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Chem. Res. Toxicol. 1997, 10, 1118-1122
Differential Effects of DNA Supercoiling on Radical-Mediated DNA Strand Breaks William A. LaMarr,† Kathleen M. Sandman,‡ John N. Reeve,‡ and Peter C. Dedon*,† Division of Toxicology, 56-787, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, and Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210 Received May 6, 1997X
Supercoiling is an important feature of DNA physiology in vivo. Given the possibility that the reaction of genotoxic molecules with DNA is affected by the alterations in DNA structure and dynamics that accompany superhelical tension, we have investigated the effect of torsional tension on DNA damage produced by five oxidizing agents: γ-radiation, peroxynitrite, Fe2+/ EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. With positively supercoiled plasmid DNA prepared by a recently developed technique, we compared the quantity of strand breaks produced by the five agents in negatively and positively supercoiled pUC19. It was observed that strand breaks produced by γ-radiation, peroxynitrite, and Fe2+/EDTA/H2O2 were insensitive to DNA superhelical tension. These results are consistent with a model in which chemicals that generate highly reactive intermediates (e.g., hydroxyl radical), but do not interact directly with DNA, will be relatively insensitive to the changes in DNA structure and dynamics caused by superhelical tension. In the case of Fe2+ and Cu2+, metals that bind to DNA, only Cu2+/H2O2 proved to be sensitive to DNA superhelical tension. Strand breaks produced by Cu2+/H2O2 in the positively supercoiled substrate occurred at lower Cu concentrations than in negatively supercoiled DNA. Furthermore, a sigmoidal Cu2+/H2O2 damage response was observed in the negatively supercoiled substrate but not in positively supercoiled DNA. The results with Cu2+ suggest that the redox activity, DNA binding orientation, or DNA binding affinity of Cu1+ or Cu2+ is sensitive to superhelical tension, while the results with the other oxidizing agents warrant further investigation into the role of supercoiling in base damage.
Introduction DNA damage resulting from exposure to endogenous reactive oxygen species and other radicals plays a role in a variety of biological processes such as mutagenesis, aging, and carcinogenesis (1, 2). Even though there is considerable understanding of radical-mediated DNA lesions in vitro, the role of genomic organization in defining the location, quantity, and chemistry of DNA damage remains elusive. To better understand how DNA physiology affects DNA damage, we have examined one feature of genomic organization, DNA superhelical tension, and the role it plays in determining strand breaks produced by γ-radiation, peroxynitrite, Fe2+/EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. Supercoiling is an important aspect of DNA physiology in both prokaryotic and eukaryotic cells and appears to be involved in gene expression (3), DNA replication (3), and DNA repair (4). While the Escherichia coli genome has a superhelical density (σ) of ∼-0.05 (supercoils/ helical turn), the genomes of Drosophila melanogaster and human cells have no net superhelical tension (5). However, as first proposed in the twin supercoiled domain model of Liu and Wang (6), the act of transcription generates localized torsional tension in genes: negative superhelical tension in the 5′-ends and positive supercoiling in the 3′-ends (6-9). * Author to whom correspondence should be addressed: e-mail,
[email protected]. † Massachusetts Institute of Technology. ‡ The Ohio State University. X Abstract published in Advance ACS Abstracts, September 15, 1997.
S0893-228x(97)00072-6 CCC: $14.00
The possibility that supercoiling-induced alterations in DNA structure and dynamics could affect the interaction of oxidizing agents with DNA served as the premise for the present studies. Torsional tension can affect DNA structure in several ways, including changes in helical repeat (twist) and writhing of the DNA helix (10) and, in the case of negative supercoiling, alteration of DNA secondary structure to form Z-DNA, cruciforms, and unpaired regions (11, 12). Although the interaction of intercalators with negatively supercoiled DNA is well defined (8, 13), the effect of superhelical tension on the activity of other DNA-binding and DNA-damaging chemicals has not been thoroughly explored. To study the role of supercoiling in DNA damage, we have developed a technique to prepare highly positively supercoiled plasmid DNA (14). The DNA has a superhelical density of ∼+0.04, while negatively supercoiled plasmid DNA, isolated from E. coli, has an average density of ∼-0.05. These levels of supercoiling are similar to those estimated to exist in vivo (3, 15). Using positively and negatively supercoiled DNA substrates, we have investigated the effect of superhelical tension on the quantity of DNA strand breaks produced by five oxidizing agents: γ-radiation, peroxynitrite, Fe2+/ EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. Fe and Cu are physiologically important metals that have been implicated in DNA damage in vivo (e.g., see refs 16, 17). In the presence of hydrogen peroxide, both metals generate hydroxyl radical-like species through Fenton chemistry, which for Cu2+ appears to involve an initial reduction to Cu1+ (18-20): © 1997 American Chemical Society
DNA Supercoiling and Radical-Mediated DNA Damage
2Cu2+ + H2O2 f 2Cu1+ + O2 + 2H+ or 2Cu2+ f Cu1+ + Cu3+ Cu1+/Fe2++ H2O2 f Cu2+/Fe3+ + OH- + •OH Supercoiling-dependent deformation of DNA structure may affect the binding of these metals and thus the DNA damage they produce. For example, Cu1+ and Cu2+ bind preferentially to the N7 of guanine, and at least Cu2+ is capable of forming chelation complexes with the O6 in the same guanine (21) and with other nearby bases (21, 22). This may account for the formation of 8-oxoG (23, 24) and strand breaks (25, 26) at runs of guanines (27). The effects may be different for Fe since it is less redox active and binds to DNA with lower affinity than Cu (28). The EDTA complex of Fe2+ presents a unique opportunity to compare Fe bound to DNA to Fe free in solution. As with free Fe2+, Fe2+/EDTA has been shown to generate a hydroxyl radical-like species in the presence of hydrogen peroxide (29, 30). However, unlike free Cu and Fe, the negative charge of the Fe2+/EDTA complex prevents it from binding to DNA phosphates or bases (30, 31), and the resulting hydroxyl radical-like species may exist in the fluid phase. This could account for the lack of sequence selectivity of strand breaks generated by the complex (30, 31). In this respect, Fe2+/EDTA may be similar to ionizing radiation in its sensitivity to DNA supercoiling. In addition to a minor contribution from direct interaction with DNA, ionizing radiation reacts with water to produce hydroxyl radicals and hydrogen atoms by homolytic fission of oxygen-hydrogen bonds and to produce hydrated electrons (29, 32). Radiation-induced hydroxyl radical, like that produced by Fe2+/EDTA/H2O2, causes both strand breaks (33, 34) and base damage (35). Both γ-radiation and Fe2+/EDTA/H2O2 are sensitive to local DNA conformation (29, 36), but the effect of superhelical tension on radiation-induced DNA damage has been controversial (37-39). Nitric oxide, an important mediator of many biological processes (40), reacts with superoxide to form peroxynitrite (ONOO-) with a rate constant near the diffusioncontrolled limit (41). Peroxynitrite and its conjugate acid, peroxynitrous acid (ONOOH), are highly reactive at physiological pH and are capable of producing base damage (42, 43) and strand breaks in DNA (43, 44). However, peroxynitrite appears to have a different reactivity than hydroxyl radical (23, 45). We have found that only the Cu2+/H2O2 system is sensitive to DNA supercoiling. A lack of sensitivity to supercoiling for γ-radiation, peroxynitrite, and Fe2+/ EDTA is consistent with damage caused by a species that does not bind to DNA prior to inducing damage, while the lack of effect with Fe2+/H2O2 suggests that Fe2+ binds to DNA by a different mechanism than Cu ions.
Experimental Procedures Chemicals. All chemicals were reagent grade. Peroxynitrite solution was generated from a reaction of ozone with sodium azide as described by Pryor et al. (46); the product was characterized spectrophotometrically in 0.1 N NaOH [302 ) 1670 M-1 cm-1 (46)] and by its ability to cause the nitration of L-tyrosine (46). Caution should be exercised in the handling of peroxynitrite as it is a strongly oxidizing species. Preparation of Supercoiled pUC19 Substrates. Positive supercoiling of plasmid pUC19 was achieved using a recombinant archaeal histone HMf (rHMf) synthesized in E. coli from
Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1119 the cloned hmfB gene from Methanothermus fervidus as described elsewhere (14). The DNA had an average superhelical density of ∼+0.04 (number of supercoils/helical turn), as determined by two-dimensional gel electrophoresis (14). Negatively supercoiled pUC19 DNA was isolated from DH5R E. coli with an average density of ∼-0.05. This DNA was subjected to the same treatment as the positively supercoiled DNA except for the addition of topoisomerase-containing chicken blood extract; sham reactions were instead performed with the dilution and storage buffer used with the chicken blood extract. Both plasmid substrates were quantitated using the Hoechst dye 33258 (Sigma) fluorescence quantitation assay (47). In both DNA substrates, care was taken to remove trace quantities of metals and other contaminants that could interfere with the radical-mediated DNA damage. To remove trace quantities of nonspecifically bound, positively charged peptides, the DNA was adjusted to 2 M NaCl and incubated at room temperature for 2 h. The DNA was then dialyzed at 4 °C against Chelex-treated phosphate buffer (50 mM, pH 7.4) containing the metal-chelating agent diethylenetriaminepentaacetic acid and finally dialyzed against phosphate buffer alone to remove the chelator. Treatment of Supercoiled pUC19 with DNA-Damaging Agents. Damage reactions were performed in 25 µL reaction volumes with 30 µg/mL pUC19 plasmid DNA at ambient temperature for 30 min. γ-Radiation and peroxynitrite damage reactions were carried out in 50 mM potassium phosphate buffer (pH 7.4). DNA was irradiated with 0-17.6 Gy in a 60Co γ-source at 4 Gy/min, and the irradiated DNA was left at ambient temperature for approximately 30 min before further processing. Peroxynitrite in 100 mM NaOH was diluted with 10 mM NaOH immediately before addition of 1 µL aliquots to the DNA solution. Fe2+/EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2 damage reactions were carried out in 50 mM phosphate buffer (pH 7.4) with 1 mM H2O2. Fe2+ and Cu2+ were added to a solution of DNA in phosphate buffer followed by addition of H2O2 to 1 mM; the Fe2+/EDTA reaction was performed as described elsewhere (48). Since oxidation of deoxyribose causes abasic sites as well as direct strand breaks, the damaged DNA was treated with putrescine (100 mM, pH 7, 1 h, 37 °C) to convert abasic sites of all types to strand breaks (49-52). As shown in previous studies, putrescine treatment does not appear to react with oxidized bases to cause formation of abasic sites and strand breaks (23). Following treatment, DNA samples were purified by passage over G-50 Sephadex spin columns. The DNA was then relaxed with chicken blood extract, to remove any superhelical-dependent biases in subsequent electrophoresis or probing; this treatment did not affect the number of strand breaks in the DNA substrates (data not shown). Finally, the DNA was purified by proteinase K digestion (100 µg/mL; 1% SDS; 2 h at 37 °C) followed by phenol/chloroform and chloroform/isoamyl alcohol extractions and ethanol precipitation. Gel Electrophoresis. DNA from each sample was resolved on a 1.5% agarose gel (Tris-borate-EDTA) containing 60 µM chloroquine (Sigma). The gels were washed extensively in water to remove the chloroquine and then stained with 0.5 µg/mL EtBr for UV photography. The gels were exposed to short wave UV light for an additional 2 min to nick the DNA and rule out superhelical-dependent effects on hybridization efficiency. The gels were subsequently dried on a conventional gel dryer for 45 min at ambient temperature and 45 min at 60 °C. Quantitation of Plasmid Topoisomers. Gels were probed with a pUC19 random primer probe according to the protocol of Lueders and Fewell (53), and the radioactivity associated with each plasmid form was determined on a phosphorimager (Molecular Dynamics). The quantity of strand breaks was calculated from the proportion of supercoiled DNA (form I) converted to nicked DNA (form II) and linear DNA (form III).
Results and Discussion Given the biological importance of DNA supercoiling and the changes in DNA conformation that accompany
1120 Chem. Res. Toxicol., Vol. 10, No. 10, 1997
superhelical tension, we have investigated the effect of DNA supercoiling on the damage produced by γ-radiation, peroxynitrite, Fe2+/EDTA/H2O2, Fe2+/H2O2, and Cu2+/H2O2. These five agents all produce DNA damage by free radical processes but differ in the mechanisms by which they associate with and damage DNA. The DNA substrates for these studies consisted of a 2686-base pair plasmid that possessed a high degree of either negative or positive supercoiling. The negatively supercoiled plasmid is estimated to have σ ∼ -0.05; this represents approximately -15 supercoils/plasmid (∆Lk ∼-15). The positively supercoiled plasmid has an average σ ∼ 0.04, with a maximal ∆Lk of +17 (14). To ensure the removal of contaminants that could interfere with the radical-induced DNA damage, the DNA was subjected to extensive dialysis against Chelex-treated phosphate buffer containing a metal chelator. The purity of the DNA is indicated by the absence of detectable nicking of the plasmid DNA in the presence of 1 mM H2O2 alone (data not shown). Furthermore, the negatively supercoiled DNA was subjected to the same conditions used to prepare the positively supercoiled DNA. Finally, while we show all of the data, we were careful to draw conclusions only from the strand break data that fell within “single-hit conditions”, which correspond to ∼3040 strand breaks in 106 bases of pUC19 DNA according to a Poisson distribution (49). Above this level of damage, the number of strand breaks is underestimated because additional nicks in an already nicked plasmid cannot be detected by topoisomer analysis. The results of the supercoiling studies are shown in Figures 1 and 2. As expected for genotoxins that have little affinity for DNA, strand breaks produced by peroxynitrite, Fe2+/EDTA/H2O2, and γ-radiation were found to be insensitive to DNA superhelical tension (Figure 1). This insensitivity is likely due to the high reactivity of the hydroxyl radical-like species thought to be responsible for the DNA damage. In any case, the results indicate that negative and positive supercoiling do not create sites of high reactivity for these agents. The present results with radiation are consistent with the findings of Milligan et al. (39) who observed no effect of supercoiling on radiation-induced strand breaks. However, their results and the results of the present study differ from the contradictory results of Miller et al. (38) and Roots et al. (37). Roots et al. concluded that there was 10-15-fold more damage in isolated salmon sperm DNA compared to negatively supercoiled SV40 DNA (37), while Miller et al. observed a direct relationship between radiation-induced strand breaks and negative superhelical density in plasmid pIBI30 (38). In both studies, the purity of the DNA was not addressed, and in the case of Miller et al., residual ethidium bromide from preparation of supercoiled DNA could interfere with radiationinduced DNA damage. We have taken great care to ensure that both supercoiled DNA substrates were free from contaminants that could either enhance or inhibit the DNA damage. Furthermore, the results with the other oxidizing agents (Figures 1 and 2) lend support to a model in which chemicals that do not bind to DNA but generate highly reactive oxidizing species (e.g., hydroxyl radical) will be relatively insensitive to the changes in DNA structure and dynamics caused by superhelical tension. The observed lack of supercoiling effects on strand breaks does not rule out small, local changes in the reactivity of DNA due to superhelical tension, such as the variation in hydroxyl radical-mediated strand break
LaMarr et al.
Figure 1. Strand breaks produced by γ-radiation, peroxynitrite, and Fe2+/EDTA/H2O2 are not sensitive to DNA supercoiling. Samples of negatively and positively supercoiled pUC19 were treated with γ-radiation, peroxynitrite, and Fe2+/EDTA/ H2O2, as described in Experimental Procedures, and gel-resolved plasmid topoisomers were quantified by phosphorimager analysis of probed gels; representative experimental results are shown in the graphs. The average ratio of strand breaks in negatively supercoiled DNA to strand breaks in positively supercoiled DNA is shown for each agent (n ) 3-5).
formation as a function of minor groove width (29, 36). Our results do suggest, however, that there are no supercoiling-dependent "hotspots" for pUC19 DNA damage produced by Fe2+/EDTA/H2O2, γ-radiation, and peroxynitrite. The two agents that bind to DNA, Cu and Fe, show different sensitivities to DNA supercoiling. Fe2+/H2O2 produced an equal number of strand breaks in both substrates (Figure 2), which suggests that supercoilingdependent changes in DNA structure and dynamics do not greatly affect the binding of Fe to DNA. With Cu2+/ H2O2, however, strand breaks in the positively supercoiled substrate occurred at lower Cu concentrations than in negatively supercoiled DNA. Furthermore, the sigmoidal damage response of the negatively supercoiled substrate, which has been observed in other studies (23,
DNA Supercoiling and Radical-Mediated DNA Damage
Figure 2. Strand breaks produced by Cu2+/H2O2, but not Fe2+/ H2O2, are sensitive to DNA supercoiling. Samples of negatively and positively supercoiled pUC19 were treated with Cu2+/H2O2 and Fe2+/H2O2, as described in Experimental Procedures, and gel-resolved plasmid topoisomers were quantified by phosphorimager analysis of probed gels; representative experimental results are shown in the graphs. The average ratio of strand breaks in negatively supercoiled DNA to strand breaks in positively supercoiled DNA is shown for each agent (n ) 3-5).
54), was absent in positively supercoiled DNA (Figure 2). The similarity of the damage profiles at high Cu concentrations (>3 µM) is likely due to the fact that the level of strand breaks exceeds single-hit conditions. There are several explanations for the supercoilingdependent changes in DNA damage produced by Cu2+/ H2O2. First, supercoiling may simply alter the affinity of Cu2+ or Cu1+ for DNA, with higher binding affinity correlated with higher levels of DNA damage. High concentrations of Cu2+ (equivalent to ∼50 µM under the present conditions) are known to denature DNA by a mechanism that may involve unwinding of the helix among other changes in DNA structure (e.g., see ref 55). This may explain the observed effects of supercoiling on Cu-induced DNA damage if the formation of DNAunwinding Cu complexes is inhibited in positively supercoiled DNA, as occurs with intercalators in positively supercoiled DNA. However, even at extremely high concentrations (g1 mM), Levy and Hecht observed that Cu2+ did not affect the topology of negatively supercoiled DNA (56), so the question of Cu-induced unwinding of DNA remains controversial. Alternatively, if the reduction of Cu2+ to Cu1+ occurs only with unbound Cu2+, then an inverse relationship may exist between Cu2+ DNA binding affinity and DNA damage. While the present studies do not address base damage, it has been proposed that Cu bound to high-
Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1121
affinity sites on bases, probably the N7 of guanine (57), causes base lesions while unbound Cu or Cu bound to lower affinity sites (e.g., backbone phosphate) produces strand breaks (54). In light of this model, the observed increase in strand breaks in positively supercoiled DNA may be accompanied by reduced base damage if positive superhelical tension limits access to high-affinity binding sites on bases. Finally, it is possible that strand breaks and base damage increase simultaneously due to improved binding site access for both lesions. Wang and Van Ness have observed that Cu2+/ascorbate/H2O2 produces site-specific, negative supercoilingdependent strand breaks in plasmid DNA (58). Our results suggest that such local changes in DNA reactivity could represent one component of a complex set of supercoiling-dependent changes in the reaction of Cu with DNA. It is possible that negative supercoiling creates sites that are hyperreactive for Cu-induced strand break formation in the setting of an overall lower reactivity compared to positively supercoiled DNA. Studies are underway to assess the sequence selectivity of both base damage and strand breaks produced by Cu under different states of DNA supercoiling. In conclusion, we have observed differential effects of superhelical tension on the reactivity of DNA with several oxidizing agents. Strand breaks produced by Cu2+/H2O2 were found to occur at lower Cu concentrations in positively supercoiled DNA, while γ-radiation, peroxynitrite, Fe2+/EDTA/H2O2, and Fe2+/H2O2 did not show significant global changes in reactivity with DNA in different states of supercoiling. The results warrant further study to define the effects of superhelical tension on both the local and global reactions of oxidants with base and sugar moieties of DNA.
Acknowledgment. The authors are grateful to Kenneth Moore, Jr. (MIT) for the synthesis of peroxynitrite. This work was supported by NIH grants CA64524 (P.C.D.), CA72936 (P.C.D.), and GM53185 (J.N.R.); the Samuel A. Goldblith Career Development Professorship (P.C.D.); and NIEHS training grant ES07020 (W.A.L.).
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