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Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. The Cu(II)-thiol system for DNA strand scission.

ABSTRACT: In the presence of Cu(II) ions, supercoiled DNA is cleaved in neutral solution by low concentrations of thiols. Supercoiled plasmid DNA is cleaved first to open circular DNA, which in turn produces linear DNA and eventually fragments. Cleavage is strongly temperature-dependent and is maximal at 0.10-0.25 M-NaCl concentration. In the presence of excess of either component of the Cu(II)-thiol pair, the extent of cleavage depended on the concentration of the limiting partner, and was easily detectable down to micromolar concentrations of limiting GSH. Scavengers of oxygen-derived species (such as hydrogen peroxide, superoxide radical ion and hydroxyl radical) indicated that the hydroxyl radical may be involved in the cleavage mechanism. DNA cleavage leads to some production of 2-thiobarbituric acid-reactive species and some of the cleavage sites, at least, had 5'-hydroxy and/or 3'-hydroxy groups. There was extensive base damage before cleavage. Studies with S1 nuclease indicated no gross sequence preference for Cu(II)-GSH cleavage of pSP64 plasmid DNA. The Cu(II)-thiol system did not appear to target special structural features in the DNA such as Z-DNA inserts, cruciform structures or left-handed (but non-Z) DNA. Cleavage might arise from a reagent generated either by the Cu(II)-thiol combination in free solution or by attack involving Cu(II) ions pre-bound to DNA. The attack of GSH plus Cu(II) ions on DNA may be a potential toxic lesion under physiological conditions unless special protective measures operate efficiently in the cell.

SUBMITTER: Reed CJ

PROVIDER: S-EPMC1150097 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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Project description:Kaempferol is a flavonoid that occurs in tea and in many vegetables and fruits, including broccoli, cabbage, beans, grapes, apples, and strawberries. The efficacy of Kaempferol has been demonstrated in the treatment of breast, esophageal, cervical, ovarian, and liver cancers and leukemia, which very likely arises from its prooxidant properties and the activation of pro-apoptotic pathways. Indeed, this matter has already been the focus of a number of published studies and reviews. The aim of the present study was to elucidate the antioxidant vs. prooxidant properties of flavonoids in the presence of the redox-active metal, copper (II) ion, by means of the Fenton reaction. The specific motivation of this work is that, since an increased level of Cu(II) ions is known to be associated with many disease states such as neurological conditions (Alzheimer's disease) and cancer, any interaction between these ions and flavonoids might affect the outcome of therapeutic uses of the latter. The structure of the Cu-kaempferol complex in DMSO was investigated by means of low temperature EPR spectroscopy, which confirmed the existence of at least two distinct coordination environments around the copper (II) ion. UV vis-spectra of kaempferol and its Cu(II) complex in DMSO revealed an interaction between the 5-OH (A ring) group and the 4-CO (C ring) group of kaempferol with Cu(II) ions. An ABTS assay confirmed that kaempferol acted as an effective radical scavenger, and that this effect was further enhanced in the form of the Cu(II)-kaempferol complex. Quantitative EPR spin trapping experiments, using DMPO as the spin trap, confirmed suppression of the formation of a mixture of hydroxyl, superoxide, and methyl radicals, in a Fenton reaction system, upon coordination of kaempferol to the redox-active Cu(II) ions, by 80% with respect to the free Cu(II) ions. A viscometric study revealed a better DNA-intercalating ability of the Cu-kaempferol complex than for free kaempferol, essential for conferring anticancer activity of these substances. The results of the viscometric measurements were compared with those from a DNA damage study of Cu-kaempferol complexes in a Fenton reaction system, using gel electrophoresis. At low concentrations of kaempferol (Cu-kaempferol ratios of 1:1 and 1:2), a very weak protective effect on DNA was noted, whereas when kaempferol was present in excess, a significant DNA-protective effect was found. This can be explained if the weakly intercalated kaempferol molecules present at the surface of DNA provide protection against attack by ROS that originate from the Fenton reaction involving intercalated Cu(II)-kaempferol complexes. Following the application of ROS scavengers, L-histidine, DMSO, and SOD, gel electrophoresis confirmed the formation of singlet oxygen, hydroxyl radicals, and superoxide radical anions, respectively. We propose that the prooxidant properties of Cu-kaempferol complexes may provide anticancer activity of these substances. When present in excess, kaempferol displays antioxidant properties under Cu-Fenton conditions. This suggests that kaempferol might prove a suitable candidate for the prevention or treatment of oxidative stress related medical conditions that involve a disturbed metabolism of redox metals such as copper, for example, Menkes disease, and neurological disorders, including Alzheimer's disease. For the potential use of kaempferol in clinical practice, it will be necessary to optimize the dose size and critical age of the patient so that this flavonoid may be beneficial as a preventive drug against cancer and neurological disorders.

Project description:BackgroundCopper nucleases as a famous class of artificial metallonucleases have attracted considerable interest in relation to their diverse potentials not only as therapeutic agents but also in genomic researches. Copper nucleases present high efficient oxidative cleavage of DNA, in which DNA strand scission occurs generally after hydrogen atom abstracted from a sugar moiety. In order to achieve the selective cleavage of DNA sequences by copper nucleases, the DNA specific recognition agents of the Dervan-type hairpin and cyclic polyamides can be considered as proper carriers of copper nucleases. Investigation of the DNA cleavage selectivity of copper nucleases assisted by the hairpin and cyclic polyamides at the molecular level has not yet been elucidated.ResultsWe carried out a series of molecular dynamics simulations for the nuclease [Cu(BPA)]2+ or [Cu(IDB)]2+ bound to the hairpin/cyclic polyamide and associated with DNA to investigate the selective DNA cleavage properties of Cu(II)-based artificial nucleases. The simulated results demonstrate that the DNA cleavage selectivity of the two nucleases assisted by the hairpin polyamide is improved efficiently. The [Cu(BPA)]2+ or [Cu(IDB)]2+ nuclease with a substrate OOH- bound to the hairpin polyamide can be stably located at the minor groove of DNA, and possibly abstracts H atom from the sugar of DNA. However, the DNA cleavage properties of the two nucleases assisted by the cyclic polyamide are significantly poor due to the rigidity of linking region between the cyclic polyamide and nuclease. With introduction of the flexible linker -CH2CH2CH2NH2, the modified cyclic polyamide can assist the two copper nucleases to improve the selective DNA cleavage properties efficiently.ConclusionA flexible linker and a proper binding site of the polyamide-type recognition agents play an important role in improving the DNA cleavage selectivity of copper nucleases. Current investigations provide an insight into the DNA cleavage specificities of chemical nucleases assisted by an appropriate nucleic acid recognition agent.

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Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. The Cu(II)-thiol system for DNA strand scission.

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