Uracil Base Research Articles

Functional Analysis on a Naturally Occurring Variant of the Staphylococcus Aureus Uracil DNA Glycosylase Inhibitor

Repair of DNA damage relies on various pathways including the base excision repair (BER) which targets erroneous bases in the DNA. Here, Uracil-DNA glycosylases (UDGs) are responsible for recognition and removal of uracil base from the DNA. Here, we characterize the interaction of Staphylococcus aureus UDG (SAUDG) with a naturally occurring variant of S. aureus uracil-DNA glycosylase inhibitor (SAUGI). This variant contains a histidine instead of a glutamate at the 24th position which affects the SAUDG:SAUGI interaction surface. We assessed the complex formation of SAUDG with these two SAUGI variants by independent biophysical methods. Our data reveal that the residue difference at the 24th position does not have a marked effect on the binding affinity, yet it confers alteration of the thermodynamics of the interaction. We propose that the E24H variant of SAUGI allows efficient complex formation, and consequently, inhibition of SAUDG.

Uracil removal-inhibited ligase reaction in combination with catalytic hairpin assembly for the sensitive and specific detection of uracil-DNA glycosylase activity.

Sensitive and specific detection of uracil-DNA glycosylase (UDG) activity is crucial in biomedical study and disease diagnosis. Here, we developed a uracil removal-inhibited ligase reaction in combination with catalytic hairpin assembly (CHA) for the sensitive and specific detection of UDG activity. A hairpin probe is specially designed, which contains two uracil bases in the loop and is extended with toehold and branch-migration domains at the ends of the stem. Two short oligonucleotides are separately hybridized to one-half of the loop of the hairpin probe to form a DNA complex with a nick. Under the action of UDG, two uracil bases in the hairpin-loop are removed to generate apurinic/apyrimidinic (AP) sites. The AP sites locating at the 3'-side of the nick inhibit the ligase reaction, leaving the toehold and branch-migration domains at the ends of the hairpin probe still adjacent. The adjacent toehold and branch-migration domains initiate CHA, producing numerous G-quadruplex (G4) structures, which interact with N-methyl-mesoporphyrin IX (NMM) to generate an enhanced fluorescence signal. The excessive probes would be masked by the ligase reaction that closes the nick and forms a long DNA strand fully complementary to the hairpin domain. The probes then get opened and the toehold/branch-migration domains are not associated, prohibiting the CHA reaction and minimizing false-positive interferences. The detection limit is as low as 0.00028 U mL-1, and UDG can be well distinguished from other DNA glycosylases. Furthermore, this method is successfully applied for detecting UDG activity from HeLa cell lysates. Additionally, the inhibition of UDG activity is analyzed, which shows inhibitor dose-dependent activity suppression. This strategy will provide a promising tool for assaying UDG activity in biomedical study and disease diagnosis.

C-to-U editing and site-directed RNA editing for the correction of genetic mutations.

Cytidine to uridine (C-to-U) editing is one type of substitutional RNA editing. It occurs in both mammals and plants. The molecular mechanism of C-to-U editing involves the hydrolytic deamination of a cytosine to a uracil base. C-to-U editing is mediated by RNA-specific cytidine deaminases and several complementation factors, which have not been completely identified. Here, we review recent findings related to the regulation and enzymatic basis of C-to-U RNA editing. More importantly, when C-to-U editing occurs in coding regions, it has the power to reprogram genetic information on the RNA level, therefore it has great potential for applications in transcript repair (diseases related to thymidine to cytidine (T>C) or adenosine to guanosine (A>G) point mutations). If it is possible to manipulate or mimic C-to-U editing, T>C or A>G genetic mutation-related diseases could be treated. Enzymatic and non-enzymatic site-directed RNA editing are two different approaches for mimicking C-to-U editing. For enzymatic site-directed RNA editing, C-to-U editing has not yet been successfully performed, and in theory, adenosine to inosine (A-to-I) editing involves the same strategy as C-to-U editing. Therefore, in this review, for applications in transcript repair, we will provide a detailed overview of enzymatic site-directed RNA editing, with a focus on A-to-I editing and non-enzymatic site-directed C-to-U editing.

Correlated target search by uracil-DNA glycosylase in the presence of bulky adducts and DNA-binding ligands

Many proteins specific for rare targets in DNA, such as transcription factors, restriction endonucleases, and DNA repair enzymes, search for their targets by one-dimensional diffusion along DNA. One of these proteins is uracil-DNA glycosylase (Ung), which excises the uracil bases formed by rare events of cytosine deamination. We have studied the ability of Ung to move along DNA with its path hindered by bulky DNA covalent adducts (fluorescein) or ligands blocking the major or minor DNA groove. The fluorescein adduct strongly inhibits translocation only along double-stranded DNA, whereas noncovalently bound ligands partly inhibit DNA cleavage but barely affect translocation. The ability of uracil-DNA glycosylase to search for its targets in the presence of molecules competing for DNA binding may be important for DNA repair in the intracellular environment.

Radiation of uracil molecules on the surface under electron beam

Background: The interaction of high-energy radiation (α, β, γ- rays or heavy ions) on living cells, basically, does not lead directly to the degradation of the biopolymer molecules, including covalent bonds in the primary structure of nucleic acids. This function is performed by the secondary electrons that are produced in large quantities during the passing of the high-energy radiation stream through biostructures. The aim of this work is to identify the luminescence of uracil molecules under electron beam in the environment where these molecules are placed on the surface of the polycrystalline matrix. Materials and methods : Research carried out using the method of electron-photon spectroscopy. Experimental samples were the planar ceramic plates with the size 10×10×2 mm. Immobilization of biomolecules performed by their precipitate from an aqueous solution on the surface of the ceramic matrix. Results: For the first time the radiation spectrum of the surface with absorbed molecules of nitrogenous base uracil NA under the electron beam energy of 600 eV under the conditions of ultrahigh vacuum P ≤ 5·10 -9 Thor was obtained. In the energy range of 200 – 800 nm three broad molecular bands with maxima at λ ~ 335 nm, λ ~ 435 nm and λ ~ 495 nm were discovered. Conclusion: It appears that the mechanisms of formation of radiation bands caused by excitation of π - electron of the uracil molecules from the ground singlet state S 0 to S n excited singlet state and excited triplet state T n , with their deactivation into S 1 and T 1 states. The radiation decay of S 1 state to the ground state generates fluorescence band with a maximum at λ ~ 335 nm. Molecular band with a maximum at λ ~ 435 nm is phosphorescence band and is caused by the radiation decay of T 1 states to the ground S 0 state

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10−5U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

Suzuki‐Miyaura cross–coupling reaction in water: facile synthesis of (hetero) aryl uracil bases using potassiumorganotrifluoroborates under microwave irradiation.

AbstractIn this paper, we report the use of (hetero) aryl potassiumorganotrifluoroborate salts as versatile nucleophilic organoboron reagents in the Suzuki‐Miyaura cross‐coupling reaction of an electron rich 6‐chloro 3‐methyl uracil in water under microwave irradiation. A comparative study of the cross‐coupling reaction of this chloro uracil analogue with different nucleophilic organoboron source revealed the superior reactivity of (hetero) aryl potassiumorganotrifluoroborates in water under microwave irradiation. Diversely functionalised (hetero) aryl uracil bases, which are known possess a wide range of applications in chemical biology, were efficiently synthesized using this optimised protocol.

Visual and light scattering spectrometric method for the detection of melamine using uracil 5′-triphosphate sodium modified gold nanoparticles

A highly selective method was presented for colorimetric determination of melamine using uracil 5′-triphosphate sodium modified gold nanoparticles (UTP-Au NPs) in this paper. Specific hydrogen-bonding interaction between uracil base (U) and melamine resulted in the aggregation of AuNPs, displaying variations of localized surface plasmon resonance (LSPR) features such as color change from red to blue and enhanced localized surface plasmon resonance light scattering (LSPR-LS) signals. Accordingly, the concentration of melamine could be quantified based on naked eye or a spectrometric method. This method was simple, inexpensive, environmental friendly and highly selective, which has been successfully used for the detection of melamine in pretreated liquid milk products with high recoveries.

Effect of 5-Substitution of Uracil Base in DNA Photocrosslinking Using 3-Cyanovinylcarbazole

Oligonucleotide containing 3-cyanovinylcarbazole (CNVK) can crosslink to a pyrimidine base in complementary DNA/RNA by a few seconds of irradiation. The effect of 5-substitution of the uracil base in DNA photocrosslinking using CNVK was evaluated. Each uracil analog base had diverse photoreactivity with CNVK, and there was good correlation with the photoreactivity and LUMO level of the target uracil base in duplex DNA.

Label-free fluorescence turn-on detection of uracil DNA glycosylase activity based on G-quadruplex formation

We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research.

Selection of Natural and Base-Modified DNA Aptamers for a Camptothecin Derivative.

Nucleic acid aptamers for small molecules are currently being developed and have a potential role in diverse applications including biosensing, diagnostics, and therapeutics involving low-molecular-weight biomarkers and drugs. To enhance and broaden their functions through chemical modification, systematic evolution of ligands by exponential enrichment (SELEX) selection has been attempted with modified DNA/RNA libraries. Recently, we demonstrated the superior efficacy of base modification for affinity enhancement and the usefulness of unnatural nucleic acid libraries for development of small-molecule aptamers. In this unit, we describe construction of a modified DNA library that includes (E)-5-(2-(N-(2-(N(6) -adeninyl)ethyl))carbamylvinyl)uracil bases and acquisition of high-affinity camptothecin-binding DNA aptamers, in addition to those of the corresponding natural DNA library and aptamers, using the SELEX method. © 2016 by John Wiley & Sons, Inc.

Comparative analysis of virus-derived small RNAs within cassava (Manihot esculenta Crantz) infected with cassava brown streak viruses

Infection of plant cells by viral pathogens triggers RNA silencing, an innate antiviral defense mechanism. In response to infection, small RNAs (sRNAs) are produced that associate with Argonaute (AGO)-containing silencing complexes which act to inactivate viral genomes by posttranscriptional gene silencing (PTGS). Deep sequencing was used to compare virus-derived small RNAs (vsRNAs) in cassava genotypes NASE 3, TME 204 and 60444 infected with the positive sense single-stranded RNA (+ssRNA) viruses cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the causal agents of cassava brown streak disease (CBSD). An abundance of 21–24nt vsRNAs was detected and mapped, covering the entire CBSV and UCBSV genomes. The 21nt vsRNAs were most predominant, followed by the 22 nt class with a slight bias toward sense compared to antisense polarity, and a bias for adenine and uracil bases present at the 5′-terminus. Distribution and frequency of vsRNAs differed between cassava genotypes and viral genomes. In susceptible genotypes TME 204 and 60444, CBSV-derived sRNAs were seen in greater abundance than UCBSV-derived sRNAs. NASE 3, known to be resistant to UCBSV, accumulated negligible UCBSV-derived sRNAs but high populations of CBSV-derived sRNAs. Transcript levels of cassava homologues of AGO2, DCL2 and DCL4, which are central to the gene-silencing complex, were found to be differentially regulated in CBSV- and UCBSV-infected plants across genotypes, suggesting these proteins play a role in antiviral defense. Irrespective of genotype or viral pathogen, maximum populations of vsRNAs mapped to the cytoplasmic inclusion, P1 and P3 protein-encoding regions. Our results indicate disparity between CBSV and UCBSV host-virus interaction mechanisms, and provide insight into the role of virus-induced gene silencing as a mechanism of resistance to CBSD.

FUdR extends the lifespan of the short-lived AP endonuclease mutant in Caenorhabditis elegans in a fertility-dependent manner.

The anticancer drug 5-fluorouracil (5-FU) and its metabolite 5-fluoro-2'-deoxyuridine (FUdR) inhibit thymidylate synthase and induce uracil bases in DNA. FUdR is commonly used for inhibiting fertility when measuring the lifespan of the nematode Caenorhabditis elegans. However, it is not known whether DNA damage induced by FUdR affects lifespan. EXO-3 is an apurinic/apyrimidinic endonuclease in C. elegans, and we reported previously that deletion of the exo-3 gene causes reproductive abnormalities and decreased lifespan. In this study, we found that FUdR extended the lifespan of exo-3 mutants. We measured the lifespan of multiple germline mutants to examine whether this lifespan extension effect was dependent on fertility. In the presence of a fem-1 mutation, which causes a deficiency in sperm production, FUdR did not extend the lifespan of the exo-3 mutant. In glp-1 mutants, which do not develop gonads, the exo-3 mutant was not short-lived, and FUdR did not extend its lifespan. These results suggest that the lifespan extension effect of FUdR depends on fertility and the presence of gonads. fem-3 mutants, which do not produce oocytes, had increased lifespan in the presence of FUdR, independent of the exo-3 mutation. It is possible that the fem-3 mutant was susceptible to the lifespan extension effect of FUdR. From these results, we suggest that FUdR affects the lifespan of C. elegans in two ways: by interfering with fertility, which extends lifespan, and by inducing DNA base damage, which reduces lifespan.

Neomycin B-cyclen conjugates and their Zn(II) complexes as RNA-binding agents

Three new conjugates featuring the aminoglycoside antibiotic neomycin B linked to the 1,4,7,10-tetraazacyclododecane (cyclen) macrocycle via alkyl chains of varying lengths were synthesized from suitably protected derivatives of these precursors via conventional peptide coupling protocols. The final products were characterized by 1H NMR spectroscopy, mass spectrometry, and elemental analysis. FRET-based measurements examining the ability of the compounds to displace coumarin-labelled kanamycin A or neomycin B from Dy547-labelled prokaryotic ribosomal A-site RNA revealed that they bind to the A-site with slightly higher affinities than the parent aminoglycoside (e.g., IC50 at pH7=1.42–2.30μM vs. 2.35μM for neomycin B). This is attributed to the higher overall positive charge of the conjugates, resulting from protonation of the macrocylic amines. Consistent with a predominantly electrostatic mode of interaction, the binding affinities of the conjugates were found to increase with decreasing pH, reflecting a greater degree of protonation at lower pH. The zinc(II) complexes of the neomycin B-cyclen conjugates were found to bind to A-site RNA with even higher affinities (IC50=0.85–1.32μM), due to the Zn(II)-cyclen motif forming coordinative (and/or electrostatic) interactions with the uracil bases and/or phosphate groups within the A-site. These results highlight the potential for the nucleic acid-binding properties of aminoglycosides to be tuned via the covalent attachment of metal complexes, which could ultimately prove useful to the development of new anti-bacterial and anti-viral agents.

G-quadruplex DNA structures can interfere with uracil glycosylase activity in vitro.

Genome sequences that contain tandem repeats of guanine can form stable four-stranded structures known as G-quadruplex, or G4 DNA. While the molecular mechanisms are not fully defined, such guanine-rich loci are prone to mutagenesis and recombination. Various repair pathways function to reduce the potential for genome instability by correcting base damage and replication errors; however, it is not yet fully defined how well these processes function at G4 DNA. One frequent form of base damage occurs from cytidine deamination, resulting in deoxyuracil and UG mismatches. In duplex and single-stranded DNA, uracil bases are recognised and excised by uracil glycosylases. Here, we tested the efficiency of uracil glycosylase activity in vitro on uracil bases located directly adjacent to guanine repeats and G4 DNA. We show that uracil excision by bacterial UDG and human hUNG2 is reduced at uracils positioned directly 5' or 3' of a guanine tetrad. Control reactions using oligonucleotides disrupted for G4 formation or reaction conditions that do not favour G4 formation resulted in full uracil excision activity. Based on these in vitro results, we suggest that folding of guanine-rich DNA into G4 DNA results in a DNA conformation that is resistant to uracil glycosylase-initiated repair and this has the potential to increase the risk of instability at guanine repeats in the genome.

A novel and label-free biosensors for uracil-DNA glycosylase activity based on the electrochemical oxidation of guanine bases at the graphene modified electrode

Uracil-DNA glycosylase (UDG) as an important base excision repair enzymes is widely distributed in organism, and it plays a crucial role in sustaining the genome integrity. Therefore, it is significant to carry out the analysis of UDG activity. In this present work, a novel and label-free electrochemical sensing platform for the sensitive detection of uracil DNA glycosylase (UDG) activity has been developed. Herein, the graphene modified glassy carbon (GC) electrode was prepared. And two complementary DNA strands were hybridized to form dsDNA (P1P2). In the presence of UDG, the uracil bases in P1P2 were specifically hydrolyzed, inducing the unwinding of the DNA duplex, and accompanied by the release of P1. Thus, the released P1 was adsorbed onto the graphene/GC electrode surface via π–π stacking. By investigating the electrochemical behavior of P1 at the graphene/GC electrode, the electrochemical oxidation of guanine bases in P1 was obviously observed. Therefore, using the current responses of guanine base in P1 as a signal indicator, UDG activity can be simply determined with high sensitivity, and the detectable lowest concentration is 0.01U/mL. This present design does not need covalent attachment of redox indicator to DNA, preventing participation of redox labels in the background. Meanwhile, the proposed strategy for the assay of UDG activity also has a remarkable sensitivity due to the excellent properties of graphene, which could increase both the immobilization amount of released ssDNA and the conductivity of the sensing system. All these elucidate that this developed protocol may lay a potential foundation for the sensitive detection of UDG activity in clinical diagnosis.

Enzymatic synthesis and RNA interference of nucleosides incorporating stable isotopes into a base moiety

Thymidine phosphorylase was used to catalyze the conversion of thymidine (or methyluridine) and uracil incorporating stable isotopes to deoxyuridine (or uridine) with the uracil base incorporating the stable isotope. These base-exchange reactions proceeded with high conversion rates (75–96%), and the isolated yields were also good (64–87%). The masses of all synthetic compounds incorporating stable isotopes were identical to the theoretical molecular weights via EIMS. 13C NMR spectra showed spin–spin coupling between 13C and 15N in the synthetic compounds, and the signals were split, further proving incorporation of the isotopes into the compounds. The RNA interference effects of this siRNA with uridine incorporating stable isotopes were also investigated. A 25mer siRNA had a strong knockdown effect on the MARCKS protein. The insertion position and number of uridine moieties incorporating stable isotopes introduced into the siRNA had no influence on the silencing of the target protein. This incorporation of stable isotopes into RNA and DNA has the potential to function as a chemically benign tracer in cells.

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.

ChemInform Abstract: Modification of Purine and Pyrimidine Nucleosides by Direct C—H Bond Activation

AbstractReview: 81 refs.