Repair of 8-methoxypsoralen photoinduced cross-links in yeast

Summary The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D₇ and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced crosslinks. In accordance with previous reports, the D₇ rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D₇ rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D₇ RAD⁺ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D₇ rad18-1 than in D₇ RAD⁺. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.     

DNA and RNA strand scission by copper, zinc and manganese superoxide dismutases

Copper/zinc (Cu/ZnSOD) and manganese (MnSOD) superoxide dismutases which catalyze the dismutation of toxic superoxide anion, O _inf2 ^sup– , to O₂ and H₂O₂, play a major role in protecting cells from toxicity of oxidative stress. However, cells overexpressing either form of the enzyme show signs of toxicity, suggesting that too much SOD may he injurious to the cell. To elucidate the possible mechanism of this cytotoxicity, the effect of SOD on DNA and RNA strand scission was studied. High purity preparations of Cu/ZnSOD and MnSOD were tested in an in vitro assay in which DNA cleavage was measured by conversion of phage X174 supercoiled double-stranded DNA to open circular and linear forms. Both types of SOD were able to induce DNA strand scission generating single- and double-strand breaks in a process that required oxygen and the presence of fully active enzyme. The DNA strand scission could be prevented by specific anti-SOD antibodies added directly or used for immunodepletion of SOD. Requirement for oxygen and the effect of Fe(II) and Fe(III) ions suggest that cleavage of DNA may be in part mediated by hydroxyl radicals formed in Fenton-type reactions where enzyme-bound transition metals serve as a catalyst by first being reduced by superoxide and then oxidized by H₂O₂. Another mechanism was probably operative in this system, since in the presence of magnesium DNA cleavage by SOD was oxygen independent and not affected by sodium cyanide. It is postulated that SOD, by having a similar structure to the active center of zinc-containing nucleases, is capable of exhibiting non-specific nuclease activity causing hydrolysis of the phosphodiester bonds of DNA and RNA. Both types of SOD were shown to effectively cleave RNA. These findings may help explain the origin of pathology of certain hereditary diseases genetically linked to Cu/ZnSOD gene.     

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Near-ultraviolet light-induced strand breaks in DNA pretreated with the carcinogen, N-acetoxy-2-acetylaminofluorene.

Neutral sucrose gradients of supercoiled DNA (phiX-174 RF I) were used to measure the in vitro production of strand breaks by the carcinogen, N-acetoxy-2-acetylaminofluorene (AcO-AAF). Treatment with AcO-AAF in 10% dimethyl sulfoxide did not directly yield strand breaks. Breaks in relatively low yield appeared after alkali treatment (pH 13, 60 min) of the RF I previously reacted with AcO-AAF. The DNA treated with AcO-AAF was sensitive to single-strand breakage by 303 nm near-ultraviolet light under neutral conditions. The greater the prior AcO-AAF treatment, the greater the sensitivity to 303 nm light. Post-irradiation alkali treatment greatly enhanced the light-induced strand breakage.     

Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage

The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu²⁺ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled ØX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17β significantly decreased the formation of single and double strand breaks in DNA induced by H₂O₂ alone or with Cu²⁺. Equilin (an equine estrogen) was more effective than estradiol-17β at the doses tested. Arachidonic acid in the presence of Cu²⁺ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.     

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes

Phorbol ester treatment of granulocytes triggers release of superoxide (O) and a concomitant burst of DNA strand breaks. The relationship between the amount of O and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O generated over a 40-min period by 1 × 10⁶ granulocytes/mL, a discontinuous “10-min pulse” method employing cytochrome c was used; 140 nmol O per 1 × 10⁶ cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1–10 mM) and staurosporine (2–10 nM) both inhibited O production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O. Zinc chloride (50–200 μM) inhibited both O and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O production more effectively than it inhibited DNA breaks. O dismutes to H₂O₂, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H₂O₂ had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O or H₂O₂ in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. J. Cell. Biochem. 66:219–228, 1997. © 1997 Wiley-Liss Inc.     

Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage

The bleomycins (BLMs) are a family of natural glycopeptides used clinically as antitumor agents. In the presence of required cofactors (Fe²⁺ and O₂), BLM causes both single-stranded (ss) and double-stranded (ds) DNA damage with the latter thought to be the major source of cytotoxicity. Previous biochemical and structural studies have demonstrated that BLM can mediate ss cleavage through multiple binding modes. However, our studies have suggested that ds cleavage occurs by partial intercalation of BLM's bithiazole tail 3′ to the first cleavage site that facilitates its re-activation and re-organization to the second strand without dissociation from the DNA where the second cleavage event occurs. To test this model, a BLM A5 analog (CD-BLM) with β-cyclodextrin attached to its terminal amine was synthesized. This attachment presumably precludes binding via intercalation. Cleavage studies measuring ss:ds ratios by two independent methods were carried out. Studies using [³²P]-hairpin technology harboring a single ds cleavage site reveal a ss:ds ratio of 6.7 ± 1.2:1 for CD-BLM and 3.4:1 and 3.1 ± 0.3:1 for BLM A2 and A5, respectively. In contrast with BLM A5 and A2, however, CD-BLM mediates ds-DNA cleavage through cooperative binding of a second CD-BLM molecule to effect cleavage on the second strand. Studies using the supercoiled plasmid relaxation assay revealed a ss:ds ratio of 2.8:1 for CD-BLM in comparison with 7.3:1 and 5.8:1, for BLM A2 and A5, respectively. This result in conjunction with the hairpin results suggest that multiple binding modes of a single BLM can lead to ds-DNA cleavage and that ds cleavage can occur using one or two BLM molecules. The significance of the current study to understanding BLM's action in vivo is discussed.     

DNA strand breakage induced by CuII and NiII, in the presence of peptide models of histone H2B

In the present study we used the plasmid relaxation assay, a very sensitive method for detection of DNA strand breaks in vitro, in order to evaluate the role of peptide fragments of histone H2B in DNA strand breakage induced by copper and nickel. We have found that in the presence of peptides modeling the histone fold domain (H2B_32-62 and H2B_63-93) as well as the N-terminal tail (H2B_1-31) of histone H2B there is an increased DNA damage by Cu²⁺/H₂O₂ and Ni²⁺/H₂O₂ reaction mixtures. On the contrary, the C-terminal tail (H2B_94-125) seems to have a protective role on the attack of ROS species to DNA. We have rendered our findings to the interactions of the peptides with DNA, as well as with the metal.     

Characterization of DNA damage induced by 3,4-estrone-o-quinone in human cells.

The DNA damage induced in a human breast cancer cell line treated with 1,5 (10)-estradiene-3,4,17-trione (3,4-estrone-o-quinone; 3,4-EQ) has been measured qualitatively and quantitatively. Single-strand (ss) but not double-strand (ds) DNA breaks were formed in MCF-7 cells treated with 3,4-EQ. The ss DNA breaks formed in MCF-7 cells were partially repaired after incubation of cells in 3,4-EQ-free media for 2 and 4 h (i.e. 33 and 23% repair, respectively, as compared to the ss DNA breaks in cells after a 1-h exposure to 3,4-EQ without a recovery period). The formation of interstrand DNA cross-links was demonstrated in MCF-7 cells exposed to the bifunctional alkylating agent, mitomycin C, but not in those exposed to 3,4-EQ. Protein-linked DNA breaks were detected in MCF-7 cells after exposure to camptothecin and etoposide but not 3,4-EQ, suggesting that the ss DNA breaks induced by 3,4-EQ are unlikely to be mediated via topoisomerases. The induction of ss DNA breaks was detected in the estrogen receptor-negative cell line, BT-20, after exposure to 3,4-EQ. Furthermore, excess estradiol in culture media did not prevent 3,4-EQ-induced ss DNA breaks, suggesting that the DNA damage was not mediated via the estrogen receptor. Evaluation of the newly synthesized quinone analogue, 5,6,7,8-tetrahydro-1-2-naphthoquinone, in the ss DNA breakage assay revealed that the A and B ring moiety of 3,4-EQ is sufficient to produce ss DNA breaks in MCF-7 cells.     

Similarity in the acute cytotoxic response of mammalian cells to mercury (II) and X-rays: DNA damage and glutathione depletion

Pronounced strand breakage of DNA analyzed by alkaline elution techniques was produced in intact Chinese hamster ovary cells by 25 μM HgCl₂ within 1 hr or 100 μM HgCl₂ within 15 min. HgCl₂-induced strand breakage was directly proportional to concentration up to 100 μM and to time within 1 hr. Levels of reduced glutathione decreased following HgCl₂ in parallel with the induction of DNA strand breakage. Evidence is presented that this rapid and pronounced induction of DNA strand breaks and other cytotoxic responses following acute exposure to HgCl₂ resembles the cellular effects of X-rays.     

Influence of Oxidized Purine Processing on Strand Directionality of Mismatch Repair

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10⁵], and this precision is improved to about [1/10⁷] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G^O) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G^O/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G^O might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR.     

Single-stranded DNA transforms plant protoplasts

Summary The transfer of the Agrobacterium T-DNA to plant cells involves the induction of the Ti plasmid virulence genes. This induction results in the generation of linear single-stranded (ss) copies of the T-DNA inside Agrobacterium and such molecules might be directly transferred to the plant cell. A central requirement of this ss transfer model is that the plant cell must generate a second strand and integrate the resulting double-stranded (ds) molecule into its genome. Here we report that incubating plant protoplasts with ss or ds DNA under conditions favouring DNA uptake results in transformation. The frequencies of transformation are similar and analysis of ss transformants suggests that the introduced DNA becomes double stranded and integrated. Analysis of transient expression from introduced ss DNA suggests that generation of the second strand is rapid and extrachromosomal.     

Diminution of singlet oxygen-induced DNA damage by curcmin and related antioxidants

Curcumin, the natural antioxidant from turmeric, an Indian spice, and its derivatives have significant abilities to protect plasmid pBR322 against single-strand breaks induced by singlet oxygen (¹O₂), a reactive oxygen species with potential genotoxic/mutagenic properties. ¹O₂ was generated at 37°C in an aqueous buffer system by the thermal dissociation of the endoperoxide of 3,3′-(1,4-naphthylene)dipropionate (NDPO₂). Among the compounds, tested, curcumin was the most effective inhibitor of DNA damage followed by desmethoxycurcumin, bisdesmethoxycurcumin and other derivatives. The observed antioxidant activity was both time-and concentration-dependent. The protectice ability of curcumin was higher than that of the well-known biological antioxidants lipoate, α-tocopherol and β-carotene. However, the highest protective ability with saturating concentrations of curcumin did not exceed 50%. The ability of curcumin and its derivatives to protect DNA against ¹O₂ seems to be related to their structures and may at least partly explain the therapeutic and other beneficial effects of these compounds including anticarcinogenic and antimutagenic properties.     

The Role of the Human Psoralen 4 (hPso4) Protein Complex in Replication Stress and Homologous Recombination

Psoralen 4 (Pso4) is an evolutionarily conserved protein that has been implicated in a variety of cellular processes including RNA splicing and resistance to agents that cause DNA interstrand cross-links. Here we show that the hPso4 complex is required for timely progression through S phase and transition through the G₂/M checkpoint, and it functions in the repair of DNA lesions that arise during replication. Notably, hPso4 depletion results in delayed resumption of DNA replication after hydroxyurea-induced stalling of replication forks, reduced repair of spontaneous and hydroxyurea-induced DNA double strand breaks (DSBs), and increased sensitivity to a poly(ADP-ribose) polymerase inhibitor. Furthermore, we show that hPso4 is involved in the repair of DSBs by homologous recombination, probably by regulating the BRCA1 protein levels and the generation of single strand DNA at DSBs. Together, our results demonstrate that hPso4 participates in cell proliferation and the maintenance of genome stability by regulating homologous recombination. The involvement of hPso4 in the recombinational repair of DSBs provides an explanation for the sensitivity of Pso4-deficient cells to DNA interstrand cross-links.     

Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae.

Mutants of Diplococcus pneumoniae that lack a membrane-localized DNAase are defective in transformation because entry of DNA into the cell is blocked. Such mutants still bind DNA on the outside of the cell. The bound DNA is double-stranded and its double-stranded molecular weight is unchanged. Its sedimentation behavior in alkali, however, shows that it has undergone single-strand breakage. The breaks are located randomly in both strands of the bound DNA at a mean separation of 2 × 10⁶ daltons of single-stranded DNA. Both binding and single-strand breakage occur in the presence of EDTA. Single-strand breaks are similarly formed on binding of DNA to normally transformable cells in the presence of EDTA. The single-strand breaks appear to be a consequence of attachment. DNA may be bound to the cell surface at the point of breakage.A mutant that is partially blocked in entry also binds DNA mainly on the outside of the cell. In the presence of EDTA, DNA bound by this mutant undergoes only single-strand breaks. In the absence of EDTA, however, double-strand breaks occur, apparently as a result of the initiation of entry. It is possible that the double-strand breaks arise from additional single-strand breaks opposite those that occurred on binding. The double-strand breaks presumably result from action of the membrane DNAase as it begins to release oligonucleotides from one strand segment while drawing the complementary strand segment into the cell.     

A role for single-strand breaks in bacteriophage phi-X174 genetic recombination

Formation of genetic recombinants in bacteriophage φX174 is stimulated up to 50-fold in host cells carrying the recA⁺ allele by subjecting the virus particles to ultraviolet irradiation before infection, or by starving the host cell for thymine during infection; in recA host strains no such increases are observed.φX174 replicative form DNA molecules formed in vivo from ultraviolet-irradiated bacteriophage consist of an intact, circular full-length viral (+) strand and a partially complete complementary (?) strand extending from the point of origin of complementary strand DNA synthesis to an ultraviolet lesion. φX174 replicative form DNA molecules formed in thymine-deficient host strains during thymine starvation have nearly complete circular viral (+) and complementary (?) strands, which contain random single-strand nicks or gaps.Correlation of these structures with the observed increases in recombination suggests that single-strand “breaks” are aggressive intermediate structures in the formation of φX174 genetic recombinants mediated by the host recA⁺ gene product.     

Purification of Oat Debranching Enzyme and Occurrence of Inactive Debranching Enzyme in Cereals

The interaction of some anthracycline antibiotics (adriamycin, daunomycin, aclacinomycin-A) with bacteriophage ?X174 was investigated. Adriamycin and daunomycin inactivated the infectivity of both free ?X174 phage and naked single-stranded ?X174 DNA without DNA strand scission, but aclacinomycin-A did not show this action. The phage inactivation reaction was reversibly inhibited by Superoxide dismutase, catalase or other oxygen radical scavengers. The inactivation of ?X174 by adriamycin and aclacinomycin-A was stimulated by the addition of Cu²⁺, while the ?X174 inactivation by daunomycin was inhibited by the addition of Cu²⁺. The ?X174 inactivation by adriamycin and aclacinomycin-A in the presence of Cu²⁺ was caused by degradation of DNA, and this inactivation reaction was inhibited irreversibly by oxygen radical scavengers. These results indicate that anthracycline antibiotics bind to ?X174 DNA in the form of free radicals and that during the auto-oxidation of these antibiotics in the presence of Cu²⁺, oxygen radicals were generated to cause the degradation of ?X174 DNA.     

A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells

A method for the determination of DNA monoadducts capable of forming interstrand crosslinks in mammalian cells is described. Such monoadducts were produced by brief treatment of cells with cis-diamminedichloro-Pt(II) (cis-DDP), 1-(2-chloroethyl)-1-nitrosourea (ClEtNU), L-phenylalanine mustard (L-PAM), or diaziridinylbenzoquinone (AZQ). The method is an alkaline elution procedure in which the DNA from lysed cells is incubated on polycarbonate filters at pH 10 and 37 degrees C. During this incubation, the progressive formation of interstrand crosslinking was observed in drug-treated cells. In the case of ClEtNU and AZQ, DNA strand breaks also formed, due to the presence of labile lesions in the DNA. This made quantitation of interstrand crosslinks difficult for these drugs. For cis-DDP and L-PAM, however, there was no significant production of strand breaks and the assay for interstrand crosslinks was quantifiable.     

Ascorbic Acid Oxidation and DNA Scission Catalyzed by Iron and Copper Chelates

The asorbic acid (AH^?) auto-oxidation rates catalyzed by copper chelates of 1,10-phenanthroline (OP) or by iron chelates of bleomycin (BLM) are only slightly higher than the oxidation rates catalyzed by the metal ions. AH^? oxidation in the presence of DNA is accompanied by degradation of the DNA. The rates of DNA scission by the metal chelates are markedly higher than the rates induced by the free metal ions. AH^? oxidation is slowed down in the presence of DNA which forms ternary complexes with the chelates. The ternary complexes react slowly with AH^? but induce DNA double strand breaks more efficiently than the free metal chelates. With OP, DNA is degraded by the reaction of the ternary complex, DNA-(OP)₂Cu(I), withH₂O₂

AH^? oxidation in the presence of DNA was biphasic, showing a marked rate increase after DNA was cleaved. We suggest that this sigmoidal pattern of the oxidation curves reflects the low initial oxidative activity of the ternary complexes, accelerating as DNA is degraded.

Using O₂^?produced by pulse radiolysis as a reductant, we found that AH^? oxidation with (OP)₂Cu(II) induced more DNA double strand breaks per single strand break than bipyridine-copper.

The site specific DNA damaging reactions indicated by these results are relevant to the mechanism of cytotoxic activities of bleomycin and similar antibiotics or cytotoxic agents.     

Genotoxicity of vanadium pentoxide evaluate by the single cell gel electrophoresis assay in human lymphocytes

Vanadium compounds are extensively used in modern industry and occupational exposure to high doses of Vanadium is quite common. In this study, the genotoxicity of vanadium pentoxide (V₂O₅) was evaluated directly in whole blood leukocytes and in human lymphocyte cultures using the single-cell gel electrophoresis assay (Comet Assay) to detect DNA damage expressed as DNA strand breaks and alkali labile sites. This chemical produces a clear dose-response in DNA migration in whole blood leukocytes and a significative positive effect only with the highest tested concentration in human lymphocyte cultures. After different recovery times the level of DNA damage returned to the control values. These results indicate that V₂O₅ is capable to induce DNA single-strand breaks and/or alkali-labile damage.