Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.     

Highly accurate synthesis of the fully 2′-fluoro-modified oligonucleotide by Therminator DNA polymerases

Oligonucleotides composed of natural nucleotides are inapplicable for biotechnical and therapeutic use due to its instability under biological conditions. Therminator DNA polymerases, mutant DNA polymerases of thermophilic marine archaea, show that they can efficiently synthesize fully 2′-fluoro-modified (2′F-) oligonucleotides. Furthermore, the sequence analysis reveals that the oligonucleotide sequence is highly accurate, especially the fidelity of a 2′F-oligonucleotide synthesized by Therminator II is more accurate than that of natural RNA synthesized by conventional RNA polymerase. These finding would be helpful for the synthesis of chemically modified oligonucleotides, for the use of biotechnical or medical applications.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2′-deoxyguanosine in solutions of free 2′-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) in solutions of free 2′-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe³⁺). The oxidation of Fe²⁺ to Fe³⁺ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe²⁺ ≥phosphate concentration, the rate of Fe²⁺ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe²⁺ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe²⁺ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)–HCl.     

DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G₁-, G₂- or G₃-positions of the NarI site (5′-G₁G₂CG₃CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G₃ construct were mutants, largely G→T, compared to 18% and 24% from the G₁ and G₂ constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G₁ and G₃ constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G₂ construct. In vitro TLS using yeast pol ζ showed that it can extend G₃*:A pair more efficiently than G₃*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.     

Synthesis of γ-N-modified 8-oxo-2’-deoxyguanosine triphosphate and its characterization

8-OxodGTP is generated by the reaction between dGTP and reactive oxygen species and a considered mutagenic nucleotide. It can be incorporated into the duplex DNA during replication processes by the DNA polymerase, and thus the repair enzyme removes oxodGTP from the nucleotide pools in living cells. On the other hand, the γ-modified triphosphates show interesting properties for use as biological tools. Therefore, the γ-N-pyrenylalkyl-oxodGTP derivatives were synthesized and their effect on the enzymatic reactions were evaluated. The γ-N-pyrenylmethyl-oxodGTP was found to be accepted by the DNA polymerase just like oxodGTP, but showed a competitive inhibition property for the human oxodGTPase.     

Fluorescence Properties and Base Pair Stability of Oligonucleotides Containing 8-Aza-7-deaza-2′-deoxyisoinosine or 2′-Deoxyisoinosine

Abstract

The fluorescence and the base pairing properties of 8-aza-7-deaza-2′-deoxyisoinosine (1) are described and compared with those of 2′-deoxyisoinosine (2). The corresponding phosphoramidites (11,12) are synthesized using the diphenyl-carbamoyl (DPC) residue for the 2-oxo group protection. The nucleosides 1 and 2 base pair with 2′-deoxy-5-methylisocytidine in DNA duplexes with antiparallel chain orientation and with 2′-deoxycytidine in a parallel DNA. These base pairs are less stable than the canonical dA-dT pair and that of 2′-deoxyinosine (4) with 2′-deoxycytidine. The fluorescence of the nucleosides 1 and 2 is quenched (～95%) in duplex DNA. The residual fluorescence is used to determine the T_m-values, which are found to be the same as determined UV-spectrophotometrically.     

Harmonising measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine in cellular DNA and urine

Abstract

Levels of oxidatively damaged cellular DNA and urinary excretion of damaged 2′-deoxyribonuclosides are widely measured in biomonitoring studies examining the role of oxidative stress induced by environmental exposures, lifestyle factors and development of disease. This has promoted efforts to harmonise measurements of oxidised guanine nucleobases by the variety of analytical approaches for DNA and urinary levels of damage, in multi-laboratory trials that are centred in Europe. The large inter-laboratory variation reported of values of oxidatively damaged DNA is reduced by harmonising assay protocols. Recent attention on optimal conditions for the comet assay may lead to better understanding of the most critical steps in procedure, which generate variation in DNA damage levels between laboratories. Measurements of urinary excretion of oxidatively generated 8-oxo-7,8-dihydro-2′-deoxyguanosine also show large differences between different methods, where chromatographic techniques generally show more reliable results than antibody-based methods. In this case, standardising calibrants is aimed at improving within technique agreement.     

Analysis of DNA Sequencing Reaction Products Made with 7-Halo-7-deaza-2′-deoxyguanosine-5′-triphosphate

Abstract

7-Chloro- and 7- Iodo-7-Deaza-2′-Deoxyguanosine-5′-Triphosphates were employed as substrates replacing dGTP, dITP, or 7-Deaza-2′-Deoxyguanosine-5′-Triphosphate in sequencing reactions with Thermo Sequenase^TM. Analysis of the reaction products by denaturing gel electrophoresis indicates DNA containing these halogenated analogs can form strong secondary structures.     

Inhibition of Rac and Rac-linked functions by 8-oxo-2′-deoxyguanosine in murine macrophages

Rac is a protein involved in the various functions of macrophages (Mφ), including the production of reactive oxygen species (ROS), phagocytosis, chemotaxis and the secretion of cytokines (such as γ-INF). This study tested the effects of nucleosides containing 8-oxoguanine(8-hydroxyguanine) such as 8-oxo-2′-guanosine (8-oxoG) or 8-oxo-2′-deoxyguanosine (8-oxodG), on Rac and the above-listed Rac-associated functions of Mφ using mouse peritoneal Mφ (MpMφ). It is reported that 8-oxodG was able to effectively inhibit Rac and the Rac-associated functions of MpMφ. Compared to 8-oxodG, 8-oxoG showed negligible effects. Furthermore, normal nucleosides such as deoxyguanosine (dG), guanosine (G) and adenosine (A) did not exert any effects. These results suggest that 8-oxodG could be used as a potential tool to modulate the functions of Mφ that are intimately related to various pathological processes.     

Actions of the Klenow fragment of DNA polymerase I and some DNA glycosylases on chemically stable analogues of N7-methyl-2′-deoxyguanosine

N7-methyl-9-deaza-dG was synthesized and incorporated into oligonucleotides. Thermal melting studies showed that replacement of dG by N7-methyl-9-deaza-dG only slightly decreased DNA duplex stability. Replication of DNA templates containing N7-methyl-9-deaza-dG and the related 7-methyl-7-deaza-dG and 7-deaza-dG by the Klenow fragment of Escherichia coli DNA polymerase I was examined. The dNTP misinsertion frequencies on all three templates were comparably low, although the 7-methyl group significantly slowed down the turnover rates of the polymerase when dCTP was incorporated. The stabilities of N7-methyl-9-deaza-dG and 7-methyl-7-deaza-dG against the actions of formamidopyrimidine DNA glycosylase (Fpg) and human alkyladenine DNA glycosylase (hAAG) were also examined. N7-methyl-9-deaza-dG was stable in the presence of both enzymes. In contrast, 7-methyl-7-deaza-dG was cleaved by Fpg, and possibly by hAAG but at an extremely slow rate. This study suggests that N7-alkyl-9-deaza-dG is a better analogue than 7-alkyl-7-deaza-dG for cellular studies.     

A Serendipitous Synthesis of 8-Dimsyl-2′-deoxyguanosine

Abstract

A serendipitous synthesis of 8-dimsyl-dG (2) has been achieved along with the known 8-benzyloxy-dG (3) in a nucleophilic substitution reaction of 8-bromo-dG (1) with in situ generated dimsyl and benzyloxy sodium. Compound 3 was directly converted into the mutagenic oxidative DNA damage product, 8-oxo-dGTP (4).     

Kinetics,Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.     

Synchronization of DNA synthesis in Neurospora crassa by 2′-deoxyadenosine and spore selection

2-Deoxyadenosine (2 mM), a DNA inhibitor, was used to synchronize DNA synthesis in cultures of Neurospora crassa lys 3. The cultures recovered spontaneously from the inhibitor which had little or no effect on the synthesis of RNA, protein or carbohydrate or on the specific growth rate of the mould. The degree of synchrony of DNA synthesis obtained with 2-deoxyadenosine varied directly with the organism's specific growth rate when the latter was altered by temperature changes. A direct relationship was observed between the rate of synthesis of DNA during the S period and the organism's specific growth rate.Conidia of Neurospora crassa lys 3 were separated into different density classes using urografin gradients; the separation treatment did not have an appreciable effect on the subsequent germination or growth of conidia. Populations of large, less dense conidia produced germ tubes more rapidly and more synchronously than populations of small, dense conidia. Cultures inoculated with the large conidia displayed continuous synthesis of RNA and protein but discontinuous synthesis of DNA.     

Differential inactivation of DNA polymerases α and β by aldehyde compounds

The effects of cyclohexanecarboxaldehyde, benzaldehyde and protocatechualdehyde on the activities of DNA polymerases α, β and E. coli DNA polymerase I were investigated. On direct addition of the aldehydes to the DNA polymerase assay mixture containing activated DNA or poly(dA) (dT)_12–18 as a template, DNA polymerase α was most strongly inhibited by the aldehyde compounds, while DNA polymerases β and I were resistant to such aldehyde inhibition. On preincubation of the enzymes with aldehyde, both DNA polymerases α and β were inactivated; however, DNA polymerase β was protected from the inactivation when activated DNA was added to the preincubation mixture. The inhibition of DNA polymerase α by aldehyde was noncompetitive with regard to the substrate dNTP and competitive with regard to the template DNA. The extent of inhibition of DNA polymerase α by aldehyde was partly reduced by the addition of cysteine to the reaction mixture.     

Suppression of 8-Oxo-2′-deoxyguanosine Formation and Carcinogenesis Induced by N-Nitrosobis(2-oxopropyl)amine in Hamsters by Esculetin and Esculin

Effects of esculetin (6,7-dihydroxycoumarin) and its glycoside, esculin, on 8-oxo-2′-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were administered esculetin by gastric intubation into the stomach 30?min before BOP administration or ingestion of a diet containing esculin for 7 days before BOP administration, and killed 1 or 4?h after BOP treatment, and the contents of thiobarbituric acid-reacting substrates (TBARS) and 8-oxodG in the pancreas were determined. Both compounds suppressed significantly the BOP-induced increases in 8-oxodG and TBARS contents in hamster pancreas. We further investigated the effect of esculin on pancreatic carcinogenesis by the rapid production model induced by augmentation pressure with a choline-deficient diet, ethionine, methionine and BOP. Esculin was given ad libitum as a 0.05% aqueous solution in either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly smaller than in the control group, while esculin given during the promotion phase showed no apparent effects. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.     

Steric and Stereoelectronic Effects of 7-Deazapurine Bases on the Sugar Conformation of 2′-Deoxynucleosides

Abstract

Conformational analyses of the sugar moieties of a series of 7-(8)- substituted 7-deazapurine-2′-deoxynucleosides on the basis of vicinal [H,H] coupling constants is presented using the PSEUROT 6.0 program.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase