H2O2-Induced DNA Damage Is Enhanced by Packaging of DNA

M.W. van Gisbergen , A.M. Voets , M.H.W. Starmans , I.F.M. de Coo , R. Yadak , R.F. Hoffmann , P.C. Boutros , H.J.M. Smeets , L. Dubois , P. Lambin. M...
0 downloads 0 Views 351KB Size
Download PDF
416

Chem. Res. Toxicol. 2001, 14, 416-422

Cu(II)/H2O2-Induced DNA Damage Is Enhanced by Packaging of DNA as a Nucleosome Qi Liang† and Peter C. Dedon* Division of Bioengineering and Environmental Health, 56-787, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 Received October 25, 2000

Copper is a physiologically important, redox-active metal that may be involved in endogenous DNA damage and mutagenesis. To understand the factors that affect the location and quantity of copper-induced oxidative DNA damage in cells, we used the 5S rDNA nucleosome as a model to assess the effect of chromatin structure on DNA damage produced by Cu(II)/H2O2. Packaging of DNA into a nucleosome increased the extent of Cu(II)/H2O2-induced strand breaks by a factor of 2, while the extent of base lesions sensitive to Fpg and endo III glycosylases increased 8-fold. We also observed that Cu(II)/H2O2 caused slightly more strand breaks than base lesions in isolated 5S rDNA (ratio of base lesions to strand breaks of ∼0.6), while base lesions outnumbered strand breaks by a factor of 3-4 when the DNA was incorporated into a nucleosome. Apart from several sites of enhanced or diminished DNA damage, there were no major changes in the sequence selectivity of Cu(II)/H2O2, and there was no apparent footprinting effect associated with nucleosome structure, such as that observed with the Fe(II)-EDTA complex. Possible mechanisms for explaining these observations include (1) an increase in Cu(II) concentration in the vicinity of nucleosomal DNA caused by binding of Cu to histone proteins or (2) increased reactivity or accessibility of nucleobases caused by DNA conformational changes associated with nucleosome structure. The enhancement of Cu(II)/H2O2-induced DNA damage in nucleosomes stands in contrast to the protective effect afforded DNA by proteins in chromatin against radiation-induced DNA damage.

Introduction Copper (Cu) is a physiologically important metal that may play a role in the endogenous oxidative DNA damage associated with aging and cancer (1, 2). Studies in purified DNA have revealed that Cu-mediated Fenton chemistry produces a spectrum of both deoxyribose and base lesions in DNA (3-12). However, substantially less is known about Cu-induced DNA damage in chromatin. To this end, we have undertaken studies of DNA damage produced by Cu(II) and H2O2 in nucleosomes. The DNA damage produced by Cu(II) likely involves initial reduction to Cu(I) by any of a variety of mechanisms, including reaction with endogenous reductants or self-reduction as shown in the reactions below. In the presence of hydrogen peroxide, Cu(I) is able to perform Fenton chemistry to generate a hydroxyl radical-like species:

Cu(II) + H2O2 f Cu(I) + H2O + H+ or 2Cu(II) f Cu(I) + Cu(III) Cu(I) + H2O2 f Cu(II) + OH- + “•OH” It has been proposed that Cu(II) and Cu(I) species not bound to DNA cause mainly strand breaks while DNAbound Cu(II) and Cu(I) cause base oxidation (4). Binding * To whom the correspondence should be addressed. Telephone: (617) 253-8017. Fax: (617) 258-0225. E-mail: [email protected]. † Present address: Center for Medical and Molecular Genetics, 1413 Research Blvd., Rockville, MD 20850.

of both Cu(I) and Cu(II) to DNA occurs primarily through the N7 of guanine (13-15), which is consistent with the observed preference for base damage predominantly at guanine (3, 4, 6). While Cu(II) binds weakly to DNA (Ka ∼ 104 M-1; 16, 17), Cu(I) binds with very high affinity (Ka ∼ 109 M-1; 18). Nucleosomes represent the first level of DNA compaction in eukaryotic nuclei and consist of 146 bp of core DNA wrapped around a cluster of eight histone proteins, with 20-60 bp of linker DNA joining adjacent cores (19-21). We have studied the uniquely positioned and well-characterized nucleosome that forms on the 5S rRNA gene of Xenopus borealis (20, 22) as a model chromatin substrate for Cu oxidation. In conjunction with crystallographic studies, the 5S rDNA system has revealed important features of DNA conformation in the nucleosome. The central 30 bp of the core DNA is configured as an S-shaped jog; the helix is underwound (∼10.7 bp/turn) relative to B-DNA, while the remaining 120 bp of core DNA is overwound (∼10.0 bp/turn) (20). The DNA is also kinked or sharply bent at four sites symmetrically positioned at one and four helical turns from the dyad axis (19, 21). The altered structure and accessibility of DNA in nucleosomes could affect Cu binding and damage as it does with other DNA-targeted chemicals. For example, intercalating chemicals are inhibited from binding to core DNA due to the constraints imposed by histone-DNA contacts (23-26). While DNA cleavage produced by the Fe(II)-EDTA complex is inhibited when accessible deoxyribose hydrogen atoms face the histone core (20, 27, 28),

10.1021/tx0002278 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/16/2001

Cu-Induced DNA Damage in Nucleosomes

alkylation of the N7 position of purine bases by dimethyl sulfate is not greatly affected by incorporation of DNA into nucleosomes (29). The observation of enhanced DNA damage near a transcription factor binding site in cells treated with hydrogen peroxide suggests that DNA structure may affect the damage produced by Cu- and Fe-mediated Fenton chemistry (30). To better understand the factors that affect the location, quantity, and chemistry of Cu(II)-induced DNA damage in chromatin, we have examined the oxidative DNA damage produced by Cu(II)/H2O2 in a reconstituted nucleosome.

Experimental Procedures Materials. Plasmid pXP-10, containing the 5S rRNA gene of X. borealis, was kindly provided by A. Wolffe (NICHHD, National Institutes of Health; 20). Diethylenetriaminepentaacetic acid (DETAPAC), L-ascorbate, and thiourea were purchased from Sigma Chemical Co. Cupric chloride, EDTA, and potassium phosphate (mono- and dibasic) were obtained from Mallinckrodt. Hydrogen peroxide, ferrous ammonium sulfate, and ammonium acetate were purchased from Fisher Scientific. Chelex-100 resin was purchased from Bio-Rad Laboratories. Cellulose ester dialysis membranes with a molecular mass cutoff of 10 000 Da were obtained from Spectrum. Microdialysis chambers with cellulose ester membranes were purchased from Sialomed, Inc. The Escherichia coli DNA repair enzymes endonuclease III (endo III) and formamidopyrimidine DNA N-glycosylase (Fpg) were provided by R. Cunningham (Department of Biology, State University of New York, Albany, NY) and A. Grollman (Department of Pharmacology, State University of New York, Stonybrook, NY), respectively. Histone proteins were isolated from HeLa cells by hydroxylapatite chromatography as described elsewhere with purity assessed by both SDS-polyacrylamide and acid-urea gel electrophoresis (31). Reconstitution of Nucleosomes with 5S rDNA. The 215 bp EcoRI-DdeI fragment of pXP-10 was 5′-end- or 3′-end-labeled with [γ-32P]ATP or [R-32P]ddATP, respectively, at the EcoRI site as described previously (32). Nucleosomes were reconstituted from the labeled DNA and purified histones by dialysis from high-salt urea essentially as described elsewhere (32), except that sonicated calf thymus DNA (∼200 bp average) was used as unlabeled carrier DNA and the ratio of histone to DNA was 1:1. Both the naked and nucleosomal DNA samples were subjected to the same high-salt and urea dialysis conditions used for nucleosome reconstitution (except for the absence of histones with the naked DNA samples). Both samples were then exhaustively dialyzed against 50 mM potassium phosphate buffer (pH 7.4, pretreated with Chelex-100 resin; 9) containing 1 mM DETAPAC for 12 h at 4 °C to remove trace metal ions, and then against Chelex-100-treated 50 mM potassium phosphate buffer twice (2 and 12 h) at 4 °C. Following dialysis, the relative DNA concentration in each sample was determined by measuring the A260 of the DNA sample dissolved in 0.1 N NaOH (A260 ) 1.0 was assumed to represent a DNA concentration of 50 µg/mL). DNA concentrations were determined in this way for comparative purposes only, since the alkaline conditions prevent an absolute determination of DNA concentration due to pHsensitive changes in the absorbance properties of the DNA. The alkaline conditions ensured the disruption of nucleosome structure, while the absence of aromatic amino acids in histone proteins (33) allowed DNA concentrations to be determined by absorbance at 260 nm. DNA Damage Reactions by Cu(II)/H2O2 and the Fe(II)EDTA Complex. Reactions were performed with both 30 µg/ mL naked and nucleosomal DNA in 25 mM potassium phosphate buffer (pH 7.4, pretreated with Chelex-100 resin) in a total volume of 10 µL at ambient temperature. All reactants were freshly prepared for each experiment. Hydroxyl radical footprinting by the Fe(II)-EDTA complex in the presence of H2O2

Chem. Res. Toxicol., Vol. 14, No. 4, 2001 417 and ascorbate was performed as described elsewhere (32), except that the reaction time was 30 and 90 s for naked DNA and nucleosomal DNA, respectively, to achieve similar levels of cleavage. DNA was incubated with CuCl2 at concentrations of 0-200 µM for 10 min followed by the addition of H2O2 to a final concentration of 1 mM. The reaction was stopped after 30 min by addition of 1 µL of 10 mM EDTA [the Cu(II)-EDTA complex does not produce DNA damage in the presence of H2O2; 32]. The DNA was purified by phenol/chloroform extraction and ethanol precipitation. Purified DNA was then resuspended in water and split into two portions. One portion was further treated with endo III and Fpg enzymes at a concentration of 25 ng of enzyme/ µg of DNA to express base damage as strand breaks (35). The reaction was carried out at 37 °C for 40 min in a buffer containing 50 mM HEPES (pH 7.4), 100 mM KCl, 1 mM EDTA, 0.1 mM dithiothreitol, and 100 µg/mL bovine serum albumin. Under these conditions, there was complete conversion of base lesions to strand breaks in treated plasmid DNA (data not shown; 33). After the enzyme reaction, the DNA was ethanol precipitated and resolved on an 8% polyacrylamide gel along with Maxam-Gilbert chemical sequencing standards (36). Dried gels were subjected to phosphorimager analysis as described elsewhere (32). To compensate for lane-to-lane variation in sample loading, the radioactivity in each band was expressed as a percentage of the total radioactivity in the lane.

Results Control Experiments with Naked and Nucleosomal 5S rDNA. To better understand how chromatin structure affects Cu-induced DNA damage, we compared DNA damage produced by Cu(II)/H2O2 in naked 5S rDNA to that in the nucleosome reconstituted on the 5S rDNA. We initially performed studies of Fe(II)-EDTA cleavage in naked and nucleosomal DNA to establish nucleosomal landmarks for interpretation of the Cu data. These results are shown in the gel in Figure 1 and in the phosphorimager cleavage profiles shown in Figure 2A. The sinusoidal cleavage pattern with a ∼10 bp repeat in the nucleosomal DNA, compared to the relatively monotonic pattern in the naked DNA, is caused by inhibition of Fe(II)-EDTA-induced cleavage as the accessible deoxyribose hydrogen atoms (mainly 5′- and 4′-positions) face the histone core (28). This behavior reflects the consistent rotational positioning of the 5S rDNA on the histones. In all reconstitutions, more than 95% of the DNA was incorporated into nucleosomes (data not shown). A representative example of the damage produced by treating naked and nucleosomal DNA with varying Cu(II) concentrations and 1 mM H2O2 is shown in the gels in Figure 1 and in the line graphs in Figure 2B. Since oxidation of DNA produces both nucleobase lesions and strand breaks, the latter due to deoxyribose oxidation, we sought to differentiate these two types of DNA damage. Direct strand breaks were localized and quantified by loading the Cu-damaged DNA directly onto a sequencing gel. As in previous studies (9), we did not observe an increase in the level of strand breaks upon treatment of the DNA with putrescine (data not shown), so we conclude that abasic sites do not contribute significantly to Cu-induced damage in either naked or nucleosomal DNA. To reveal base lesions as additional strand breaks, the Cu-treated DNA was digested with Fpg and endo III. Cu-induced damage to purines consists mainly of 8-hydroxyguanine and 8-hydroxyadenine (8), both of which are substrates for Fpg. This enzyme possesses both N-glycosylase and AP-lyase activities for 8-oxoG, FaPy-G, and other oxidized purine species

418

Chem. Res. Toxicol., Vol. 14, No. 4, 2001

Liang and Dedon

Figure 1. Sequencing gel analysis of DNA damage produced by CuCl2 with 1 mM H2O2 (left panels) and the Fe(II)-EDTA complex (right panel) in purified and nucleosomal 5′-32P end-labeled 5S rDNA. CuCl2 concentrations (micromolar) are noted above each pair of lanes. The “+” and “-” symbols represent, respectively, the presence (i.e., base lesions + strand breaks) and absence (i.e., direct strand breaks) of Fpg/endo III treatment. Lanes marked GA and CT represent Maxam-Gilbert chemical sequencing standards (36). In the Fe-EDTA panel, “Na” and “Nu” denote naked and nucleosomal DNA, respectively. The sequence of the 5′-end of the 5S rDNA subjected to analysis is shown below the gel images, with the corresponding sequence numbering shown in the gel image.

(37-39) as well as at least one product of 8-oxoG oxidation (40). To a lesser extent, Cu-mediated Fenton chemistry also affects pyrimidines, mainly as cytosine glycol (8), so we used endo III to convert oxidized pyrimidines to strand breaks. Endo III has both DNA glycosylase and AP lyase activities and acts on a wide range of oxidized, hydrated, and ring-fragmented pyrimidines (37, 41). DNA samples were used for analysis only when less than 30% of the parent DNA band was

present as cleavage fragments. This ensures that the cleavage patterns reflected one damage event per DNA molecule (9). Characteristics of Cu(II)/H2O2-Induced DNA Damage in the 5S rDNA Nucleosome. The sequencing gels displayed in Figures 1 and 2 show an increase in the quantity of strand breaks and base damage induced by Cu(II)/H2O2 with increasing concentrations of Cu(II). To better visualize the DNA damage patterns, the line

Cu-Induced DNA Damage in Nucleosomes

Chem. Res. Toxicol., Vol. 14, No. 4, 2001 419

Figure 2. Line graphs of the frequency of Cu(II)/H2O2-induced DNA damage and the Fe(II)-EDTA cleavage profile along the naked and nucleosomal 5S rDNA. Lanes from the sequencing gel shown in Figure 1 were subjected to phosphorimager analysis, and the data are presented as line graphs. (A) Frequency of Fe(II)-EDTA cleavage in naked (upper tracings) and nucleosomal 5S rDNA (lower tracings). The black and gray lines represent direct strand breaks and the sum of base lesions and direct strand breaks (glycosylase treatment), respectively. The numbering depicts positions in the 5S rDNA sequence, with the dyad axis of pseudosymmetry denoted by the bar. (B) The frequency of Cu(II)/H2O2-induced DNA damage in naked (gray lines) and nucleosomal 5S rDNA (black lines). The lower and upper tracings represent direct strand breaks and the sum of base lesions and direct strand breaks (glycosylase treatment), respectively.

graphs in Figure 2B (generated from lanes in Figure 1) were prepared with Cu(II) concentrations that generated similar levels of DNA damage in nucleosomes (25 µM) and naked DNA (75 µM). The strand breaks produced by Cu(II) were relatively sequence nonselective, as observed with the Fe(II)-EDTA complex. However, neither the strand breaks nor the base lesions produced by Cu(II)/H2O2 in the nucleosomes displayed a footprint

or sinusoidal cleavage pattern as observed with the Fe(II)-EDTA complex. Given the preference of Cu species for binding to N7 of guanine in the major groove, inhibition of Cu-induced damage by histone proteins would be expected to produce a sinusoidal cleavage pattern out of phase by ∼5 bp relative to the minor groove pattern produced by the Fe(II)-EDTA complex. The results suggest that, in comparison to Fe(II)-EDTA, Cu(II)

420

Chem. Res. Toxicol., Vol. 14, No. 4, 2001

Figure 3. Dose-response curves for Cu(II)-induced base lesions (upper panel) and strand breaks (lower panel) at all positions in the naked (O) or nucleosomal (b) 5S rDNA sequence. A 1 mM concentration of H2O2 was used in all cases. Asterisks denote data sets for which the naked and nucleosomal data are significantly different from each other at P < 0.05 (Student’s t test).

has greater access to sites in nucleosomal DNA or that Cu(II)-induced DNA damage occurs by attack at both minor and major groove sites. In contrast to strand breaks, Cu(II)/H2O2-induced base damage had significant sequence selectivity in both naked and nucleosomal DNA. Base damage induced by Cu(II) oxidation occurred most frequently at guanines. This was most notable in runs of guanines, in particular, the 5′-GGGGAGG-3′ sequence highlighted in Figure 1. Though the frequency of strand breaks along the 215 bp DNA fragment was similar in the naked and nucleosomal DNA samples, the patterns of base modification were slightly affected by the folding of DNA into a nucleosome. This is illustrated by the base damage at the four contiguous guanines at positions -23 to -26 in Figures 1 and 2B. There is a larger increase in the level of base oxidation at the 3′-most guanines (positions -23 and -24) than in the 5′-guanines (positions -25 and -26) in the nucleosome than in naked DNA. Other variations in base damage frequency are distributed along the whole fragment as shown in the line graph overlay in Figure 2B. Enhancement of Cu(II)/H2O2-Induced DNA Damage in Nucleosomes. Visual inspection of the sequencing gels in Figure 1 suggests that there are more Cuinduced strand breaks and base lesions in nucleosomes than in naked DNA. This is most obvious at low Cu(II) concentrations. To quantify this phenomenon, the radioactivity representing DNA damage fragments in each lane was expressed as a percentage of the total radioactivity (parent band + damage fragments) in the lane. The combined data for several experiments, presented graphically in Figure 3, reveal several important features of Cu(II)/H2O2-induced DNA damage in nucleosomes. In general terms, there is an increase in the total quantity

Liang and Dedon

Figure 4. Dose-response curves for Cu(II)-induced base lesions (upper panel) and strand breaks (lower panel) occurring in the run of four guanines at positions -23 to -26 in naked (O) and nucleosomal (b) 5S rDNA. A 1 mM concentration of H2O2 was used in all cases. Asterisks denote data sets for which the naked and nucleosomal data are significantly different from each other at P < 0.05 (Student’s t test).

of base lesions and strand breaks in nucleosomal DNA compared to that in naked DNA. The number of strand breaks increases an average of 2.1((0.9)-fold, while the number of base lesions increases an average of 7.5((3.1)fold. The decrease in the number of base lesions and strand breaks at 100 µM Cu(II) in nucleosomal DNA (Figure 3) may be due to saturation of Cu binding sites, leading to similar levels of damage in nucleosomal and naked DNA. Though there is considerable variation in the ratios of base damage to strand breaks for naked DNA and nucleosomes, there is an increase in the ratio from ∼0.6 in naked DNA to ∼3-4 in nucleosomes. As shown in Figure 4, the damage produced by Cu(II)/H2O2 at the run of four guanines (positions -23 to -26) behaves in a similar manner, with the number of base lesions in the nucleosome increasing 4.7((1.1)-fold over that in naked DNA and a 2.4((0.6)-fold increase in the number of strand breaks.

Discussion Using a model nucleosome system, we have found that DNA damage produced by Cu(II)/H2O2 is enhanced in nucleosomes compared to purified DNA. The increase cannot be explained by the presence of small, redox-active contaminants (e.g., metals) in the nucleosome preparation since both the purified DNA and nucleosomes were subjected to identical biochemical environments and metal chelators during the preparation of the nucleosomes. Furthermore, hydrogen peroxide alone produced the same low level of background DNA damage in both DNA preparations (Figure 1), which rules out contamination by metals capable of Fenton chemistry.

Cu-Induced DNA Damage in Nucleosomes

One interesting feature of the cleavage profile produced by Cu(II)/H2O2 is the lack of an apparent footprint caused by contact of the DNA with the surface of the histone core. Such a footprint is readily apparent with the Fe(II)-EDTA complex as shown in Figure 1. The strand breaks produced by the Fe(II)-EDTA complex result from abstraction of deoxyribose hydrogen atoms according to their solvent accessibility (28). There is thus a reduction in the level of cleavage when the most accessible hydrogen atoms (e.g., 5′- and 4′-positions) are blocked by the histone protein in contact with DNA in the nucleosome, which is generally associated with the minor groove facing the histone core. Cu(II) binds to N7 of guanine in the major groove, and if access were limited when the major groove faced the histone core, one would expect a cleavage pattern offset by ∼5 bp from the Fe(II)EDTA profile. However, there is no consistent reduction in the level of Cu(II)-induced base damage, or strand breaks, in phase with the DNA helical repeat. This result suggests that DNA binding sites or target deoxyribose hydrogen atoms are accessible to Cu(II) at all sites in the nucleosome, a situation similar to that observed with dimethyl sulfate (29). While the accessibility of Fe(II) to sites in the nucleosome is unknown, it is possible that Fenton chemistry is not restricted by the compaction of DNA in chromatin. This stands in contrast to the protective effects of chromatin proteins against DNA damage produced by ionizing radiation (42-44), a situation discussed in more detail shortly. The major conclusion from these studies is that DNA damage produced by Cu(II)/H2O2 is enhanced in the 5S rDNA nucleosome compared to that in naked DNA. This applies to both strand breaks and base lesions, though the number of the latter is increased by 2-4-fold over the former. This result appears to be a general phenomenon along the length of the 5S rDNA since damage at the run of four guanines (positions -23 to -26) is increased by a similar magnitude despite the highly reactive nature of this sequence. There are several explanations for the enhancement of Cu(II)-induced damage in nucleosomes. One involves changes in DNA conformation and dynamics that make the Cu(II) binding sites more accessible. This would explain the preferential increase in the number of base lesions compared to strand breaks if base modification is caused by base-bound Cu(II)/Cu(I), while strand breaks are caused by Cu(II)/Cu(I) in solution as proposed by Drouin et al. (4). However, enhancement of Cu-induced damage due to nucleosome-dependent widening of the major groove when it faces away from the histone proteins does not explain the enhancement, since this would result in a sinusoidal behavior in the Cu(II) cleavage pattern. Stabilization of intra- and interstrand base coordination by Cu(II) could result from the constraints placed on DNA dynamics by the histone contacts. However, this does not explain the increase in the number of strand breaks in the nucleosome. Another factor contributing to the damage enhancement may be binding of Cu(II) to the histone proteins. The histone proteins are rich in lysine and arginine residues (33) that could serve as low-affinity chelation sites for Cu(II) cations. An increase in the net concentration of Cu(II) in the vicinity of the nucleosomal DNA would thus explain the generalized increase in the level of DNA damage in the nucleosome, both strand breaks and base damage. However, it does not fully explain the

Chem. Res. Toxicol., Vol. 14, No. 4, 2001 421

preferential increase in the number of base lesions. Furthermore, the affinity of the histones for Cu(II) must be quite low since competition experiments comparing naked DNA and nucleosomes did not reveal significant differences in the affinity of either for Cu(II) (data not shown). Gelagutashvili et al. have determined that Cu(II) interacts with nucleosomes with a binding constant of 4-5 × 104 M-1 (45), which is similar to the estimated binding constant of ∼104 M-1 for the binding of Cu(II) to naked DNA (16). The observed enhancement of Cu(II)/H2O2-induced DNA damage in nucleosomes stands in contrast to the protective effects of chromatin structure against DNA damage produced by ionizing radiation (43, 44). Ljungman has observed that depletion of proteins from chromatin causes a 10-15-fold increase in susceptibility to radiation-induced DNA damage, while compaction of the chromatin provides an additional 6-fold protection over unfolded chromatin (43). Our results suggest that chromatin structure may not protect DNA from oxidative insults caused by Fenton chemistry associated with Cu. Given the parallel chemistries of Cu and Fe, one might also anticipate a similar behavior with Fe.

Acknowledgment. This work was supported by NIH Grants ES09980 and CA72936.

References (1) Ames, B. N., and Gold, L. S. (1991) Endogenous mutagens and the causes of aging and cancer. Mutat. Res. 250, 3-16. (2) Ames, B. N., Shigenaga, M. K., and Hagen, T. M. (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U.S.A. 90, 7915-7922. (3) Aruoma, O. I., Halliwell, B., Gajewski, E., and Dizdaroglu, M. (1991) Copper-ion-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem. J. 273, 601-604. (4) Drouin, R., Rodriguez, H., Gao, S.-W., Gebreyes, Z., O’Connor, T. R., Holmquist, G. P., and Akman, S. A. (1996) Cupric ion/ ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications. Free Radical Biol. Med. 21, 261-273. (5) Rodriguez, H., Drouin, R., Holmquist, G. P., O’Connor, T. R., Boiteux, S., Laval, J., Doroshow, J. H., and Akman, S. A. (1995) Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J. Biol. Chem. 270, 17633-17640. (6) Sagripanti, J. L., and Kraemer, K. H. (1989) Site-specific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide. J. Biol. Chem. 264, 1729-1734. (7) Toyokuni, S., and Sagripanti, J. L. (1996) Association between 8-hydroxy-2′-deoxyguanosine formation and DNA strand breaks mediated by copper and iron. Free Radical Biol. Med. 20, 859864. (8) Dizdaroglu, M., Rao, G., Halliwell, B., and Gajewski, E. (1991) Damage to DNA bases in mammalian chromatin by hydrogen peroxide in the presence of ferric and cupric ions. Arch. Biochem. Biophys. 285, 317-324. (9) Kennedy, L. J., Moore, K., Jr., Caulfield, J. L., Tannenbaum, S. R., and Dedon, P. C. (1997) Quantitation of 8-oxoguanine and strand breaks produced by four oxidizing agents. Chem. Res. Toxicol. 10, 386-392. (10) Reed, C. J., and Douglas, K. T. (1991) Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. Biochem. J. 275, 601-608. (11) Rodriguez, H., Holmquist, G. P., D’Agostino, R. J., Keller, J., and Akman, S. A. (1997) Metal ion-dependent hydrogen peroxideinduced DNA damage is more sequence specific than metal specific. Cancer Res. 57, 2394-2403. (12) Wang, Y., and Van Ness, B. (1989) Site-specific cleavage of supercoiled DNA by ascorbate/Cu(II). Nucleic Acids Res. 17, 6915-6926. (13) Gao, Y. G., Sriram, M., and Wang, A. H. J. (1993) Crystallographic studies of metal ion-DNA interactions: different binding modes of cobalt(II), copper(II) and barium(II) to N7 of guanines in Z-DNA and a drug-DNA complex. Nucleic Acids Res. 21, 4093-4191.

422

Chem. Res. Toxicol., Vol. 14, No. 4, 2001

(14) Geierstanger, B. H., Kagawa, T. F., Chen, S.-L., Quigley, G. J., and Ho, P. S. (1991) Base-specific binding of copper(II) to Z-DNA. The 1.3-Å single-crystal structure of d(m5CGUAm5CG) in the presence of CuCl2. J. Biol. Chem. 266, 20185-20191. (15) Kagawa, T. F., Geierstanger, B. H., Wang, A. H. J., and Ho, P. S. (1991) Covalent modification of guanine bases in double-stranded DNA. The 1.2-Å Z-DNA structure of d(CGCGCG) in the presence of CuCl2. J. Biol. Chem. 266, 20175-20184. (16) Bach, D., and Miller, I. R. (1967) Polarographic investigation of binding of Cu2+ and Cd2+ by DNA. Biopolymers 5, 161-172. (17) Bryan, S. E., and Frieden, E. (1967) Interaction of copper(II) with deoxyribonucleic acid below 30 degrees. Biochemistry 6, 27282734. (18) Pru¨tz, W. A., Butler, J., and Land, E. J. (1990) Interaction of copper(I) with nucleic acids. Int. J. Radiat. Biol. 58, 215-234. (19) Richmond, T. J., Finch, J. T., Rushton, B., Rhodes, D., and Klug, A. (1984) Structure of the nucleosome core particle at 7 Å resolution. Nature 311, 532-537. (20) Hayes, J. J., Tullius, T. D., and Wolffe, A. P. (1990) The structure of DNA in a nucleosome. Proc. Natl. Acad. Sci. U.S.A. 87, 74057409. (21) Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 Å resolution (see comments). Nature 389, 251260. (22) Rhodes, D. (1985) Structural analysis of a triple complex between the histone octamer, a Xenopus gene for 5S RNA and transcription factor IIIA. EMBO J. 4, 3473-3482. (23) Yu, L., Goldberg, I. H., and Dedon, P. C. (1994) Enediynemediated DNA damage in nuclei is modulated at the level of the nucleosome. J. Biol. Chem. 269, 4144-4151. (24) Xu, J., Wu, J., and Dedon, P. C. (1998) DNA damage produced by enediynes in the human phosphoglycerate kinase gene in vivo: Esperamicin A1 as a nucleosome footprinting agent. Biochemistry 37, 1890-1897. (25) Kuo, M. T. (1978) Bleomycin causes release of nucleosomes from chromatid and chromosomes. Nature 271, 83-84. (26) Moore, C. W. (1988) Internucleosomal cleavage and chromosomal degradation by bleomycin and phleomycin in yeast. Cancer Res. 48, 6837-6843. (27) Dixon, W. J., Hayes, J. J., Levin, J. R., Weidner, M. F., Dombrowski, B. A., and Tullius, T. D. (1991) Hydroxyl radical footprinting. Methods Enzymol. 208, 380-413. (28) McGhee, J. D., and Felsenfeld, G. (1979) Reaction of nucleosome DNA with dimethyl sulfate. Proc. Natl. Acad. Sci. U.S.A. 76, 2133-2137. (29) Rodriguez, H., Drouin, R., Holmquist, G. P., and Akman, S. A. (1997) A hot spot for hydrogen peroxide-induced damage in the human hypoxia-inducible factor 1 binding site of the PGK 1 gene. Arch. Biochem. Biophys 338, 207-212. (30) Liang, Q., Choi, D.-J., and Dedon, P. C. (1997) Calicheamicininduced DNA damage in a reconstituted nucleosome is not

Liang and Dedon

(31)

(32) (33)

(34)

(35)

(36) (37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

affected by histone acetylation: Role of drug structure in the target recognition process. Biochemistry 36, 12653-12659. Yu, L., Salzberg, A. A., and Dedon, P. C. (1995) New insights into calicheamicin-DNA interactions derived from a model nucleosome system. Bioorg. Med. Chem. 3, 729-741. van Holde, K. E. (1989) Chromatin, Springer-Verlag, New York. Kobayashi, S., Ueda, K., and Komano, T. (1990) The effects of metal ions on the DNA damage induced by hydrogen peroxide. Agric. Biol. Chem. 54, 69-76. Tretyakova, N. Y., Burney, S., Pamir, B., Wishnok, J. S., Dedon, P. C., Wogan, G. N., and Tannenbaum, S. R. (2000) Peroxynitriteinduced DNA damage in the supF gene: correlation with the mutational spectrum. Mutat. Res. 447, 287-303. Maxam, A., and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499560. Cunningham, R. P. (1997) DNA glycosylases. Mutat. Res. 383, 189-196. Krokan, H. E., Standal, R., and Slupphaug, G. (1997) DNA glycosylases in the base excision repair of DNA. Biochem. J. 325, 1-16. Tchou, J., Bodepudi, S., Shibutani, S., Antoshechkin, S., Miller, J., Grollman, A. P., and Johnson, F. (1994) Substrate specificity of Fpg protein. J. Biol. Chem. 269, 15318-15324. Burney, S., Niles, J. C., Dedon, P. C., and Tannenbaum, S. R. (1999) DNA damage in deoxynucleosides and oligonucleotides treated with peroxynitrite. Chem. Res. Toxicol. 12, 513-20. Demple, B., and Harrison, L. (1994) Repair of oxidative damage to DNA: Enzymology and biology. Annu. Rev. Biochem. 63, 915948. Balasubramanian, B., Pogozelski, W. K., and Tullius, T. D. (1998) DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc. Natl. Acad. Sci. U.S.A. 95, 9738-9743. Nygren, J., Ljungman, M., and Ahnstrom, G. (1995) Chromatin structure and radiation-induced DNA strand breaks in human cells: soluble scavengers and DNA-bound proteins offer a better protection against single- than double-strand breaks. Int. J. Radiat. Biol. 68, 11-18. Ljungman, M. (1991) The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers. Radiat. Res. 126, 58-64. Ljungman, M., and Hanawalt, P. C. (1992) Efficient protection against oxidative DNA damage in chromatin. Mol. Carcinog. 5, 264-269. Gelagutashvili, E. S., Sigua, K. I., and Sapojnikova, N. A. (1998) Binding and the nature of Cu(II) ion interaction with nucleosomes. J. Inorg. Biochem. 70, 207-210.

TX0002278