DNA Nucleosides Research Articles

The Cyclobutane Dimers of 2′‐Deoxyuridine, 2′‐Deoxycytidine, 5‐Methyl‐2′‐Deoxycytidine and 5‐Bromo‐2′‐Deoxyuridine

Irradiation of DNA and RNA pyrimidine nucleosides with UV light in frozen aqueous solution or in solution with acetone often results in the formation of cyclobutane dimers (CBDs). Many of these photodimers have not been characterized. We present here the results of work designed to achieve the isolation, spectroscopic characterization and determination of the stereochemical nature of a number of little studied or previously unstudied CBDs of four 2'-deoxyribonuclesides. These nucleosides are 2'-deoxyuridine (dUrd), 2'-deoxycytidine (dCyd), 5-methyl-2'-deoxycytidine (5-MedCyd) and 5-bromo-2'-deoxyuridine (5-BrdUrd). In particular, we have isolated and characterized six dUrd CBDs, five dCyd CBDs, five 5-MedCyd CBDs and four 5-BrdUrd CBDs. Photoproducts were studied by UV spectroscopy, mass spectrometry, proton NMR spectroscopy and via chemical approaches. Also presented are results from less definitive studies of a number of (6-4) (or 5-4) photoadducts of these nucleosides. In addition, results from exploratory photochemical studies of other 2'-deoxyribonucleosides in frozen solution, as well as some mixtures of two nucleosides, are given. The latter results indicate that 5-iodo-2'-deoxyuridine (5-IdUrd), 5-bromo-2'-deoxycytidine and 5-iodo-2'-deoxycytidine each form putative CBDs and that 5-BrdUrd is capable of forming putative mixed CBDs and (6-4) and/or (5-4) adducts with thymidine (Thd); 5-IdUrd similarly forms a (6-4) (or (5-4)) adduct with Thd.

Vitamins at physiological levels cause oxidation to the DNA nucleoside deoxyguanosine and to DNA--alone or in synergism with metals

Vitamins with antioxidant properties have the ability to act as pro-oxidants, inducing oxidative damage and oxidative stress as opposed to preventing it. While vitamin supplements are commonly consumed, the scientific evidence for their health beneficial effects is inconclusive. In fact, even harmful effects have been reported. The present study aimed to investigate and compare pro-oxidant properties of different antioxidants and vitamins commonly found in dietary supplements, at concentrations of physiological relevance, alone or in combination with metals also found in supplements. Focus was on damages related to DNA. The vitamins' chemical oxidation potencies were studied by measuring the amount of the oxidation product 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formed from the DNA nucleoside deoxyguanosine (dG) after vitamin exposure, using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. To study the vitamins' ability to cause DNA damage to cultured cells, promyelocytic leukemia cells (HL-60) were exposed to vitamins, and strand breaks, alkali-labile sites and oxidative DNA lesions, i.e. formamido pyrimidine DNA glycosylase-sensitive sites, were detected using the comet assay. Vitamins A and C chemically induced oxidation of dG, alone and in synergism with iron or copper, whereas only vitamin C and copper induced DNA damage in cultured cells. Contrary, vitamins B1, B2, B3, B6 and B12, β-carotene, folic acid, α-tocopherol, δ-tocopherol or γ-tocopherol did not induce oxidative damage to dG, while lycopene induced a weak dose-response increase. Taken together, vitamin C and copper stood out with the strongest oxidative potency, which is of potential concern since both substances are commonly found in multivitamins.

Electronic Properties of DNA Nucleosides Adsorbed on a Au(100) Surface

The geometrical properties and electronic structure of single DNA nucleosides (deoxyadenosine, deoxythymidine, deoxyguanosine, deoxycytidine) adsorbed on a metallic surface of Au(100) are determined using density functional theory computations. We investigate multiple adsorption geometries and the resulting molecule–surface interaction mechanisms. For adenosine, we found negligible differences between the binding energy in the two configurations investigated by us, while for guanosine this difference reaches the maximum value among the four nucleosides (i.e., 0.38 eV). The projected density of states indicates that the physisorption is the main cause of the binding energy. Nevertheless, for the adsorbed deoxycytosine (dC), we point out the presence of the chemical interaction too. While the absolute values of the molecule–surface charge transfer are small, they are qualitatively dependent on the orientation of the nucleosides to the surface. If the DNA bases are oriented perpendicular to the surface, the ...

Common Inherited Mitochondrial Dna Mutations and Nucleoside Reverse Transcriptase Inhibitor-Induced Severe Hyperlactataemia in HIV-Infected Adults: An Exploratory Study

Genetic predisposition to dideoxynucleoside-induced mitochondrial dysfunction might be related to mitochondrial DNA (mtDNA) polymorphisms. Severe hyperlactataemia is probably the best model to assess such a predisposition. For this exploratory study in White European and Black African HIV-infected adults, hypervariable region 1 of mtDNA samples from peripheral blood mononuclear cells or buccal smears of patients who have developed confirmed severe hyperlactataemia was sequenced. Additionally, 21 single nucleotide polymorphisms and a 9 bp deletion were genotyped to assign mtDNA haplogroups. Finally, entire mtDNA sequencing was performed in a subset of European samples. Samples were obtained from Black African cases and controls recruited from a single centre in Johannesburg, South Africa and from white European cases from Amsterdam, London and Zurich. A total of 40 cases and 38 controls from Johannesburg were included. All of the cases and 33 controls were receiving stavudine-based therapy at the time of the index date (P=0.024). The distribution of mtDNA haplotypes was not different between cases and controls (P=0.137), and neither were the predicted haplogroups (P=0.751). In total, 11 of the 12 European cases were on stavudine and/or didanosine at the time of the event. No hypervariable region 1 haplotype was consistently found in the European cases. Sequencing of the entire mtDNA from three of these cases supported the absence of any shared mutations other than major alleles frequently seen in the mtDNA database. We did not find an association between homoplasmic inherited mtDNA polymorphisms and severe hyperlactataemia. Our data do not support the existence of non-synonymous mtDNA mutations that explain an increased predisposition to dideoxynucleoside-induced mitochondrial dysfunction.

Synergistic cytotoxicity of the DNA alkylating agent busulfan, nucleoside analogs and suberoylanilide hydroxamic acid in lymphoma cell lines

Hematopoietic stem cell transplant (HSCT) is a promising treatment for lymphomas. Its success depends on effective pre-transplant conditioning regimens. We previously reported on the efficacy of DNA alkylating agent–nucleoside analog (NA) combinations for conditioning in acute myeloid leukemia (AML). We hypothesized that a similar combinatory approach can be used for lymphomas. A combination of busulfan (Bu) with two NAs - clofarabine (Clo), fludarabine (Flu) or gemcitabine (Gem) - resulted in synergistic cytotoxicity in lymphoma cell lines. We demonstrated that the [2 NAs + Bu] combination activates a DNA damage response through the ATM-CHK2 and ATM-CHK1 pathways, leading to cell cycle checkpoint activation and apoptosis. Histone modifications and KAP1 phosphorylation are indicative of chromatin relaxation mediated by the nucleoside analogs, which sequentially increase Bu alkylation. Addition of suberoylanilide hydroxamic acid (SAHA) enhanced chromatin relaxation through increased histone acetylation and further augmented the cytotoxicity of [2 NAs + Bu]. Our results provide a preclinical basis for a clinical trial on using [2 NAs + Bu ± SAHA] combinations as conditioning therapy for patients with chemotherapy-refractory lymphoma undergoing HSCT.

Risk Factors of Gene-Resistant Mutations in Different Nucleosides

To explore the risk factors of gene-resistant mutations during nucleotide treatment. A total of 320 patients with CHB were randomly divided into lamivudine (LAM, 107 cases), adefovir (ADV, 106 cases) and entecavir (ETV, 107 cases) groups, and P gene mutations of HBV were regularly detected. Kaplan-Meier and Cox regression analysis were performed. There was no statistical significance in baseline data between the three groups. The gene-resistant mutation rates were 20.0%, 0.0% and 0.0% one year later, 37.1%, 3.8% and 0.0% two years later, 42.8%, 9.5% and 0.9% three years later, and 64.7%, 25.7% and 0.9% four years later in LAM, ADV and ETV groups, respectively. In LAM group, rtL180M combined with rtM204V mutations accounted for 32.7% and in ADV group, rtN236T mutation accounted for 12.3%. The gene-resistant mutation rate was the most strong in LAM group. With an extension of time, gene-resistant mutation rates are increased, but it is the highest in LAM group and the lowest in ETV group. Family history, the negative conversion time of HBV DNA and different nucleosides are independent risk factors of gene-resistant mutations.

Association between nitric oxide and 8-hydroxydeoxyguanosine levels in semen of diabetic men

The incidence of diabetes mellitus is rapidly increasing in the world. One of the complications of diabetes includes disturbance of the reproductive tract, such as infertility, erectile dysfunction, and endocrine disruption. Nitric oxide (NO) is a free radical produced by most cells including the human male and female reproductive tracts. NO has a dual role where low concentrations are essential for homeostatic cellular biology and physiology, but high levels have detrimental effects relating to cellular damage from this reactive oxygen species (ROS). 8-hydroxydeoxyguanosine (8-OHdG) is an oxidized nucleoside of DNA that is currently used as a biomarker of cellular oxidative stress, where urinary levels can correlate with diabetic nephropathy and retinopathy. Our aim was to investigate the relationship between nitrate/nitrite levels and 8-OHdG levels in the semen of diabetic and non-diabetic men. Concentrations of nitrate/nitrite and 8-OHdG were examined in seminal plasma of 32 diabetic and 35 non-diabetic men. The level of nitrate/nitrite was assayed by colorimetric reaction and 8-OHdG was measured by ELISA. Our results showed that the seminal plasma nitrate/nitrite levels were significantly higher in the diabetic group (p < 0. 01). There were also significantly higher 8-OHdG levels in diabetic men compared to non-diabetic men (p < 0.05). Regression analysis indicated that in diabetic men, nitrate/nitrite levels correlated well with 8-OHdG levels (r = 0.64, p < 0.001). A significant trend between nitrate/nitrite and sperm parameters was not observed. Our data suggests that high levels of nitrate/nitrite in the semen of diabetic men is suggestive of reactive oxygen species induced DNA damage that is correlated with 8-OHdG levels but not sperm parameters. These results support the further investigation of NO and 8-OHdG as biomarkers for assessing male infertility.

Nucleoside-Assisted Self-Assembly of Oligo(p-phenylenevinylene)s at Liquid/Solid Interface: Chirality and Nanostructures

The formation of DNA nucleoside-assisted π-conjugated nanostructures was studied by means of scanning tunneling microscopy (STM) and force field simulations. Upon adsorption of the achiral oligo(p-phenylenevinylene) (OPV) derivative at the liquid/solid interface, racemic conglomerates with mirror related rosettes are formed. Addition of the DNA nucleosides D- and L-thymidine, which act as "chiral handles", has a major effect on the supramolecular structure and the expression of chirality of the achiral OPV molecules. The influence of these "chiral handles" on the expression of chirality is probed at two levels: monolayer symmetry and monolayer orientation with respect to the substrate. This was further explored by tuning the molar ratio of the building blocks. Molecular modeling simulations give an atomistic insight into the monolayer construction, as well as the energetics governing the assembly. Thymidine is able to direct the chirality and the pattern of OPV molecules on the surface, creating chiral lamellae of π-conjugated dimers.

Initial Excited-State Structural Dynamics of 2′-Deoxyguanosine Determined via UV Resonance Raman Spectroscopy

The resonance Raman spectra of 2'-deoxyguanosine, a DNA nucleoside, were measured in aqueous solution at wavelengths throughout its 260 nm absorption band. Self-consistent analysis of the resulting resonance Raman excitation profiles and absorption spectrum using a time-dependent wave packet formalism with two electronic states yielded the initial excited-state structural dynamics in both states. The vibrational modes containing the N(7)═C(8) stretching and C(8)-H bending internal coordinates were found to exhibit significant initial structural dynamics upon photoexcitation to either state and are coincident with the photochemical reaction coordinate involving the formation of the 2'-deoxyguanosine cation radical.

Intramolecular CH…O Hydrogen Bonds in the AI and BI DNA-like Conformers of Canonical Nucleosides and their Watson-Crick Pairs. Quantum Chemical and AIM Analysis

The aim of this work is to cast some light on the H-bonds in double-stranded DNA in its AI and BI forms. For this purpose, we have performed the MP2 and DFT quantum chemical calculations of the canonical nucleoside conformers, relative to the AI and BI DNA forms, and their Watson-crick pairs, which were regarded as the simplest models of the double- stranded DNA. Based on the atoms-in-molecules analysis (AIM), five types of the CH…O hydrogen bonds, involving bases and sugar, were detected numerically from 1 to 3 per a conformer: C2′H…O5′, C1′H…O2, C6H…O5′, C8H…O5′, and C6H…O4′. The energy values of H-bonds occupy the range of 2.3–5.6 kcal/mol, surely exceeding the kT value (0.62 kcal/mol). The nucleoside CH…O hydrogen bonds appeared to “survive” turns of bases against the sugar, sometimes in rather large ranges of the angle χ values, pertinent to certain conformations, which points out to the source of the DNA lability, necessary for the conformational adaptation in processes of its functioning. The calculation of the interactions in the dA·T nucleoside pair gives evidence, that additionally to the N6H…O4 and N1…N3H canonical H-bonds, between the bases adenine and thymine the third one (C2H…O2) is formed, which, though being rather weak (about 1 kcal/mol), satisfies the AIM criteria of H-bonding and may be classified as a true H-bond. The total energy of all the CH…O nontraditional intramolecular H-bonds in DNA nucleoside pairs appeared to be commensurable with the energy of H-bonds between the bases in Watson-Crick pairs, which implies their possible important role in the DNA shaping.

Vitamin A and C compounds permitted in supplements differ in their abilities to affect cell viability, DNA and the DNA nucleoside deoxyguanosine

In accordance with the European Parliament and Council's directive, vitamin A and C supplements can include any of four (vitamin A) or five (vitamin C) specified compounds. This study focuses on these compounds and compares their abilities to affect the DNA and viability of cells in culture, but also their potencies to chemically oxidise the DNA nucleoside deoxyguanosine (dG). To study the vitamins' strict chemical oxidation potencies, dG was exposed to vitamin solution and the amount of the oxidation product 8'-hydroxydeoxyguanosine (8-oxodG) formed was estimated using a high-performance liquid chromatography system with electrochemical and ultraviolet detection. The vitamin's ability to cause DNA damage to promyelocytic leukaemia cells (HL-60), as detected by strand breaks, alkaline labile sites and formamido pyrimidine DNA glycosylase (FPG)-sensitive sites was, after vitamin exposure, measured using the comet assay and cytotoxicity was estimated using trypan blue staining. The results highlight that vitamin A and C compounds found in supplements do have different properties, chemically as well as in a cellular system. Among the vitamin C compounds, ascorbic acid, sodium ascorbate and calcium ascorbate stood out causing both oxidation to dG and cytotoxicity to cells. The vitamin A compounds retinol, retinyl acetate and retinal (a breakdown product found in vivo) caused oxidation of dG, while retinal was the only compound causing cytotoxicity, giving rise to an almost complete cell death. β-carotene caused, as the only vitamin compound, a small increase in FPG-sensitive sites. It is concluded that even though the compounds are found under the same name (vitamin A or C), they do have different properties linked to oxidation, cytotoxicity and DNA damage.

Structure-Function Analysis of the OB and Latch Domains of Chlorella Virus DNA Ligase

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.

758 OXIDATIVE STRESS-INDUCED SYSTEMIC AND CAVERNOSAL MOLECULAR ALTERATIONS – THEIR ROLE IN THE PROGRESSION OF DIABETIC ERECTILE DYSFUNCTION

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 2011758 OXIDATIVE STRESS-INDUCED SYSTEMIC AND CAVERNOSAL MOLECULAR ALTERATIONS – THEIR ROLE IN THE PROGRESSION OF DIABETIC ERECTILE DYSFUNCTION Angela Castela, Pedro Gomes, Pedro Coelho, Pedro Vendeira, Ronald Virag, and Carla Costa Angela CastelaAngela Castela Porto, Portugal More articles by this author , Pedro GomesPedro Gomes Porto, Portugal More articles by this author , Pedro CoelhoPedro Coelho Porto, Portugal More articles by this author , Pedro VendeiraPedro Vendeira Porto, Portugal More articles by this author , Ronald ViragRonald Virag Paris, France More articles by this author , and Carla CostaCarla Costa Porto, Portugal More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1783AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Erectile Dysfunction (ED) is a common complication of diabetes. Increased oxidative stress (OS) contributes to corporeal deterioration, altering diabetic penile tissue homeostasis. However, it remains unclear the effects of OS in diabetic corpus cavernosum (CC) with the progression of the disease and its role in the development of ED. We aimed to evaluate/quantify systemic and cavernosal alterations caused by OS in early and established diabetes. METHODS Male Wistar rats were divided into groups (n=5/group): 2 and 8 weeks of streptozotocin-induced diabetes and age-matched controls. Blood was withdrawn, 24-hour urine was collected and penises were harvested. All samples were processed for the evaluation/quantification of OS-induced injuries using established biomarkers. Systemic OS was assessed on blood samples by the chromatographic detection of oxidized glutathione (GSSG)/reduced glutathione (GSH) and in urine by evaluating the production of hydrogen peroxide (H2O2). Locally, in CC, the noxious effects of OS were analyzed by: quantifying the production of H2O2, evaluating protein structural alterations by immunohistochemistry for 3-nitrotyrosine (3-NT) and by assessing DNA-induced oxidative lesions through 8-hydroxydeoxyguanosine (8-OHdG) immunodetection. The expression of 3-NT and 8-OHdG was also evaluated in human non-diabetic and diabetic cavernosal samples. Immunohistochemical quantification of these markers was performed using ImageJ. RESULTS Our results showed that particularly at 8-week of diabetes there was an increase in blood ratio of GSSG/GSH and of urinary H2O2 levels, indicative of systemic OS augmentation. In CC, H2O2 production was only significant in the established group. Concordantly, 3-NT and 8-OHdG expression was increased in 8-week diabetic erectile tissue, as well as, in human diabetic CC, indicating that more severe alterations, as protein nitration and DNA nucleoside modification, occur at a later stage of the disease. CONCLUSIONS Our study suggested that systemic and penile OS effects are detected mostly in established diabetes. Locally, OS-induced penile protein and DNA lesions present in advanced diabetes may be responsible for hampering functional molecular and cellular processes, culminating with the progression of diabetic-associated ED. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e305 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Angela Castela Porto, Portugal More articles by this author Pedro Gomes Porto, Portugal More articles by this author Pedro Coelho Porto, Portugal More articles by this author Pedro Vendeira Porto, Portugal More articles by this author Ronald Virag Paris, France More articles by this author Carla Costa Porto, Portugal More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins.

Desulfurization of 2-thiouracil nucleosides: Conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside ( H 2o 4U ) and 4-pyrimidinone 2′-deoxyribofuranoside ( dH 2o 4U ) were synthesized by the oxidative desulfurization of parent 2–thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H 2o 4U and dH 2o 4U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2′- endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H 2o 4U , which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH 2o 4U stabilizes the sugar moiety conformation in the C3′- endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH 2o 4U reveals a similar population of the C2′- endo conformation (65%) to that of 2′-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H 2o 4U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.

Quenching Enhancement of the Singlet Excited State of Pheophorbide-a by DNA in the Presence of the Quinone Carboquone

Changes in the emission fluorescence intensity of pheophorbide-a (PHEO) in the presence of carboquone (CARBOQ) were used to obtain the association constant, the number of CARBOQ molecules interacting with PHEO, and the fluorescence quantum yield of the complex. Excitation spectra of mixtures of PHEO and CARBOQ in ethanol (EtOH) show an unresolved doublet in the red-most excitation band of PHEO, indicating the formation of a loose ground-state complex. The 1:1 CARBOQ-PHEO complex shows a higher fluorescence quantum yield in EtOH (0.41 ± 0.02) than in buffer solution (0.089 ± 0.002), which is also higher than that of the PHEO monomer (0.28). Quenching of the PHEO fluorescence by DNA nucleosides and double-stranded oligonucleotides was also observed and the bimolecular quenching rate constants were determined. The quenching rate constant increase as the oxidation potential of the DNA nucleoside increases. Larger quenching constants were obtained in the presence of CARBOQ suggesting that CARBOQ enhances DNA photo-oxidation, presumably by inhibiting the back-electron-transfer reaction from the photoreduced PHEO to the oxidized base. Thus, the enhanced DNA-base photosensitized oxidation by PHEO in the presence of CARBOQ may be related to the large extent by which this quinone covalently binds to DNA, as previously reported.

Evaluating the Effects of the Nonplanarity of Nucleic Acid Bases on NMR, IR, and Vibrational Circular Dichroism Spectra: A Density Functional Theory Computational Study

The pyramidalizations of N9/1 glycosidic nitrogens in DNA and RNA nucleosides, recently discovered and analyzed in their ultrahigh-resolution X-ray crystal structures ( Sychrovský ; et al. Nucleic Acid Res. 2009 , 37 , 7321. ), were found to have significant effects on the structural interpretation of the (3)J(C4/2-H1') and (3)J(C8/6-H1') NMR scalar couplings in purine/pyrimidine nucleosides. The calculated effects on IR and vibrational circular dichroism (VCD) spectra were only minor. The calculated structural deformations in nucleosides, depending on sugar-to-base orientation, gave rise to corrections in the phase shift of the Karplus equations for the (3)J(C8/6-H1') and (3)J(C4/2-H1') couplings ranging from -26° to +25° and from -5.7° to +2.0°, respectively. The sign alternation of this correction in syn and anti nucleosides arises from the stereoinversion of the N9/1 glycosidic nitrogen occurring upon reorientation of the glycosidic torsion. The effect was calculated consistently in the dG, dA, dC, dT, rA, and rG nucleosides. Utilization of the calculated phase-shift corrections in the design of Karplus equations for the (3)J couplings was suggested, and the effects on structural interpretation of the experimental couplings were evaluated.

Photoemission Study of Thymidine Adsorbed on Au(111) and Cu(110)

The adsorption geometry of the DNA nucleoside thymidine on Au(111) and Cu(110) surfaces has been determined from experimental results of soft X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. The XPS of C, N, and O 1s as well as the absorption spectra at the N and O K-edges were measured for monolayer thymidine films, and the nature of the bonding with the two metal surfaces has been determined. The NEXAFS data at the N and O K-edge show a strong angular dependence of the π*/σ* intensity ratios. We conclude that the thymine moiety is lying nearly parallel to the Au(111) surface, while on the Cu(110) surface, it adsorbs at a steep angle.

Electronic signatures of all four DNA nucleosides in a tunneling gap.

Nucleosides diffusing through a 2 nm electron-tunneling junction generate current spikes of sub-millisecond duration with a broad distribution of peak currents. This distribution narrows 10-fold when one of the electrodes is functionalized with a reagent that traps nucleosides in a specific orientation with hydrogen bonds. Functionalizing the second electrode reduces contact resistance to the nucleosides, allowing them to be identified via their peak currents according to deoxyadenosine > deoxycytidine > deoxyguanosine > thymidine, in agreement with the order predicted by a density functional calculation.

APOBEC3 proteins mediate the clearance of foreign DNA from human cells

Bacteria evolved restriction endonucleases to prevent interspecies DNA transmission and bacteriophage infection. Here, we show that human cells possess an analogous mechanism. APOBEC3A is induced by interferon following DNA detection, and it deaminates foreign double-stranded DNA cytidines to uridines. These atypical DNA nucleosides are converted by the uracil DNA glycosylase UNG2 to abasic lesions, which lead to foreign DNA degradation. This mechanism is evident in cell lines and primary monocytes, where up to 97% of cytidines in foreign DNA are deaminated. In contrast, cellular genomic DNA appears unaffected. Several other APOBEC3s also restrict foreign gene transfer. Related proteins exist in all vertebrates, indicating that foreign DNA restriction may be a conserved innate immune defense mechanism. The efficiency and fidelity of genetic engineering, gene therapy, and DNA vaccination are likely to be influenced by this anti-DNA defense system.