PHOTOLYSIS OF PHOSPHODIESTER BONDS IN PLASMID DNA BY HIGH INTENSITY UV LASER IRRADIATION

Abstract— The cleavage of phosphodiester bonds in DNA exposed to high intensity UV laser pulses in aerated aqueous solution has been investigated using a krypton fluoride excimer laser (248 nm) and bacterial plasmid DNA. The dependence of strand breakage on fluence and intensity has been studied in detail and shows that the process is non-linear with respect to intensity. The relationship between the quantum yield for strand breakage and intensity shows that the strand breakage reaction involves two-photon excitation of DNA bases. The quantum yield rises with intensity from a lower value of 7 times 10^-5 until a maximum value of 4.5 times 10^-4 is attained at intensities of 10¹¹ W m^-2 and above. This value is approximately fifty-fold higher than the quantum yield for strand breakage induced by exposure to low density UV irradiation (254 nm, 12 W m^-2). DNA sequencing experiments have shown that strand breakage occurs by the specific cleavage of the phosphodiester bond which lies immediately 3' to guanine residues in the DNA, leaving some alkali-labile remnant attached to the terminal phosphate. A mechanism for DNA strand breakage which involves the generation of guanine radical cations is proposed.     

Synthesis and DNA strand breakage activity of some 1,4-diazepines

Reactions of 1,3-propanediamine with alpha-dicarbonyl compounds (1a-e) were examined and various condensed heterocyclic compounds such as 1,4-diazepines (2) and 3-pyrimidine derivatives (3) were obtained. Some of 1,4-diazepines (2) showed DNA strand breakage activity.     

Irradiation of DNA with 193 nm Light Yields Formamidopyrimidine-DNA Glycosylase (Fpg) Protein-Sensitive Lesions

Abstract— Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample φ_ssb= [1.5 ± 0.1] ± 10⁻⁴, argon-saturated sample φ_ssb= [0.9 ± 0.1] ± 10⁻⁴). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimi-dine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand breakage (air-saturated sample φ_fpg= [33.1 ± 3.1] ± 10⁻⁴, argon-saturated sample φ_fpg= [23.8 ± 2.6] × 10 ⁴). This result indicates that 193 nm light induces other modification) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.     

ALKALINE ELUTION STUDIES OF HEMATOPORPHYRIN-DERIVATIVE PHOTOSENSITIZED DNA DAMAGE AND REPAIR IN CHINESE HAMSTER OVARY CELLS

Abstract We have used alkaline elution to study DNA damage produced by the photosensitizer hematoporphyrin derivative (HPD) in cultured Chinese hamster cells. Dosimetry was performed by measuring fluence and calculating photon absorption by intracellular HPD. HPD photosensitization causes DNA strand breakage. These breaks are repaired by the cell, although their fractional rate of repair is smaller than that for X-ray induced strand breaks at equivalent levels of strand breakage. The combined DNA polymerase inhibitors cytosine arabinoside and hydroxyurea suppress the repair of HPD-photosensitized breaks more strongly than they suppress repair of X-ray induced breaks. Addition of novobiocin to the aforementioned inhibitors causes almost total suppression of photosensitized break repair. A nucleotide excision repair system with inhibitor susceptibility similar to that of the system which removes pyrimidine dimers thus does not act upon HPD-photosensitized damage. The repair rate and inhibitor sensitivity findings together suggest biologically important differences in the chemical nature of X-ray induced and HPD-photosensitized strand breaks. In addition to strand breaks, HPD photosensitization produces covalent DNA-protein crosslinks, some of which persist through at least 90 min incubation, but which are repaired within 180 min.     

A PHOTOSYSTEM I-PHENOSAFRANINE SOLAR CELL

Abstract Bacteriophage λ_vir was inactivated when it was irradiated with near-UV light in the presence of chlorpromazine. DNA strand breakage in the treated phage was indicated by alkaline sucrose gradient centrifugation. The number of the breaks was increased with increasing fluence. Although the inactivation rate was enhanced with a decreasing salt concentration in the reaction mixture and under a nitrogen atmosphere, the number of the strand breaks was not altered in either case. Therefore, the DNA strand breakage is not a sole lethal damage in the treated phage. The addition of NaN₃ repressed the inactivation and the reaction in a D₂O medium enhanced the inactivation even if the reaction mixture was irradiated under anaerobic conditions. Under anaerobic conditions, the inactivation occurs presumably via a radical mechanism.     

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

Resonant Formation of Strand Breaks in Sensitized Oligonucleotides Induced by Low‐Energy Electrons (0.5–9 eV)

Halogenated nucleobases are used as radiosensitizers in cancer radiation therapy, enhancing the reactivity of DNA to secondary low‐energy electrons (LEEs). LEEs induce DNA strand breaks at specific energies (resonances) by dissociative electron attachment (DEA). Although halogenated nucleobases show intense DEA resonances at various electron energies in the gas phase, it is inherently difficult to investigate the influence of halogenated nucleobases on the actual DNA strand breakage over the broad range of electron energies at which DEA can take place (<12 eV). By using DNA origami nanostructures, we determined the energy dependence of the strand break cross‐section for oligonucleotides modified with 8‐bromoadenine (^8BrA). These results were evaluated against DEA measurements with isolated ^8BrA in the gas phase. Contrary to expectations, the major contribution to strand breaks is from resonances at around 7 eV while resonances at very low energy (<2 eV) have little influence on strand breaks.     

SINGLE-STRAND BREAKS INDUCED IN DNA BY VACUUM-ULTRAVIOLET RADIATION

Abstract— The efficiency of vacuum u.v. for producing single-strand breaks in DNA was determined for wavelengths between 58 and 254 nm (corresponding to photon energies of 21·2 and 4·9 eV, respectively) by using the supertwisted RF-DNA of bacteriophage φX174. The cross-section for production of single-strand breaks increases continuously by about 5 orders of magnitude between 5 and 10 eV photon energy, whereas from 11 to 21 eV the number of strand breaks produced per unit of incident radiation energy is approximately constant. Thus, absorption of a 10-eV photon causes DNA strand breaks with maximum efficiency. In addition, the number of electrons liberated from DNA by photons below 10 eV is one or two orders of magnitude higher than the frequency of strand breaks, demonstrating that in this energy range only a small fraction of the ionizations leads to strand breakage in DNA.     

Sensitizing DNA Towards Low‐Energy Electrons with 2‐Fluoroadenine

2‐Fluoroadenine (^2FA) is a therapeutic agent, which is suggested for application in cancer radiotherapy. The molecular mechanism of DNA radiation damage can be ascribed to a significant extent to the action of low‐energy (<20 eV) electrons (LEEs), which damage DNA by dissociative electron attachment. LEE induced reactions in ^2FA are characterized both isolated in the gas phase and in the condensed phase when it is incorporated into DNA. Information about negative ion resonances and anion‐mediated fragmentation reactions is combined with an absolute quantification of DNA strand breaks in ^2FA‐containing oligonucleotides upon irradiation with LEEs. The incorporation of ^2FA into DNA results in an enhanced strand breakage. The strand‐break cross sections are clearly energy dependent, whereas the strand‐break enhancements by ^2FA at 5.5, 10, and 15 eV are very similar. Thus, ^2FA can be considered an effective radiosensitizer operative at a wide range of electron energies.     

Enhanced radical-scavenging activity of a planar catechin analogue

A catechin analogue in which the geometry was constrained to be planar was synthesized. The planar catechin showed excellent radical-scavenging ability, comparable to that of quercetin, and efficient protection against DNA strand breakage induced by the Fenton reaction.     

The action spectrum (313-435 nm) for killing Hoechst 33258 treated Chinese hamster ovary cells containing bromodeoxyuridine substituted DNA

Abstract— The action spectrum (313–435 nm) for killing Chinese hamster ovary cells containing bromo-deoxyuridine substituted DNA and treated with Hoechst 33258 was very similar to the absorption spectrum of the dye bound to chromatin, indicating that sensitization was mediated through direct absorption of radiation by the dye. The ratio of sensitization cross sections for 365 nm (plus dye) to 313 nm (no dye) was approx. 30 while this ratio for strand breakage was about one. These results are in agreement with the hypothesis that strand breaks are not the major class of lethal photoproducts induced via Hoechst 33258 sensitization.     

ALKALI-LABILE PHOTOLESIONS MAPPING TO PURINE SITES IN ULTRAVIOLET-IRRADIATED DNA

Gel sequencing experiments with the 5'- and 3'-end-labeled oligonucleotides d(A₃GA₄GA₅GA₆GA₃G) and d(AT) ₁₀ have demonstrated that dimeric adenine photoproducts and thymine-adenine photoadducts constitute alkali-labile lesions in UV-irradiated DNA. On treatment with hot piperidine, DNA strand breakage occurs predominantly at the sites of 5'-adenines in the dimeric photoproducts and of 3'-adenines in the thymine-adenine photoadducts. With 5'-end-labeled oligonucleotides of mixed sequence, major UV-induced loci for alkaline cleavage map to purine bases flanked on their 5'-side by two pyrimidines. This behavior does not arise from enhanced photoreactivity of purines in this sequence context as has been inferred from photofootprinting studies. Instead, as shown by 3'-labeling and selective substitution with 5-methylcytosine, it results from the anomalous electrophoretic mobility of 5'-end-labeled fragments produced by alkaline cleavage of DNA at adjacent pyrimidine (6-4) pyrimidone photoproducts.     

Sequence dependence of DNA radioprotection by the thiols WR-1065 and WR-151326

Sequence-dependent variations of DNA structure modulate radiation-induced strand breakage. Thiols reduce breakage by scavenging damaging radiolytic OH ^. and repairing sugar radicals. As shown by sequencing gel electrophoresis, WR-1065 radioprotection is modulated by sequence, whereas that of WR-151326, a larger thiol, is more evenly distributed. Molecular modelling was performed on complexes of a 53 bp oligonucleotide (belonging to a natural restriction fragment) with one molecule of WR-1065 or WR-151326. Energy minimised structures exhibit a broadening of the minor groove of an AAATT motif upon WR-1065 binding, and a narrowing of the groove upon WR-151326 binding. Consequently, the accessibility to OH^˙ of H4′ (whose abstraction leads to strand breakage) increases near WR-1065, whereas it decreases near WR-151326. This modifies locally the otherwise homogeneous radioprotection. The effect of WR-151326 strengthens the protection at all tested binding sites, whereas that of WR-1065 diminishes it in some regions, in good agreement with the observed radioprotection distribution. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 11 November 1998     

DNA targeted UVA photosensitization: characterization of an extremely photopotent iodinated minor groove binding DNA ligand

Previous studies have described UVA-induced DNA strand breakage at the binding sites of iodinated DNA minor groove binding bisbenzimidazoles. The DNA breakage, presumably mediated by the carbon-centred ligand radical produced by photodehalogenation, was also shown to be cytotoxic. The earlier studies included a comparison of three ligand isomers, designated ortho-, meta- and para-iodoHoechst, and the efficiency of photo-induction of strand breaks in plasmid DNA proved to be much higher for the ortho-isomer. We have now extended the comparison of the three isomers with respect to photo-induced cytotoxicity in K562 cells. Although the relationship between the extent of nuclear uptake and the concentration of the ligand in the medium was similar for the three isomers, assay of in situ dehalogenation in drug-treated cells indicated that the apparent cross-section for dehalogenation of the ortho-isomer was greater than 5-fold higher than that for the meta- and para-isomers. Also, analysis of clonogenic survival data showed that the dehalogenation event associated with ortho-iodoHoechst was a more efficient mediator of UVA-induced cytotoxicity in K562 cells than that for meta- or para-iodoHoechst. The number of dehalogenation events associated with 50% cell-kill for ortho-iodoHoechst (1.23+/-0.04 x 10(4)) was less than that for the para- (3.92+/-0.29 x 10(4)) and meta- (11.6+/-0.90 x 10(4)) isomers. Thus it is concluded that the photopotency of ortho-iodoHoechst, which is an important feature in the context of its potential use in clinical phototherapy, is due not only to more efficient UVA-mediated dehalogenation of the ligand, but also to greater cytotoxic potency per dehalogenation event.     

PRODUCTION OF BREAKS IN SINGLE- AND DOUBLE-STRANDED FORMS OF BACTERIOPHAGE øx174 DNA BY PROFLAVINE AND LIGHT TREATMENT

Abstract— Irradiation at 440 + 360 nm and a fluence rate of 3.8 kJm^-2 min^-1, of both complexes previously formed between proflavine and either øX circular single-stranded (ss) DNA or øX supercoiled duplex (RFI)DNA, induces single-strand scissions in the two DNAs under consideration. Linear øXSS DNA molecules are detected by sedimentation through alkaline sucrose gradients. After treatment of the øXRFI DNA, however, the degree of degradation is the same whether it is measured under neutral or alkaline conditions, indicating that alkaline-labile bonds are not created; moreover, double-strand breaks can only be detected after accumulation of single-strand breaks. In addition to the amount of proflavine bound to the DNA and the duration of irradiation, the following factors are shown to influence the nicking activity of the treatment: (1) the DNA structure (the øXRFI DNA is much more sensitive than the øXss DNA); (2) the ionic strength of the medium during irradiation (a high value of 0.5 leads to a markedly increased efficiency); (3) the addition of cysteamine (this latter compound decreases the reaction rate) and (4) the irradiation wavelength (after irradiation at 440 nm alone, the reaction occurs at a reduced rate and is sensitive to NaN₃). The kinetics of the nicking reaction does not follow a single-hit curve showing that at least one primary lesion occurs prior to strand breakage. On the other hand, strand scission cannot be detected after irradiation of the proflavine-DNA complexes at the low fluence rate causing a decrease in the infectivity of both øXSS and øXRFI DNAs. Similarly. the sedimentation pattern of the DNA extracted from treated øx174 phages 99.9% inactivated, is identical to that of the control ss DNA, although more drastic treatments are susceptible to induce single strand breaks inside the phage head. Finally, the unknown lesion (s) that is biologically important does not prevent the treated DNAs from penetrating into the hostcells.     

D-氨基葡萄糖与Sm^3＋和Eu^3＋离子配合物的合成和表征

D-氨基葡萄糖(2氨-基-2脱-氧-D葡-萄糖,简称GLCN)是甲壳素或壳聚糖的最终水解产物。由于其分子内含有多个反应中心(-OH,-NH2),且无毒、水溶性好,易与多种金属离子配位,而用于贵金属回收、工业废水处理等生态和环境保护方面;由于其与金属离子形成的配合物水溶性好、低毒、甚至无     

Modeling the action of an antitumor drug: a density functional theory study of the mechanism of tirapazamine

Density functional theory methods are employed to investigate experimentally proposed mechanisms by which the antitumor drug tirapazamine may react with a DNA sugar-C(1)' radical to give the sugar derivative deoxyribonolactone, with concomitant DNA strand breakage. For the previously proposed minor pathway, ionization of the sugar-C(1)' radical by tirapazamine, the calculated ionization energy, and the electron affinity of the models of the sugar-C(1)' radical of DNA and tirapazamine suggest that tirapazamine must be protonated to be able to oxidize the sugar-C(1)' radical. The preferred mechanism for reaction of tirapazamine with a sugar-C(1)' radical, in agreement with experimental observations, is found to proceed by direct attack of an N-oxide oxygen of tirapazamine at the sugar-C(1)' position, followed by homolytic cleavage of the N-O bond of the drug moiety. Possible alternative mechanisms are also investigated.     

193 NM LIGHT INDUCES SINGLE STRAND BREAKAGE OF DNA PREDOMINANTLY AT GUANINE

Irradiation of DNA with 193 nm light results in monophotonic photoionization, with the formation of a base radical cation and a hydrated electron (φ_P1 = 0.048–0.065). Although >50% of the photoionization events initially occur at guanine in DNA, migration of the “hole” from the other bases to guanine occurs to yield predominantly its radical cation or its deprotonated form. From sequence analysis, the data reveal that 193 nm light induces single strand breaks (ssb) in double-stranded DNA preferential 3’ to a guanine residue. However, it has previously been reported that 193 nm light yields very low yields of ssb (<2% of the yield of eaq). The distribution of these ssb at guanine is nonrandom, showing a dependence on the neighboring base moiety. The efficiency of ssb formation at nonguanine sites is estimated to be at least one order of magnitude lower. The preferred cleavage at guanine is consistent with migration and localization of the electron loss center at guanine. It is argued that singlet oxygen and the photoionized phosphate group of the sugar moiety are not major precursors to ssb. At present, the mechanisms of strand breakage are not known although a guanine radical or one of its products remain potential precursors.     

PHOTOINITIATED DNA DAMAGE BY MELANOGENIC INTERMEDIATES IN VITRO

Cysteinyldopas, metabolic by-products of activated melanocytes, are photochemically unstable in the presence of biologically relevant ultraviolet radiation (i.e. wavelengths > 300 nm). Initial photochemical processes involve free radical production; continued photolysis yields polymeric photoproducts. Radicals produced during 5SCD photolysis were trapped by 5,5-dimethyl-l-pyrrolidine-l-oxide (DMPO) and identified by their ESR spectra. Further characterization by use of nitroso spin trap (2-methvl-2-nitrosopropane-MNP) demonstrated that homolytic cleavage of the -S-CH₂ bond of the 5SCD cysteinyl side chain is a significant photochemical pathway. The potential photobiological significance of these reactive intermediates was investigated in vitro using isolated nucleic acids. Radiolabeled 5-[³⁵S]-cysteinyldopa was found to photobind to calf thymus DNA with 300 nm light activation. Under similar conditions, 5-S-cysteinyldopa also induced single strand breakage of ³H-radiolabeled superhelical, circular pBR322 plasmid DNA. The implications of the 5SCD photoinitiated DNA damage and the production of highly reactive free radicals in this process are discussed with respect to the etiology of various skin cancers, particularly malignant melanoma.     

Damage to model DNA fragments from very low-energy (<1 eV) electrons

Although electrons having enough energy to ionize or electronically excite DNA have long been known to cause strand breaks (i.e., bond cleavages), only recently has it been suggested that even lower-energy electrons (most recently 1 eV and below) can also damage DNA. The findings of the present work suggest that, while DNA bases can attach electrons having kinetic energies in the 1 eV range and subsequently undergo phosphate-sugar O-C sigma bond cleavage, it is highly unlikely (in contrast to recent suggestions) that electrons having kinetic energies near 0 eV can attach to the phosphate unit's P=O bonds. Electron kinetic energies in the 2-3 eV range are required to attach directly to DNA's phosphate group's P=O pi orbital and induce phosphate-sugar O-C sigma bond cleavages if the phosphate groups are rendered neutral (e.g., by nearby counterions). Moreover, significant activation barriers to C-O bond breakage render the rates of both such damage mechanisms (i.e., P=O-attached and base-attached) slow as compared to electron autodetachment and to other damage processes.