs), the primer chain gets access to its complementary sequence of the stem part and then triggers a polymerization of the DNA strand, leading to the release of mRNA and starting the next polymerization cycle. Finally, the composite of PDNA-covered and ADNA-covered (embedded with Ru(bpy)32+) gold nanoparticles (hereafter called AuNPs@(PDNA+ADNA-Ru(bpy)32+) is added, and the ECL intensity is recorded. Because of the polymerization cycle and the aggregation of the illuminator of Ru(bpy)32+, the detected signal is amplified significantly. The results showed that the corresponding ECL signal has a good linear relationship with a logarithm of target mRNA concentration in the range of 1 × 10-16 to 1 × 10-7 mol/L, with a detection limit of 4.3 × 10-17 mol/L. The mRNA spiked in the human serum sample is determined, and the recoveries are from 97.2 to 102.0%. This sensor demonstrates good selectivity, stability, and reproducibility. Graphical abstract ᅟ.

DNA repair in plant mitochondria - a complete base excision repair pathway in potato tuber mitochondria.

Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg2+ dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA.

Revealing the role of the product metal in DNA polymerase ß catalysis.

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase ß, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.

The Establishment of an Assay to Measure DNA Polymerase-Catalyzed Repair of UVB-Induced DNA Damage in Skin Cells and Screening of DNA Polymerase Enhancers from Medicinal Plants.

An in vitro assay method was established to measure the activity of cellular DNA polymerases (Pols) in cultured normal human epidermal keratinocytes (NHEKs) by modifying Pol inhibitor activity. Ultraviolet (UV) irradiation enhanced the activity of Pols, especially DNA repair-related Pols, in the cell extracts of NHEKs. The optimal ultraviolet B (UVB) exposure dose and culture time to upregulate Pols activity was 100 mJ/cm² and 4-h incubation, respectively. We screened eight extracts of medicinal plants for enhancement of UVB-exposed cellular Pols activity using NHEKs, and found that rose myrtle was the strongest Pols enhancer. A Pols' enhancement compound was purified from an 80% ethanol extract of rose myrtle, and piceatannol was isolated by spectroscopic analysis. Induction of Pol activity involved synergy between UVB irradiation and rose myrtle extract and/or piceatannol. Both the extract and piceatannol reduced UVB-induced cyclobutane pyrimidine dimer production, and prevented UVB-induced cytotoxicity. These results indicate that rose myrtle extract and piceatannol, its component, are potential photo-protective candidates for UV-induced skin damage.

Rose myrtle (Rhodomyrtus tomentosa) extract and its component, piceatannol, enhance the activity of DNA polymerase and suppress the inflammatory response elicited by UVB­induced DNA damage in skin cells.

A number of naturally occurring agents are hypothesized to protect against ultraviolet (UV)­induced skin damage. The present study screened >50 plant extracts for inhibitors of UVB­induced cytotoxicity, using cultured normal human epidermal keratinocytes (NHEK), and identified that the fruit of rose myrtle (Rhodomyrtus tomentosa) was the most marked inhibitor of cell death. The protective effect of rose myrtle extract and the two key components, piceatannol and piceatannol­4'­O­ß­D­glucopyranoside, on UVB­induced damage and inflammation in cultured NHEK was investigated. The 80% ethanol extract from rose myrtle fruit with piceatannol exhibited protection of UVB­induced cytotoxicity in NHEK; however, piceatannol­4'­O­ß­D­glucopyranoside exhibited no protection, as determined by a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide assay. This extract and piceatannol reduced the production of UVB­induced cyclobutane pyrimidine dimers and enhanced the cellular enzyme activity of the DNA polymerases in UVB­irradiated NHEK, suggesting that UVB­stimulated DNA damage was repaired by the polymerases. In addition, the secretion of prostaglandin E2, which is an inflammatory mediator, was decreased. These results indicated that rose myrtle fruit extract and its key constituent, piceatannol, are potential photoprotective candidates for UV­induced skin damage.

Discovery of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.

N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides.

Protected N-branched nucleoside phosphonates containing adenine and thymine bases were prepared as the monomers for the introduction of aza-acyclic nucleotide units into modified oligonucleotides. The phosphotriester and phosphoramidite methods were used for the incorporation of modified and natural units, respectively. The solid phase synthesis of a series of nonamers containing one central modified unit was successfully performed in both 3'→5' and 5'→3' directions. Hybridization properties of the prepared oligoribonucleotides and oligodeoxyribonucleotides were evaluated. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a considerable destabilizing effect of the introduced units. We also examined the substrate/inhibitory properties of aza-acyclic nucleoside phosphono-diphosphate derivatives (analogues of nucleoside triphosphates) but neither inhibition of human and bacterial DNA polymerases nor polymerase-mediated incorporation of these triphosphate analogues into short DNA was observed.

Cycloartenyl trans-ferulate, a component of the bran byproduct of sake-brewing rice, inhibits mammalian DNA polymerase and suppresses inflammation.

During the screening of selective DNA polymerase (pol) inhibitors, we isolated cycloartenyl trans-ferulate (CAF), which is a major component of γ-oryzanol, which is a byproduct formed during the production of Japanese rice wine "sake". CAF selectively inhibited the activity of mammalian A, B, and X pol families, but Y family pols were not affected. CAF did not influence the activities of plant or prokaryotic pols, nor the activity of other DNA metabolic enzymes tested. Individual chemical components of CAF, including cycloartenol (CA) and ferulic acid (FA), did not inhibit pol enzyme activities. CAF suppressed TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation in the mouse ear, but CA and FA did not. The ability to inhibit mammalian pol enzymes in vitro was positively correlated with their propensity to suppress inflammation in vivo. These results suggest that this byproduct formed during the sake-brewing process is useful as an anti-inflammatory agent.

Ribonucleotides and manganese ions improve non-homologous end joining by human Polµ.

Human DNA polymerase mu (Polµ), a family X member involved in DNA repair, has both template-directed and terminal transferase (template-independent) activities. In addition to their ability to incorporate untemplated nucleotides, another similarity between Polµ and terminal deoxynucleotidyl transferase (TdT) is their promiscuity in using ribonucleotides (NTPs), whose physiological significance is presently unknown. As shown here, Polµ can use NTPs instead of deoxynucleotides (dNTPs) during non-homologous end joining (NHEJ) of non-complementary ends, a Polµ-specific task. Moreover, a physiological concentration of Mn(2+) ions did benefit Polµ-mediated NHEJ by improving the efficiency and accuracy of nucleotide insertion. Analysis of different mutations in the 'steric gate' of the active site indicated that Polµ is taking advantage of an open active site, valid for selecting alternative activating metal ions and nucleotides as substrates. This versatility would allow ad hoc selection of the most appropriate nucleotide/metal ion combination for individual NHEJ events to gain efficiency without a cost in terms of fidelity, thus widening the spectrum of available solutions to position a discontinuous template strand in proper register for connection.