Non-covalent intercalative binding of 7,8-dihydroxy-9,10-epoxybenzo(a)pyrene to DNA

When the benzo(a)pyrene diol epoxide (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) is mixed into a DNA solution, a 10nm red shift in the absorption maximum of BPDE appears at 354nm which is due to a non-covalent intercalation complex. The major reaction pathway at this intercalation site is the hydrolysis of BPDE to its tetraol which is accompanied by a decrease in the absorbance and a shift from 354 to 353nm (the latter is due to intercalated tetraol). The non-covalent binding constants are approximately 8200M^?1 for BPDE and 3300M^?1 for the tetraol at 25°C, pH 7.0. Covalent adduct formation between BPDE and DNA occurs either at another, external binding site, or after some rearrangement of the intercalated BPDE, since covalent adducts display a 345nm absorption maximum (2nm red shift only).     

Linear dichroism characteristics of ethidium-and proflavine-supercoiled DNA complexes

A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide (EB) and proflavine (PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured at the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB, or PF, respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04-0.05, and then decreases as r is increased further, mimicking the behavior of the sedimentation coefficients, viscosities, and gel electrophoresis patterns measured by other workers at similar values of r. However, LD/A at 520 nm, which is due to DNA-bound EB molecules, is constant within the range of r values of 0.02-0.06 in which the magnitude of LD/A determined at 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r, both exhibiting pronounced maxima at r = 0.05; this parallel behavior is expected according to a simple intercalation model in which the DNA bases and drug molecules are stacked on top of one another, and in which both are oriented to similar extents in the flow gradient. The unexpected differences in the dependencies of (LD/A)258 and (LD/A)520 on r in the case of EB bound to supercoiled DNA, are attributed to differences in the net overall alignment of the EB molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound EB molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound EB molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

Picosecond and microsecond pulse laser studies of exciton quenching and exciton distribution in spinach chloroplasts at low temperatures

Studies of the fluorescence quantum yield and decay times, determined at the emission maxima of 685 and 735 nm, using picosecond laser pulses for excitation, indicate that the pigments which are responsible for the 735 nm emission derive their energy by transfer of singlet excitons from the light-harvesting pigments and not by direct absorption of photons. Microsecond pulse laser studies of the fluorescence quantum yields at these two fluorescence wavelengths indicate that long lived quenchers (most probably triplet states), which quench singlet excitons, accumulate preferentially within the long wavelength pigment system which gives rise to the 735 nm emission band.     

Intrastrand G-U cross-links generated by the oxidation of guanine in 5'-d(GCU) and 5'-r(GCU)

It has been suggested that carbonate radical anions are biologically important because they may be produced during the inflammatory response. The carbonate radicals can selectively oxidize guanine in DNA and RNA by one-electron transfer mechanisms and the guanine radicals thus formed decay by diverse competing pathways with other free radicals or nucleophiles. Using a photochemical method to generate CO(3)(-) radicals in vitro, we compare the distributions of products initiated by the one-electron oxidation of guanine in the trinucleotides 5'-r(GpCpU) and 5'-d(GpCpU) in aqueous buffer solutions (pH 7.5). Similar distributions of stable end products identified by LC-MS/MS methods were found in both cases. The guanine oxidation products include the diastereomeric pair of spiroiminodihydantoin (Sp) and 2,5-diamino-4H-imidazolone (Iz). In addition, intrastrand cross-linked products involving covalent bonds between the G and the U bases (GCU) were also found, although with different relative yields in the 2'-deoxy- and the ribotrinucleotides. The positive-ion MS/MS spectra of the 5'-r(GpCpU) and 5'-d(GpCpU) products clearly indicate the presence of covalently linked G-U products that have a mass smaller by 2 Da than the sum of the G and U bases in both types of trinucleotides. The 5'-d(GCU) cross-linked product was further characterized by 1D and 2D NMR methods that confirm its cyclic structure in which the guanine C8 atom is covalently linked to the uracil N3 atom.     

Accumulation of polyunsaturated fatty acids by cladocerans: effects of taxonomy,temperature and food

1. Zooplankton are important in transferring dietary nutrients, including polyunsaturated fatty acids (PUFA), up through aquatic food webs. 2. We tested the hypothesis that the taxonomic composition of zooplankton affects the retention and subsequent transfer of PUFA from upwards through the food web. Using laboratory experiments, we investigated dietary PUFA accumulation and bioconversion capacities of six cladoceran species (Ceriodaphnia sp., Daphnia longispina, Daphnia magna, Daphnia pulex, Scapholeberis mucronata and Simocephalus vetulus) fed on two diets (Scenedesmus obliquus and Cryptomonas sp.) that differed in their PUFA profiles. We performed experiments at two different temperatures (14 and 20 °C) to assess the role of temperature in the trophic transfer of PUFA. 3. There was little variation in the concentrations of PUFA in these cladocerans which were controlled by dietary PUFA supply. Moreover, as expected, the concentrations of PUFA in all cladoceran species were higher at low temperature. 4. However, even if the composition of PUFA in the cladoceran species generally corresponded to that in their diet, preferential accumulation of some PUFA was recorded in all these taxa. When fed on a highly unsaturated fatty acid‐deficient diet, all the cladocerans showed some ability to convert C18‐PUFA into arachidonic acid and eicosapentaenoic acid. Interspecific variation in the ability to accumulate and bioconvert PUFA in cladocerans was more pronounced at low temperature (14 °C) for both diets. 5. Our results strongly suggest that in heterogeneous habitats with food partitioning between co‐existing cladocerans, foraging behaviour may affect the transfer of PUFA more strongly than interspecific variation in accumulating and/or bioconverting dietary PUFA.     

Short‐term effects of CO2 and O2 on citrate metabolism in illuminated leaves

Although there is now a considerable literature on the inhibition of leaf respiration (CO₂ evolution) by light, little is known about the effect of other environmental conditions on day respiratory metabolism. In particular, CO₂ and O₂ mole fractions are assumed to cause changes in the tricarboxylic acid pathway (TCAP) but the amplitude and even the direction of such changes are still a matter of debate. Here, we took advantage of isotopic techniques, new simple equations and instant freeze sampling to follow respiratory metabolism in illuminated cocklebur leaves (Xanthium strumarium L.) under different CO₂/O₂ conditions. Gas exchange coupled to online isotopic analysis showed that CO₂ evolved by leaves in the light came from ‘old’ carbon skeletons and there was a slight decrease in ¹³C natural abundance when [CO₂] increased. This suggested the involvement of enzymatic steps fractionating more strongly against ¹³C and thus increasingly limiting for the metabolic respiratory flux as [CO₂] increased. Isotopic labelling with ¹³C₂‐2,4‐citrate lead to ¹³C‐enriched Glu and 2‐oxoglutarate (2OG), clearly demonstrating poor metabolism of citrate by the TCAP. There was a clear relationship between the ribulose‐1,5‐bisphosphate oxygenation‐to‐carboxylation ratio (v_o/v_c) and the ¹³C commitment to 2OG, demonstrating that 2OG and Glu synthesis via the TCAP is positively influenced by photorespiration.     

Structure of a high fidelity DNA polymerase bound to a benzo[a]pyrene adduct that blocks replication

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.     

Spiroiminodihydantoin lesions derived from guanine oxidation: structures, energetics, and functional implications

Reactive oxygen species present in the cell generate DNA damage. One of the major oxidation products of guanine in DNA, 8-oxo-7,8-dihydroguanine, formed by loss of two electrons, is among the most extensively studied base lesions. The further removal of two electrons from this product can yield spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G --> T and G --> C transversions. Hence, they are of interest as examples of endogenous DNA damage that may initiate cancer. To interpret the mutagenic properties of the Sp lesions, an understanding of their structural properties is needed. To elucidate these structural effects, we have carried out computational investigations at the level of the Sp-modified base and nucleoside. At the base level, quantum mechanical geometry optimization studies have revealed exact mirror image symmetry of the R and S stereoisomers, with a near-perpendicular geometry of the two rings. At the nucleoside level, an extensive survey of the potential energy surface by molecular mechanics calculations using AMBER has provided three-dimensional potential energy maps. These maps reveal that the range and flexibility of the glycosidic torsion angles are significantly more restricted in both stereoisomeric adducts than in unmodified 2'-deoxyguanosine. The structural and energetic results suggest that the unusual geometric, steric, and hydrogen bonding properties of these lesions underlie their mutagenicity. In addition, stereoisomer-specific differences indicate the possibility that their processing by cellular replication and repair enzymes may be differentially affected by their absolute configuration.     

Oxidative DNA damage associated with combination of guanine and superoxide radicals and repair mechanisms via radical trapping

In living tissues under inflammatory conditions, superoxide radicals (O(2)*)) are generated and are known to cause oxidative DNA damage. However, the mechanisms of action are poorly understood. It is shown here that the combination of O(2)* with guanine neutral radicals, G(-H)* in single- or double-stranded oligodeoxyribonucleotides (rate constant of 4.7 +/- 1.0 x 10(8) m(-1) s(-1) in both cases), culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo-7,8-dihydroguanine). The G(-H)* and O(2)* radicals were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of guanine in the 5'-d(CC[2AP]-TCGCTACC) strands and the trapping of the ejected electrons by molecular oxygen (Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and Geacintov, N. E. (2000) Phys. Chem. Chem. Phys. 2, 4399-4408). The addition of Cu,Zn-superoxide dismutase, known to react rapidly with superoxide, dramatically enhances the life-times of guanine radicals from 4 to 7 ms to 0.2-0.6 s in the presence of 5 microm superoxide dismutase. Oxygen-18 isotope labeling experiments reveal two pathways of 8-oxo-7,8-dihydroguanine formation including either addition of O(2)* to the C-8 position of G(-H)* (in the presence of oxygen), or the hydration of G(-H)* (in the absence of oxygen). The formation of the guanine lesions via combination of guanine and superoxide radicals is greatly reduced in the presence of typical antioxidants such as trolox and catechol that rapidly regenerate guanine by the reductive "repair" of G(-H)* radicals. The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed.     

Energy migration and exciton trapping in green plant photosynthesis

The possible origins of the different fluorescence decay components in green plants are discussed in terms of a random walk and Butler's bipartite model. The interaction of the excitations with the photosystem II reaction centers and, specifically, the regeneration of theses excitations by charge recombination within the reaction centers, are considered. Based on comparisons between fluorescence decay profiles, time-dependent exciton annihilation and photoelectric phenomena, it appears that the fast 200 ps decay component corresponds to primary energy transport from the antenna to the reaction centers and is dominant in filling the photosystem II reaction centers.