Inhibition of Pyrimidine Dimer Formation in DNA by Cationic Molecules: Role of Energy Transfer

In addition to the acridine dyes, acridine orange and proflavine, we find that three other cationic molecules which bind to DNA-ethidium bromide, chloroquine, and methyl green-inhibit the production of cyclobutyl pyrimidine dimers by ultraviolet radiation. Intercalation is not necessary for dimer inhibition. The long range nature of the inhibition implies that energy transfer is responsible. The transfer is between the lowest excited singlet state of DNA and the acceptor singlet, and seems to involve the F?rster mechanism.     

Evidence for Excision of Ultraviolet-Induced Pyrimidine Dimers from the DNA of Human Cells In Vitro

Within 12-24 hr after human cells were irradiated with ultraviolet light, approximately 50% of the ultraviolet-induced pyrimidine dimers were lost from the DNA. Pyrimidine dimers were found in the TCA-soluble fraction of ultraviolet-irradiated cells at 24 hr. Excess thymidine, caffeine, or hydroxyurea had no effect on the loss of pyrimidine dimers from the DNA of ultraviolet-irradiated cells.     

Mechanisms of Inhibition of Pyrimidine Dimer Formation in Deoxyribonucleic Acid by Acridine Dyes

The ultraviolet (UV)-induced formation of cyclobutyl pyrimidine dimers in Escherichia coli deoxyribonucleic acid (DNA) in vitro has been investigated in terms of the mechanism of inhibition by acridine dyes, the effect on dimer yield of specific singlet and triplet quenchers, and the mechanism of dimer formation. Our results indicate that (a) energy transfer is important in dimer reduction by acridines, (b) this transfer occurs from the singlet (S₁) of DNA, and (c) at room temperature triplet quenchers do not reduce dimer yield in DNA.     

Interactions of Polyoma and Mouse DNAs II. Polyoma-Induced Mouse DNA Replication and Pseudovirion Formation

Secondary mouse embryo (ME) cultures which had been grown prior to infection in the presence of 5-bromodeoxyuridine (BUdR) and 5-fluorodeoxy-uridine were found to be permissive for polyoma virus (16). The DNA extracted from the progeny virus yielded two bands on CsCl isopycnic centrifugation. The light band (LL) contained supercoiled circular (polyoma DNA I), open circular (polyoma DNA II), and linear (polyoma DNA III) molecules, as was seen by electron microscopy. The hybrid band (HL) contained exclusively linear molecules. This DNA was pure, density - labeled, pseudovirion DNA, i.e., fragmented HL mouse DNA. The quantitative comparison of HL and LL polyoma DNA III from six different virus preparations always revealed an excess of HL DNA, the ratio of HL/LL being between 1.2 and 2.2. These results led to the conclusion that in BUdR-prelabeled, polyoma-infected ME cells pseudovirion DNA is excised both from unreplicated and newly replicated regions of mouse DNA.     

DNA Repair Effect of Traditional Sweet Pepper Fushimi-togarashi: Seen in Suppression of UV-induced Cyclobutane Pyrimidine Dimer in Human Fibroblast

The aqueous fraction of Fushimi sweet pepper increased the repair effect of the solvent control against UV-induced cyclobutane pyrimidine dimers in human fibroblast to 150%, but ordinary sweet pepper did not have a statistically significant effect. When Fushimi sweet pepper was boiled, the activity of the aqueous fraction was elevated to 209% of the control (p<0.05), while that of the grilled state was decreased to 125% of the control. The repair activity of a dialyzate (MW<12,000) of the aqueous fraction from Fushimi sweet pepper showed 191% of the control (p<0.05). The dialyzate was contained 1.9% in the weight of the fresh fruit body of Fushimi sweet pepper, and the activity can be stable in its boiling state, and it might be therefore considered to be the worthy source for expecting the DNA repair activity in human diet.     

The Interactions of Enzyme and Chemical Probes with Inverted Repeats in Supercoiled DNA

Abstract

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as SI, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endo- nucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.     

The Class III Cyclobutane Pyrimidine Dimer Photolyase Structure Reveals a New Antenna Chromophore Binding Site and Alternative Photoreduction Pathways

Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.     

NaCI胁迫对UV-B辐射诱导的绿豆环丁烷嘧啶二聚体和紫外吸收物质含量变化的影响

将2个对UV-B敏感性不同的绿豆品种‘秦豆-20’和‘中绿-1’幼苗放在培养室内,进行0.4W/m~2 UV-B辐射和0.4%NaCl胁迫的单独或复合处理,研究了NaCl胁迫对UV-B辐射诱导的DNA伤害和修复的影响。结果显示:在NaCl胁迫下,(1)在光下抗UV-B的品种‘中绿-1’的环丁烷嘧啶二聚体(CPD)累积量降低,而敏感品种‘秦豆-20’的CPD累积量未发生变化;(2)两品种CPD形成量均比无NaCl胁迫时低;(3)抗UV-B品种DNA的光、暗修复能力均比无NaCl胁迫时高:(4)而敏感品种DNA的光修复能力比无NaCl胁迫时低、暗修复能力未发生变化。另外,CPD形成量与紫外吸收物含量间具有明显的负相关性。说明NaCl胁迫不仅影响2个绿豆品种幼苗的CPD形成量,而且影响DNA的光、暗修复能力,进而导致了CPD累积量发生变化,由此影响了幼苗的UV-B敏感性。结果也暗示CPD形成量的变化是由于紫外吸收物质含量的不同所导致的。     

Stereoselective Differentiation between Ribonucleosides and Deoxynucleosides by Reaction with the Copper(II) Acetate Dimer

METAL ions are involved in the distinction between ribonucleosides and deoxynucleosides in biochemical processes. For example, RNA polymerase incorporates only ribonucleotides into RNA in the presence of magnesium ions, whereas manganese ions may bring about the misincorporation of deoxy-nucleotides¹. Therefore it is of interest to examine ways in which metal ions can distinguish between these two types of molecules.     

Molecular Mechanisms of Pyrimidine Dimer Excision in Saccharomyces cerevisiae: Incision of Ultraviolet-Irradiated Deoxyribonucleic Acid In Vivo

A group of genetically related ultraviolet (UV)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD⁺ strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.     

DNA Duplexes Containing Altered Sugar Residues as Probes of EcoRII and MvaI Endonuclease Interactions with Sugar-Phosphate Backbone

Abstract

Oligonucleotides containing 1-(β-D-2′-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(β-D-2′-deoxy-threo-pentofuranosyl)thymine (dT_x) in place of dC and dT residues in the EcoRII and MvaI recognition site CC^A/_TGG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2′-deoxyxylosyl moieties of dC_x and dT_x, 3′-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2′-deoxyxylo-syl moiety into a dC·dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA·dT pair the effect of a 2′-deoxyxylose incorporation was much more pronounced. Multiple dC_x modifications and their combination with dT_x did not enhance the destabilization effect. Hydrolysis of dC_x-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dT_x residue was cleaved by the enzyme, but k_cat/K_M was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.     

Toxic Effects of Copper on Photosystem II of Spinach Chloroplasts

The room temperature fluorescence induction of chloroplasts was utilized as a probe to locate the site of inhibition on PSII by copper. It was found that, while the initial fluorescence yield was hardly affected, the variable fluorescence yield was lowered without significant change in its kinetics. Addition of DCMU, or abolishing oxygen evolution capability by Tris treatment, did not alter this basic inhibition pattern. Copper was also found to lower the fluorescence yield of chloroplasts treated with linolenic acid which inhibited the secondary electron transport on both oxidizing and reducing sides of PSII. The data indicate that copper adversely affects the primary charge separation at the PSII reaction center. We suggest that the inhibition is due to creation of a lesion close to the reaction center, leading to increased dissipation of incoming excitation energy to heat.     

Copper(II) complexes of diclofenac: Spectroscopic studies and DNA strand breakage

Copper(II) complexes of diclofenac with interesting anti-inflammatory profiles have been prepared and studied by infrared and electronic spectroscopy. In the solid state and in polar and coordinating solvents, all the complexes are solvated binuclear carboxylato-bridged complexes, [Cu(L)²(S)]², where L is monodeprotonated diclofenac and S is the axially bonded solvent. The effect of the copper(II) complexes on the in vitro DNA strand breakeage was studied by agarose gel electrophoresis. Relaxation or double stranded scissions of pDNA were observed leading to the formation of linear pDNA. Treatment of pDNA with high concentrations of these compounds caused a disappearance of pDNA. For the parent drug, sodium diclofenac, no effect on the pDNA was observed. This study presents some indications that the binuclear copper(II) complexes, [Cu(L)²(S)]², could have some relevance in the treatment of tumor cell lines.     

Three-Dimensional Structure of the Photosystem II Core Dimer of Higher Plants Determined by Electron Microscopy

Here we report the first three-dimensional structure of a higher plant photosystem II core dimer determined by electron crystallography at a resolution sufficient to assign the organization of its transmembrane helices. The locations of 34 transmembrane helices in each half of the dimer have been deduced, 22 of which are assigned to the major subunits D1 (5), D2 (5), CP47 (6), and CP43 (6). CP47 and CP43, located on opposite sides of the D1/D2 heterodimer, are structurally similar to each other, consisting of 3 pairs of transmembrane helices arranged in a ring. Both CP47 and CP43 have densities protruding from the lumenal surface, which are assigned to the loops joining helices 5 and 6 of each protein. The remaining 12 helices within each half of the dimer are attributed to low-molecular-weight proteins having single transmembrane helices. Comparison of the subunit organization of the higher plant photosystem II core dimer reported here with that of its thermophilic cyanobacterial counterpart recently determined by X-ray crystallography shows significant similarities, indicative of a common evolutionary origin. Some differences are, however, observed, and these may relate to variations between the two classes of organisms in antenna linkage or thermostability.     

Copper(II) facilitates bleomycin-mediated unwinding of plasmid DNA

The unwinding of plasmid DNA by bleomycin A2 (BLM A2) was investigated by use of two-dimensional gel electrophoresis. It was found that Cu2+ ions greatly facilitated the unwinding of topoisomers of plasmid DNA by BLM A2 at concentrations where cupric ions alone had no effect on DNA supercoiling. The concentration of BLM A2 required for observable unwinding was reduced at least 100-fold in the presence of equimolar Cu2+. A plot of [Cu2+] vs extent of DNA unwinding in the presence of 10(-4) M BLM A2 gave a curve consistent with the action of cupric ions on BLM in an allosteric fashion, possibly rearranging the drug into a conformation that facilitates DNA unwinding. The participation of the metal center in enhancing DNA unwinding via direct ionic interaction with one or more negatively charged groups on the DNA duplex also seems possible. Further analysis of the structural factors required for BLM-mediated DNA unwinding was carried out with Cu2+ + BLM demethyl A2, the latter of which differs from BLM A2 only in that it lacks a methyl group, and associated positive charge, at the C-terminus. Cu(II).BLM demethyl A2 was found to be much less effective than Cu(II).BLM A2 as a DNA unwinding agent, emphasizing the strong dependence of this process on the presence of positively charged groups within the BLM molecule. These findings constitute the first direct evidence that the metal center of BLM can participate in DNA interaction, as well as in the previously recognized role of oxygen binding and activation.     

Effects of Dietary Copper (II) Sulfate and Copper Proteinate on Performance and Blood Indexes of Copper Status in Growing Pigs

160 crossbred (Duroc × Landrace ×Yorkshire) gilts averaged 21.25 kg body weight were used to study the effects of dietary copper (II) sulfate (CuSO₄) and copper proteinate (Cu-Pr) on growth performance, plasma Cu concentration, ceruloplasmin activity, and erythrocyte Cu/Zn-superoxide dismutase (SOD) activity. All pigs were allotted to four treatments and fed with basal diets supplemented with 0 (control), 250 mg /kg Cu as CuSO₄, and 50 and 100 mg/kg Cu as Cu-Pr. Growth performance was determined based on two growth phase (phase 1: days 0 to 15, phase 2: days 15 to 30). After 30 days of the treatment, 16 pig blood samples (four per treatment) were collected for indexes of copper status determination. The experimental results showed that compared with control, pigs fed with 250 mg Cu/kg as CuSO₄ and 100 mg Cu/kg as Cu-Pr had higher average daily gain and average daily feed intake in the whole growth phase (d 0 to 30). In addition, 250 mg Cu/kg as CuSO₄ and 100 mg/kg Cu as Cu-Pr enhanced plasma ceruloplasmin activity (P < 0.05), and 100 mg/kg Cu as Cu-Pr increased erythrocyte Cu/Zn-SOD activity (P < 0.01) compared with the control. There was no obvious treatment response on plasma Cu concentration in the present study.     

Copper(II) complexes of 1,10-phenanthroline-derived ligands: studies on DNA binding properties and nuclease activity

A series of copper(II) complexes of the type [Cu(L)]2+, where L = N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine and R = methyl (L1), n-propyl (L2), isopropyl (L3), sec-butyl (L4), or tert-butyl (L5) group, have been synthesized. The interaction of the complexes with DNA has been studied by DNA fiber electron paramagnetic resonance (EPR) spectroscopy, emission, viscosity and electrochemical measurements and agarose gel electrophoresis. In the X-ray crystal structure of [Cu(HL2)Cl2]NO3, copper(II) is coordinated to two ring nitrogens and one of the two secondary amine nitrogens of the side chains and two chloride ions as well and the coordination geometry is best described as trigonal bipyramidal distorted square based pyramidal (TBDSBP). Electronic and EPR spectral studies reveal that all the complexes in aqueous solution around pH 7 possess CuN3O2 rather than CuN4O chromophore with one of the alkylamino side chain not involved in coordination. The structures of the complexes in aqueous solution around pH 7 change from distorted tetragonal to trigonal bipyramidal as the size of the alkyl group is increased. The observed changes in the physicochemical features of the complexes on binding to DNA suggest that the complexes, except [Cu(L5)]2+, bind to DNA with partial intercalation of the derivatised phen ring in between the DNA base pairs. Electrochemical studies reveal that the complexes prefer to bind to DNA in Cu(II) rather than Cu(I) oxidation state. Interestingly, [Cu(L5)]2+ shows the highest DNA cleavage activity among all the present copper(II) complexes suggesting that the bulky N-tert-butyl group plays an important role in modifying the coordination environment around the copper(II) center, the Cu(II)/Cu(I) redox potential and hence the formation of activated oxidant responsible for the cleavage. These results were compared with those for bis(1,10-phenanthroline)copper(II), [Cu(phen)2]2+.