TOXlCOLOCYANDAPPLIEDPHARMACOLOCY
Interaction JOSE-LUIS Division
110,477-485
(1991)
of Copper with DNA and Antagonism SAGRIPANTI,’
PETER L. GOERING,
by Other Metals
AND ANTHONY
LAMANNA
of Li$e Sciences. OJice of Science and Technology, Center for Devices and Radiological Food and Drug Administration, Rockville, Maryland 20857
Received
April
15. 1991; accepted
Health,
June 10, 1991
Interaction of Copper with DNA and Antagonism by Other Metals. SAGRIPANTI, J.-L., GOERING, P. L.. AND LAMANNA, A. (1991). Toxicol. Appl. Pharmacol. 110, 477-485. Copper [Cu(II)] has been shown to enhance DNA damage in several biological systems. Binding of copper to DNA may be a key step in producing these lesions. The results of this study indicate that the DNA double helix contains at least two kinds of binding sites for copper. One site is present once every four nucleotides, has high affinity, and shows a cooperative effect.The other is an intercalating site for copper that is present in every base pair. This site is saturable, has a dissociation constant (Kd) for Cu(I1) of 4 I PM. In single-stranded DNA, we found an average copper binding site every three nucleotides with lower affinity than in dsDNA. The binding of copper to DNA shows an unexpected high specificity when studied in the presence of other metallic ions. The relative efficacy of several divalent cations to antagonize Cu(II) binding was: Ni = Cd = Mg 9 Zn = Hg > Ca > Pb + Mn, while Cr(V1) enhanced Cu(I1) binding to DNA. We hope this study will broaden the understanding of copper-DNA interactions, particularly as they relate to treatment modalities for diseasesassociated with disruption of copper homeostasis and potential development of copper antitumor agents.
Copper is an essential element and its concentration in serum and tissues is under homeostatic control. The normal level of copper in human blood is approximately 16 FM. The human fetal liver copper concentration is 30 pg/g and decreases 6 months postpartum to 5 to 10 pg/g (Aaseth and Norseth, 1986). Some human diseases, including Wilson’s disease and Menke’s syndrome (Sternlieb, 1980; Bremner, 1987) are associated with a disruption of copper homeostasis, resulting in elevated blood copper levels. These hereditary disorders are characterized by severe hepatocellular injury and a marked increase in the liver copper concentration. The mechanism by which physiological copper imbalance relates to disease is not well understood but may ’ To whom correspondence should be addressed at Dr. J. L. Sagripanti (HFZ-113) CDRH, FDA, 12709 Twinbrook Pkwy, Rockville, MD 20857.
include alterations of cell membrane integrity, enzyme inhibition, and reduced stability of DNA (Schilsky et al., 1989). Copper serves as a cofactor for enzymes, including ferrooxidases, cytochrome oxidase, superoxide dismutase, and amine oxidases (Aaseth and Norseth, 1986) and plays a role in gene regulation as a cofactor in a DNAbinding “copper fist” protein (Furst and Hamer, 1989). Copper at micromolar concentrations has been shown to enhance DNA damage in several biological systems, a property which has been exploited in cancer chemotherapy. Bleomycin, streptonigrin (Sugiura et al., 1985, 1984), and uv-photoactivated camptothecin (Kuwuhara et al., 1986) were shown to require copper for their antitumor action. In other studies, copper also enhanced the lethal effect of ionizing radiation (Samuni et al., 1984) and was involved in the DNA
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SAGRIPANTI,
GOERING,
damage produced by microwaves (Sagripanti et al., 1987). Binding of copper to DNA was proposed to be the cause of sequence-specific DNA damage produced by copper and peroxide (Sagripanti and Kraemer, 1989). The interaction of copper with nucleic acids may be key to understanding the development of several metabolic diseases and the mechanism of action of important chemotherapeutic drugs. Early studies showed that nucleic acids isolated from a variety of sources contain large amounts of firmly bound metals. RNA obtained from different animal species contained from 80 to 190 pg of bound copper per gram of RNA (Wacker and Vallee, 1959) and chromatin was found to contain 0.4 pmol of copper tightly bound per gram of DNA (Bryan et al., 1981). Jendryczko et al. (1986) found more copper in tumors than in surrounding tissue. Due to base pairing of nucleic acid residues, only a limited number of possible binding sites are available in double-stranded DNA (dsDNA). Of the available sites, interaction of metals with the phosphate moiety is considered to stabilize DNA, while binding to the bases is considered to destabilize the double helix (Eichorn and Shin, 1968). Based upon early experiments, metal ions were placed into two categories, those that bind to phosphate and those that bind to bases (for a review, see Izatt et al., 1971). Proton NMR data implicated binding of Cu(I1) to the phosphate moieties. Melting temperature and viscosity data indicated that Cu(I1) binds mainly to bases in DNA (Eichorn and Shin, 1968). Absorption spectral data demonstrated binding of Cu(I1) to the pyrimidine moieties of DNA. Despite these varied findings, laser Raman spectroscopy has shown that copper has a pronounced effect on DNA structure (Tajmir et a(., 1988). The biochemical nature of copper-DNA binding sites and how these interactions relate to the biological properties of this metal are not yet known. It is clear however that before copper can be available to bind DNA in a living organism, it will compete for the target site with a variety of other essential cations. The
AND LAMANNA
presence of nonessential metals due to environmental pollution or leaching from implantable medical devices could also alter copper binding to DNA. The present study was undertaken to examine the binding of Cu(I1) to dsDNA and single-stranded DNA (ssDNA). The data presented in this study indicate the existence of two kinds of copper binding sites in DNA. In addition, we have also studied the effect of nine metal competitor ions on Cu(II)-DNA interactions. We observed that DNA shows an unexpected high specificity for copper when compared with other metal ions. We hope a better understanding of the interaction of copper with DNA will further elucidate the mechanism for copper regulation at the DNA level as it is related to copper-mediated diseases and therapies.
METHODS Reagents. The following reagents were used: calfthymus DNA, double- and single-stranded DNA-cellulose, DNAfree cellulose, zinc (Zn(I1)) chloride, copper (Cu(I1)) chloride, mercuric (Hg(I1)) chloride (Sigma Chemical Co., St. Louis, MO), calcium (Ca(I1)) chloride and magnesium (Mg(I1)) monophosphate (Fisher Scientific Co., Fairlawn, NJ), cadmium (Cd(I1)) chloride, nickel (Ni(I1)) acetate, sodium acetate, acetic acid: and ammonium hydroxide (Mallinckrodt, Paris, KY), lead (Pb(I1)) acetate and potassium (Cr(V1)) chromate (J. T. Baker Co., Phillipsburg, NJ). Buffered ethanol was prepared by adding 2 ml 3 M sodium acetate buffer (pH adjusted to 5.0-5.2 with glacial acetic acid) to 1 liter of 95% ethanol. All reagents were analytical grade and were prepared in double-distilled, deionized water (dd water) with a pH of 6.9 and a conductivity of 0.05 j&. Copper determination. Samples were analyzed for Cu(I1) by flame atomic absorption spectrophotometry (FAAS) (Perkin-Elmer Model 305B) using a wavelength of 325 nm. The samples were compared to Cu(I1) standards ranging from 10 to 750 PM. Samples with various concentrations of Cu(I1) but without DNA were processed as blanks for the ethanol precipitation assay.Even at 1000 PM Cu(II), background absorbance after assay of these blanks remained unchanged. Therefore, 95% ethanol, pH 5.4, did not precipitate Cu(I1) in the absence of DNA at the highest Cu(I1) concentration studied. Phosphate and
COPPER-DNA Tris buffers, in contrast, interfered with Cu(II) detection by FAAS and use of these reagents was avoided. Binding to DNA-cellulose. Slurries of dsDNA- and ssDNA-cellulose and free cellulose were prepared in dd water. The amount of DNA attached to cellulose was estimated by the method of Albert and Herrick (197 1). Aliquots of 1 ml slurry were placed in a l&ml microcentrifuge tube and incubated at room temperature with various concentrations (50 to 700 PM) of Cu(I1) for 1 hr with periodic mixing. After incubation, slurries were centrifuged at 15,OOOgfor 15 min. Supematants were analyzed for ., free Cu(I1) as described above. Binding to DNA in solution. A DNA stock solution was prepared by dissolving calf thymus dsDNA in dd water at a concentration of 0.54 to 0.58 mg/ml. The concentration of DNA was estimated spectrophotometrically (260 nm) assuming 1 OD unit was equal to 50 and 40 pg/ml for dsDNA and ssDNA, respectively (Maniatis et al., 1982). The absorption ratio at 260 nm over 280 nm of the DNA solutions ranged from 1.872 to 1.894. The average molecular weight per nucleotide in DNA was considered to be 331 (Maniatis et al., 1982). Double-stranded DNA (56 rg) was incubated in a volume of 1 ml at room temperature with various concentrations of Cu(I1) (10 to 400 pM) for I hr and was mixed periodically. Following incubation, free and DNA-bound Cu(I1) were separated by ethanol precipitation (Sagripanti et al., 1987). To each incubate was added 5 ml of buffered ethanol, followed by vortexing and chilling at -9°C for 1 hr. The samples were then centrifuged at 2750g for 2 hr at - 10°C. Supernatants containing free Cu(I1) were decanted and the tubes inverted to drain onto absorbent paper for 30 min. The tubes containing the pellet of DNA-bound Cu(I1) were then placed in a 37°C heating block for 30 min to evaporate the remaining ethanol. The DNA pellets were then reconstituted in 1 ml 1.OM ammonium hydroxide and allowed to stand at room temperature for 60 min. At the end of this period, the samples were vortexed and analyzed for Cu(I1) as described above. In certain experiments ssDNA was produced by heating dsDNA for 10 min in a boiling water bath and chilling afterwards on ice. Binding antagonism. The ability of several physiologically essential and nonessential metals to antagonize Cu(II)-DNA binding in solution was determined. Incubates consisted of 150 pM Cu(I1) in the presence and absenceof Ca(II), Cd(II), Hg(II), Mg(II), Ni(I1). Zn(II), Pb(II), and Cr(V1) (50-10,000 pM). All metals were added to incubates prior to the DNA additions. The copper concentration was chosen because it was approximately two times the minimal amount necessary to saturate the dsDNA binding sites present in the incubates (see Fig. 2). Incubation and separation of bound and free Cu(I1) were performed by ethanol precipitation as described above. Dam were analyzed using log regression analysis to obtain an IC50 (concentration of inhibitor at which Cu(I1) binding is ilalf-maximal) for each inhibitor.
479
INTERACTIONS
Data analysis. Cu(I1) binding to DNA was analyzed by the graphic method of Scatchard (1949) with the mathematical considerations described by Sagripanti et al. ( 1984). The molar ratio of Cu(I1) to nucleotide bases in DNA was calculated assuming an average molecular weight of a DNA nucleotide is 33 1. The apparent dissociation constant (&) is the reciprocal of the apparent affinity constant (K,) which was estimated from the slope of the Scatchard plot using data obtained in seven independent experiments.
RESULTS The binding of Cu(I1) to DNA-cellulose was studied at 20°C after reaching equilibrium (Fig. 1). Binding of Cu(I1) to blank cellulose was studied in parallel. The dsDNA-cellulose consisted of 107 pg of dsDNA (323 nmol nucleotide) attached to 14.3 mg of cellulose (Fig. 1A). Binding of Cu(I1) to the same amount of blank cellulose is also displayed. There are 236 nmol copper binding sites in the dsDNA-cellulose. The saturation curve for cellulose indicates that there are 5.6 nmol of copper binding sites per milligram of cellulose. The specific binding of Cu(I1) to dsDNA is obtained by subtracting the copper bound to cellulose from the copper bound to dsDNA-cellulose. The presence of 156 nmol of Cu(I1) binding sites in the 323 nmol nucleotide present in the incubation mixture can be estimated by considering the copper specifically bound to dsDNA. This corresponds to 1 Cu(I1) binding site per 2.1 bases (or 1.46 nmol/pg of dsDNA). The binding of Cu(I1) to ssDNA was studied in a similar fashion and is displayed in Fig. 1B. The total binding of copper to 100 pg of ssDNA (302 nmol nucleotide) attached to 17.9 mg of cellulose was 19 1 nmol. The specific binding to ssDNA was 97 nmol of copper binding sites per 302 nmol of nucleotide. This corresponds to 1 site per 3.1 nucleotide (or 0.97 nmol sites/pg of ssDNA). Saturation of 156 nmol of sites in dsDNA was achieved with 300 nmol of Cu(I1); 500 nmol of Cu(I1) was necessary to saturate 97 nmol of ssDNA. This suggests a higher affinity of Cu(I1) for dsDNA sites than for SSDNA sites.
SAGRIPANTI,
480
Double-Stranded
I-
GGERING,
AND LAMANNA
The saturation curve for Cu(I1) binding sites in dsDNA in solution is shown in Fig. 2. The nonsaturable component of copper binding was considered to be nonspecific. The specific DNA bound copper was calculated by subtracting the nonspecific component from the total binding. A maximal specific binding capacity value corresponding to 80 PM Cu(I1) for a base concentration of 170 I.LM in dsDNA was obtained. This corresponds to approximately one copper binding site per two nucleotides. The ethanol precipitation method showed that the 56 pg of dsDNA present in the in-
DNA
/
120
100
Single-Stranded
DNA 60
0
200
400
600
600
1'
Total Cu &M)
FIG. 1. Analysis of copper bound to double- and singlestranded DNA-cellulose and DNA-free cellulose. Concentrations of total DNA-cellulose-bound Cu(I1) (closed circles), specific DNA-bound Cu(I1) (open circles), and nonspecific cellulose-bound Cu(I1) (triangles) as a function of the total copper concentration in the incubation mixture are shown. Various concentrations of Cu(I1) (50 to 700 FM) were incubated with either double- or single-stranded DNA-cellulose in a 1.0 ml total volume. Cu(I1) was incubated with an aliquot of dsDNA-cellulose consisting in 107 pg of dsDNA (323 nmol nucleotide) attached to 14.3 mg of cellulose. The ssDNA was aliquoted in 100 pg of dsDNA (302 nmol nucleotide) attached to 17.9 mg of cellulose. Each point represents the mean from four independent determinations and standard deviations were < 10% of the means.
The binding of copper to free cellulose was a deterrent to further binding studies, particularly in the range of low Cu(I1) concentrations. Binding of Cu(I1) to DNA in solution with subsequent separation of copper bound to DNA from free copper by ethanol precipitation was more successful and was used throughout the remainder of the studies.
5
z aa
60
40
I
20
0 L 0
r/ 100
200
Non-specific
300
400
Total Cu @M)
FIG. 2. Analysis of copper bound to double-stranded DNA in solution. Concentrations of total DNA-bound Cu(I1) (closed circles), specific DNA-bound Cu(I1) (open circles), and nonspecific Cu(I1) binding (triangles) as a function of the total copper concentration in the incubation mixture are shown. Various concentrations of Cu(I1) (10 to 400 pM) were incubated with 56 fig of dsDNA (170 nmol nucleotide) in 1.O ml total volume. Free and DNAbound copper were separated by an ethanol precipitation procedure described under Methods and copper was analyzed by flame atomic absorption spectrophotometry. Each point represents the mean from seven independent determinations and standard deviations were < 10% of the means.
COPPER-DNA
cubation mixture was saturated with 80 PM Cu(I1). This agrees closely with the results obtained with the method employing dsDNAcellulose (Fig. 1A), where 107 pg of dsDNA was saturated in the presence of 156 PM Cu(I1). Therefore, both methods indicate that saturation of copper binding sites is achieved with approximately 1.4 PM Cu(I1) per microgram of dsDNA. Scatchard analysis (Scatchard, 1949) of copper-DNA interactions over a broad range of Cu(I1) concentrations from seven independent experiments exhibited several interesting features which are depicted in Fig. 3. At a low proportion of occupied sites corresponding up to one copper molecule bound per four bases (=G40 PM Cu(I1) in the figure), a positive slope was observed, indicative of positive cooperativity of Cu(I1) binding to DNA. When the proportion of bound copper molecules per base fell in the range between 1:4 and 1:2, (40 to 80 PM in the figure), an approximate linear relationship with negative slope was obtained. Analysis of the regions of the Scatchard plot showing a negative slope revealed that the concentration of copper binding sites was 86 I.LM for 170 PM of nucleotides in dsDNA, corresponding to one site per two bases. The best fit slope for the descending portion of the binding curve revealed an affinity constant (&) of 2.4 x lo4 M-‘. Although ethanol separation of DNAbound and free Cu(I1) was useful for Scatchard analysis and the studies with metal antagonists that will be discussed later, the method appeared suboptimal for kinetic studies. No binding differences could be detected between binding at equilibrium and the shortest incubation time that could be achieved by this technique (1 min). No differences in binding between Cu(I1) and dsDNA could be detected for incubations carried out in a range of temperature between 4 and 37°C. The capacity of nine metals to antagonize Cu(I1) binding to DNA was determined and is shown in Fig. 4. None of the metals interfered with the FAAS detection method for
481
INTERACTIONS
IO
Bound
Cu (JLM)
FIG. 3. The binding of copper to DNA as analyzed by the method of Scatchard ( 1949). Analysis was conducted using values for specific DNA-bound Cu(II) shown in Fig. 2. Each point represents the mean from sevenindependent determinations.
copper. In all cases, the background absorbance was unchanged by the antagonist metals even at the highest concentration analyzed (10,000 PM). The displacement of Cu(I1) was approximately a linear function of the log antagonist concentration in the range tested. All metal antagonists demonstrated a negative displacement trend except for Cr(VI), which showed an apparent enhancement of copper binding to DNA. The concentration of metal which displaces 50% of bound Cu(I1) (IC50) can be compared in Table 1. The ability to displace Cu(I1) from DNA was Ni = Cd = Mg b Zn = Hg > Ca > Pb p Mn. In the presence of 150 FM Cu(II), 50% displacement of copper was achieved by addition of 3- to 5-fold excess of Ni(II), Cd(II), and Mg(I1). A 20- to 25-fold excess of Zn(I1) and Hg(II), and an approximately 40-fold excess of Ca(I1) and Pb(I1) were required to achieve a 50% copper displacement. In the presence of 150 PM Cu(II), 456 PM Ni(I1) was able to displace 50% bound copper. Assuming
482
SAGRIPANTI,
GOERING,
Yit......
=
loo
-
Q E 8 8o B E 6o z i Li 4o a
.
k
. . . WI .
’ Mn CA,’ ’
20 t
FIG. 4. Concentration-dependent effect of various metals on copper binding to DNA. The Cu(I1) concentration in alI incubates was 150 pM and 100% represents DNA-bound Cu(II) in the absence of competing ions. Each point represents the mean of five to nine independent determinations.
that both metals compete for the same chemical structures, this result indicates that Ni(I1) ions have about half the affinity of Cu(I1) for the copper binding sites. Mn(I1) essentially did not displace Cu(I1) from DNA; the IC50 was 14,55 1 PM. Cr(V1) apparently enhanced copper binding to DNA. DISCUSSION Copper has been shown to be deleterious to chromosomes and increases the frequency of
AND LAMANNA
noncomplementary nucleotides in DNA (Aaseth and Norseth, 1986). Copper in the presence of peroxide produces DNA lesions located specifically in polyguanosine sequences. This work suggested that the key event in producing DNA damage is the binding of copper to specific sites within DNA (Sagripanti and Kraemer, 1989). The present study attempted to extend these findings by characterizing the copper-DNA interaction and the extent of the specificity of the binding sites for the metal. Our saturation studies indicate that there is an average of one Cu(I1) molecule bound for every two nucleotides equivalent to 1.50 pmol copper per milligram of dsDNA. Bryan et al. (198 1) found 0.4 nmol of copper tightly bound per milligram of DNA in chromatin. Taken together, these results indicate that only 1 in 3700 copper sites are actually occupied in DNA in vivo. Unsaturated binding sites may allow for a regulatory role of copper at the DNA level. In ssDNA there is an average copper binding site every three nucleotides as compared with one average Cu(I1) site every two bases in dsDNA (Fig. 1). This indicates that appropriate DNA base-pairing accounts for some of the Cu(I1) binding. We found the interaction of Cu(I1) with ssDNA to be of lower affinity TABLE 1 ANTAGONISM
OF COPPER BINDING VARIOUS METALS
TO DNA
Antagonist
1C50 (PM)”
Ni(I1) Cd(I1) M&II) Zn(I1) WIU Ca(I1) WI) Mn(I1) ctiw
456 670 709 3,407 3,557 5.361 7,008 14,551 Enhanced binding
BY
u Concentrations of various metals that displace 50% of Cu(I1) bound to dsDNA under the conditions described in the legend to Fig. 4.
COPPER-DNA
than with dsDNA (Fig. 1); however, the implications of copper binding to ssDNA as found in open transcribing chromatin could be significant for gene regulation. Scatchard plots with marked deviation from linearity and with positive slopes at low occupancy of DNA sites have been shown for carcinogenic divalent metals such as Hg(II), Cd(II), and Ni(I1) (Simpson, 1964; Waalkes and Poirier, 1984; Kasprzak et al., 1986). The steep positive slope we observed for copper could indicate a very high affinity between this metal and the low abundance sites in DNA (Fig. 3). The presence of one site per four nucleotides in dsDNA agrees with a previous report about the formation of a DNA base-copper complex with a stoichiometry of one complex per four bases (Minchenkova and Ivanov, 1967). One copper binding site per four nucleotides can be explained in one of two ways. Either Cu(I1) binds to four bases in two adjacent base pairs, or Cu(I1) shows preferential binding for one of the four bases. A previous report (Sagripanti and Kraemer, 1989) describing a copper-mediated lesion specific for guanosine suggests that the low abundance binding site could be base-specific. For the Cu(I1) binding sites in high abundance we obtained an apparent stability constant (&) of 2.4 X lo4 M-’ (Fig. 3). This value is comparable to those obtained previously by spectroscopic analysis under different ionic strengths (1.1 X 10m4 M-’ in 0.25 M NaCl to 1.8 X lo4 M-’ in 0.001 M NaCl; Sayenko et al., 1986). Stacking of complementary bases in DNA and electrostatic binding of Na(1) to phosphates as ionic strength increases most likely makes the penetration of partially hydrated Cu(I1) into the double helix more difficult. Our slightly higher value of K, can be explained by the fact that we carry out our binding studies in water to maximize the access of copper to all potential DNA binding sites. The relatively more abundant copper binding site is present on average once every two
INTERACTIONS
483
nucleotides. The frequency of appearance of the high abundance site is similar to that observed upon the release of Cu(I1) during DNA renaturation (Richard et al., 1973). However, we do not consider this copper binding site simply as counterion neutralization of phosphate charges. The stability constant reported for the interaction of Cu(I1) and the phosphate in cytidine-monophosphate was 7 X 10’ Me1 (Siegel, 1989). Binding to phosphate alone could not explain the higher affinity we found for Cu(II)-DNA. The low competition we have observed for other divalent cations also indicates a more specific interaction between copper and DNA. We interpret the high abundance site in dsDNA as including groups from two nucleotides in complementary strands. It is well known that various metals will antagonize the effects of other metals both in vitro and in vivo. For example, cadmium binding to DNA in vitro was antagonized by zinc, magnesium, and calcium (Waalkes and Poitier, 1984). Magnesium, manganese, and calcium reduced nickel binding to DNA in vitro (Kasprzak et al., 1986). The relative capacities of these metals to antagonize binding of cadmium and nickel to DNA correlates remarkably with their abilities to prevent cadmium and nickel carcinogenesis (Waalkes and Poitier, 1984; Kasprzak et al., 1986). Other studies have shown that zinc administration attenuates copper toxicity in Wilson’s disease (Shilsky et al., 1989; Brewer et al., 1989). In the present study, several physiologically essential and nonessential cations were found to compete with binding of Cu(I1) to DNA. The relative efficacy of these metals in antagonizing Cu(I1) binding was: Ni(I1) = Cd(I1) = Mg(I1) >> Zn(I1) = Hg(I1) > Ca(I1) > Pb(I1) B Mn(I1). The low antagonism of Zn(I1) observed in this study suggests that the mitigating effect of zinc in anticopper therapy for Wilson’s disease (Schilsky et al., 1989; Brewer et al., 1989) is not mediated by displacement of Cu(I1) from DNA binding sites.
484
SAGRIPANTI,
GOERING,
The value for the apparent stability constant of 2.4 X lo4 M-t we obtained for Cu(I1) binding sites in high abundance is lower than the reported values for cadmium (4 X lo4 M-l; Waalkes and Poirier, 1984) and for nickel ( 19 X lo4 M-t; Kasprzak et al., 1986). The fact that metals with higher affinity constants for DNA, such as cadmium and nickel, displaced Cu(I1) minimally, leads us to speculate that the primary binding site for copper is different than the binding site for the other metals. The distorted octahedral coordination sphere proposed for Cu(I1) and the strong tendency to coordinate donor atoms equatorially (Siegel and Martin, 1982) may be responsible for the specific interaction with DNA. The biological mechanisms underlying copper toxicity could be rooted in the binding of copper to specific sites within DNA. In spite of the body of data accumulated regarding metal-DNA interactions, the characteristics and biological consequences of the binding of copper to DNA remains an exciting field to be explored. ACKNOWLEDGMENT The authors recognize Edward Gordon for performing the log regression analyses in this report.
REFERENCES ALBERTS, B., AND HERRICK, G. (1971). DNA-cellulose chromatography. In Methods in Enzymology (L. Grossman and K. Moldave, Eds.), Vol. 21, pp. 198-217. Academic Press, San Diego. AASETH, J., AND NORSETH, T. (1986). Copper. In Handbook on the Toxicology of Metals (L. Friberg, G. F. Nordberg, and V. B. Vouk, Eds.), Vol. 2, pp. 233-254. Elsevier, New York. BREMNER, I. (1987). Nutritional and physiological significance of metallothionein. Experientia Suppl. 52, 8 l107. BREWER, G. J., YUZBASIYAN-GURKAN, V., LEE, D.-Y., AND APPELMAN, H. (1989). Treatment of Wilson’s disease with zinc. VI. Initial treatment studies. J. Lab. Clin. Med. 114,633-638. BRYAN, S. E., VIZARD, D. L., BEARY, D. A., LABICHE, R. A., AND HARDY, K. J. (198 1). Partitioning of zinc
AND LAMANNA
and copper within subnuclear nucleoprotein particles. Nucleic Acids Res. 9, 581 l-5823. EICHORN, L. E., AND SHIN, Y. A. (1968). Interaction of metal ions with polynucleotides and related compounds. XII. The relative effect of various metal ions on DNA helicity. J. Am. Chem. Sot. 90, 7323-7328. FURST,P., AND HAMER, D. (1989). Cooperative activation of a eukariotic transcription factor: Interaction between Cu(I) and yeast ACE1 protein. Proc. Natl. Acad. Sci. USA 86,5267-527 1. IZATT, R. M., CHRISTENSEN,J. J., AND RYTTING, J. H. (197 1). Sites and thermodynamic quantities associated with proton and metal ion interaction with RNA, DNA and their constituent bases,nucleosides, and nucleotides. Chem. Rev. 71,439-48 1. JENDRYCZKO,A., DROZDZ, M., TOMALA, J., AND MAGNER, K. (1986). Copper and zinc concentrations, and superoxide dismutase activities in malignant and nonmalignant tissue of female reproductive organs. Neoplasma 33,239-244. KASPRZAK, K. S., WAALKES, M. P., AND POIRIER, L. A. (1986). Antagonism by essential divalent metals and amino acids of nickel(II)-DNA binding in vitro. Toxicol. Appl. Pharmacol. 82,336-343. KUWAHARA, J., SUZUKI, T., FUNAKOSHI, K., AND SUGIURA, Y ( 1986). Photosensitive DNA cleavageand phage inactivation by copper(camptothecin. Biochemistry 25, 1216-1221. MANIATIS, T., FRITSCH,E. F., AND SAMBROOK. J. (1982). Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. MICHENKOVA, L. E., AND IVANOV, V. I. (1967). Influence of reductants upon optical characteristics of the DNACu(I1) complex. Biopolymers 5, 6 15-625. RICHARD, H., SCHREIBER,J. P., AND DUANE, M. (1973). Interactions of metallic ions with DNA. V. DNA renaturation mechanism in the presence of Cu(I1). Biopolymers 12, 1- 10. SAGRIPANT~, J.-L., AND KRAEMER, K. H. (1989). Sitespecific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide. J. Biol. Chem. 264, 1729-1734.
SAGRIPANTI, J. L., SWICORD. M. L., AND DAVIS, C. C. (1987). Microwave effects on plasmid DNA. Radiat. Res. 110, 219-231. SAGRIPANTI, J. L.. SANTACOLOMA, T. A., AND CALVO, J. C. (1984). A simple computer program for Scatchard plot analysis of hormone receptors including statistical analysis on a low cost desktop calculator. Acta Fisiol. Latinoam. 34,45-53. SAMUNI, A., CHEVION, M., AND CZPASKI,G. (1984). Roles of copper and O(2) in the radiation-induced inactivation of T7 bacteriophage. Radiat. Res. 99, 562-572. SAYENKO, G. N., BABII, A. P., AND BAGAVEYEV. I. A. (1986). Change in the ultraviolet spectra of copper(R)
COPPER-DNA complexes with native DNA with the ionic strength of solution. Biophysics 31,45 l-456. SCATCHARD, G. (1949). The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51,660612.
SCHILSKY, M. L., BLANK, R. R., CZAJA, M. .I., ZERN, M. A., SCHEINBERG, I. H., STOCKERT, R. J., AND STERNLIEB,I. (1989). Hepatocellular copper toxicity and its attenuation by zinc. J. Clin. Invest. 84, 1562-l 568. SIEGEL, H. (1989). Metal nucleotide interactions. In Metal DNA Chemistry. pp. 158-203. American Chemical Society, Washington, DC. SIEGEL, H., AND MARTIN, B. R. (1982). Coordinating properties of the amine bond. Stability and structure of metal ion complexes of peptides and related ligands. Chem. Rev. 82,385-426. SIMPSON, R. B. ( 1964). Association constants of methylmercuric and mercuric ions with nucleosides. J. Amer. Chem. Sot. 86,2059-2065.
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STERNLIEB, I. (1980). Copper and the liver. Gastroenterology78, 1615-1628. SUGIURA, Y., TAKITA, T., AND UMEZAWA, H. (1985). Bleomycin antibiotics: Metal complexes and their biological action. Metal Ions Biol. Syst. 19, 8 l-108. S~G~JRA, Y., Kuwm, J., AND Suzrrru, T. (1984) DNA interaction and nucleotide sequence cleavage of copperstreptonigrin. Biochem. Biophys. Acta 782,254-26 1. TAJMIR, H. A., LANGLAIS, M., AND SAVOIE, R. (1988). A laser Raman spectroscopic study of the interaction of calf thymus DNA with Cu(II) and Pb(II) ions: Metal binding and DNA conformational changes. Nucl. Acid Res. 16, 15 I-762. WAALKES, M. P., AND POIRIEQ L. A. (1984). In vitro cadmium-DNA interactions: Cooperativity of cadmium binding and competitive antagonism by calcium, magnesium, and zinc. Toxicol. Appl. Pharmacol. 75,539-546. WACKER, W. E., AND VALLEE, B. L. (1959). Nucleic acids and metals. J. Biol. Chem. 234,3257-3262.
Interaction JOSE-LUIS Division
110,477-485
(1991)
of Copper with DNA and Antagonism SAGRIPANTI,’
PETER L. GOERING,
by Other Metals
AND ANTHONY
LAMANNA
of Li$e Sciences. OJice of Science and Technology, Center for Devices and Radiological Food and Drug Administration, Rockville, Maryland 20857
Received
April
15. 1991; accepted
Health,
June 10, 1991
Interaction of Copper with DNA and Antagonism by Other Metals. SAGRIPANTI, J.-L., GOERING, P. L.. AND LAMANNA, A. (1991). Toxicol. Appl. Pharmacol. 110, 477-485. Copper [Cu(II)] has been shown to enhance DNA damage in several biological systems. Binding of copper to DNA may be a key step in producing these lesions. The results of this study indicate that the DNA double helix contains at least two kinds of binding sites for copper. One site is present once every four nucleotides, has high affinity, and shows a cooperative effect.The other is an intercalating site for copper that is present in every base pair. This site is saturable, has a dissociation constant (Kd) for Cu(I1) of 4 I PM. In single-stranded DNA, we found an average copper binding site every three nucleotides with lower affinity than in dsDNA. The binding of copper to DNA shows an unexpected high specificity when studied in the presence of other metallic ions. The relative efficacy of several divalent cations to antagonize Cu(II) binding was: Ni = Cd = Mg 9 Zn = Hg > Ca > Pb + Mn, while Cr(V1) enhanced Cu(I1) binding to DNA. We hope this study will broaden the understanding of copper-DNA interactions, particularly as they relate to treatment modalities for diseasesassociated with disruption of copper homeostasis and potential development of copper antitumor agents.
Copper is an essential element and its concentration in serum and tissues is under homeostatic control. The normal level of copper in human blood is approximately 16 FM. The human fetal liver copper concentration is 30 pg/g and decreases 6 months postpartum to 5 to 10 pg/g (Aaseth and Norseth, 1986). Some human diseases, including Wilson’s disease and Menke’s syndrome (Sternlieb, 1980; Bremner, 1987) are associated with a disruption of copper homeostasis, resulting in elevated blood copper levels. These hereditary disorders are characterized by severe hepatocellular injury and a marked increase in the liver copper concentration. The mechanism by which physiological copper imbalance relates to disease is not well understood but may ’ To whom correspondence should be addressed at Dr. J. L. Sagripanti (HFZ-113) CDRH, FDA, 12709 Twinbrook Pkwy, Rockville, MD 20857.
include alterations of cell membrane integrity, enzyme inhibition, and reduced stability of DNA (Schilsky et al., 1989). Copper serves as a cofactor for enzymes, including ferrooxidases, cytochrome oxidase, superoxide dismutase, and amine oxidases (Aaseth and Norseth, 1986) and plays a role in gene regulation as a cofactor in a DNAbinding “copper fist” protein (Furst and Hamer, 1989). Copper at micromolar concentrations has been shown to enhance DNA damage in several biological systems, a property which has been exploited in cancer chemotherapy. Bleomycin, streptonigrin (Sugiura et al., 1985, 1984), and uv-photoactivated camptothecin (Kuwuhara et al., 1986) were shown to require copper for their antitumor action. In other studies, copper also enhanced the lethal effect of ionizing radiation (Samuni et al., 1984) and was involved in the DNA
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$3.00
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SAGRIPANTI,
GOERING,
damage produced by microwaves (Sagripanti et al., 1987). Binding of copper to DNA was proposed to be the cause of sequence-specific DNA damage produced by copper and peroxide (Sagripanti and Kraemer, 1989). The interaction of copper with nucleic acids may be key to understanding the development of several metabolic diseases and the mechanism of action of important chemotherapeutic drugs. Early studies showed that nucleic acids isolated from a variety of sources contain large amounts of firmly bound metals. RNA obtained from different animal species contained from 80 to 190 pg of bound copper per gram of RNA (Wacker and Vallee, 1959) and chromatin was found to contain 0.4 pmol of copper tightly bound per gram of DNA (Bryan et al., 1981). Jendryczko et al. (1986) found more copper in tumors than in surrounding tissue. Due to base pairing of nucleic acid residues, only a limited number of possible binding sites are available in double-stranded DNA (dsDNA). Of the available sites, interaction of metals with the phosphate moiety is considered to stabilize DNA, while binding to the bases is considered to destabilize the double helix (Eichorn and Shin, 1968). Based upon early experiments, metal ions were placed into two categories, those that bind to phosphate and those that bind to bases (for a review, see Izatt et al., 1971). Proton NMR data implicated binding of Cu(I1) to the phosphate moieties. Melting temperature and viscosity data indicated that Cu(I1) binds mainly to bases in DNA (Eichorn and Shin, 1968). Absorption spectral data demonstrated binding of Cu(I1) to the pyrimidine moieties of DNA. Despite these varied findings, laser Raman spectroscopy has shown that copper has a pronounced effect on DNA structure (Tajmir et a(., 1988). The biochemical nature of copper-DNA binding sites and how these interactions relate to the biological properties of this metal are not yet known. It is clear however that before copper can be available to bind DNA in a living organism, it will compete for the target site with a variety of other essential cations. The
AND LAMANNA
presence of nonessential metals due to environmental pollution or leaching from implantable medical devices could also alter copper binding to DNA. The present study was undertaken to examine the binding of Cu(I1) to dsDNA and single-stranded DNA (ssDNA). The data presented in this study indicate the existence of two kinds of copper binding sites in DNA. In addition, we have also studied the effect of nine metal competitor ions on Cu(II)-DNA interactions. We observed that DNA shows an unexpected high specificity for copper when compared with other metal ions. We hope a better understanding of the interaction of copper with DNA will further elucidate the mechanism for copper regulation at the DNA level as it is related to copper-mediated diseases and therapies.
METHODS Reagents. The following reagents were used: calfthymus DNA, double- and single-stranded DNA-cellulose, DNAfree cellulose, zinc (Zn(I1)) chloride, copper (Cu(I1)) chloride, mercuric (Hg(I1)) chloride (Sigma Chemical Co., St. Louis, MO), calcium (Ca(I1)) chloride and magnesium (Mg(I1)) monophosphate (Fisher Scientific Co., Fairlawn, NJ), cadmium (Cd(I1)) chloride, nickel (Ni(I1)) acetate, sodium acetate, acetic acid: and ammonium hydroxide (Mallinckrodt, Paris, KY), lead (Pb(I1)) acetate and potassium (Cr(V1)) chromate (J. T. Baker Co., Phillipsburg, NJ). Buffered ethanol was prepared by adding 2 ml 3 M sodium acetate buffer (pH adjusted to 5.0-5.2 with glacial acetic acid) to 1 liter of 95% ethanol. All reagents were analytical grade and were prepared in double-distilled, deionized water (dd water) with a pH of 6.9 and a conductivity of 0.05 j&. Copper determination. Samples were analyzed for Cu(I1) by flame atomic absorption spectrophotometry (FAAS) (Perkin-Elmer Model 305B) using a wavelength of 325 nm. The samples were compared to Cu(I1) standards ranging from 10 to 750 PM. Samples with various concentrations of Cu(I1) but without DNA were processed as blanks for the ethanol precipitation assay.Even at 1000 PM Cu(II), background absorbance after assay of these blanks remained unchanged. Therefore, 95% ethanol, pH 5.4, did not precipitate Cu(I1) in the absence of DNA at the highest Cu(I1) concentration studied. Phosphate and
COPPER-DNA Tris buffers, in contrast, interfered with Cu(II) detection by FAAS and use of these reagents was avoided. Binding to DNA-cellulose. Slurries of dsDNA- and ssDNA-cellulose and free cellulose were prepared in dd water. The amount of DNA attached to cellulose was estimated by the method of Albert and Herrick (197 1). Aliquots of 1 ml slurry were placed in a l&ml microcentrifuge tube and incubated at room temperature with various concentrations (50 to 700 PM) of Cu(I1) for 1 hr with periodic mixing. After incubation, slurries were centrifuged at 15,OOOgfor 15 min. Supematants were analyzed for ., free Cu(I1) as described above. Binding to DNA in solution. A DNA stock solution was prepared by dissolving calf thymus dsDNA in dd water at a concentration of 0.54 to 0.58 mg/ml. The concentration of DNA was estimated spectrophotometrically (260 nm) assuming 1 OD unit was equal to 50 and 40 pg/ml for dsDNA and ssDNA, respectively (Maniatis et al., 1982). The absorption ratio at 260 nm over 280 nm of the DNA solutions ranged from 1.872 to 1.894. The average molecular weight per nucleotide in DNA was considered to be 331 (Maniatis et al., 1982). Double-stranded DNA (56 rg) was incubated in a volume of 1 ml at room temperature with various concentrations of Cu(I1) (10 to 400 pM) for I hr and was mixed periodically. Following incubation, free and DNA-bound Cu(I1) were separated by ethanol precipitation (Sagripanti et al., 1987). To each incubate was added 5 ml of buffered ethanol, followed by vortexing and chilling at -9°C for 1 hr. The samples were then centrifuged at 2750g for 2 hr at - 10°C. Supernatants containing free Cu(I1) were decanted and the tubes inverted to drain onto absorbent paper for 30 min. The tubes containing the pellet of DNA-bound Cu(I1) were then placed in a 37°C heating block for 30 min to evaporate the remaining ethanol. The DNA pellets were then reconstituted in 1 ml 1.OM ammonium hydroxide and allowed to stand at room temperature for 60 min. At the end of this period, the samples were vortexed and analyzed for Cu(I1) as described above. In certain experiments ssDNA was produced by heating dsDNA for 10 min in a boiling water bath and chilling afterwards on ice. Binding antagonism. The ability of several physiologically essential and nonessential metals to antagonize Cu(II)-DNA binding in solution was determined. Incubates consisted of 150 pM Cu(I1) in the presence and absenceof Ca(II), Cd(II), Hg(II), Mg(II), Ni(I1). Zn(II), Pb(II), and Cr(V1) (50-10,000 pM). All metals were added to incubates prior to the DNA additions. The copper concentration was chosen because it was approximately two times the minimal amount necessary to saturate the dsDNA binding sites present in the incubates (see Fig. 2). Incubation and separation of bound and free Cu(I1) were performed by ethanol precipitation as described above. Dam were analyzed using log regression analysis to obtain an IC50 (concentration of inhibitor at which Cu(I1) binding is ilalf-maximal) for each inhibitor.
479
INTERACTIONS
Data analysis. Cu(I1) binding to DNA was analyzed by the graphic method of Scatchard (1949) with the mathematical considerations described by Sagripanti et al. ( 1984). The molar ratio of Cu(I1) to nucleotide bases in DNA was calculated assuming an average molecular weight of a DNA nucleotide is 33 1. The apparent dissociation constant (&) is the reciprocal of the apparent affinity constant (K,) which was estimated from the slope of the Scatchard plot using data obtained in seven independent experiments.
RESULTS The binding of Cu(I1) to DNA-cellulose was studied at 20°C after reaching equilibrium (Fig. 1). Binding of Cu(I1) to blank cellulose was studied in parallel. The dsDNA-cellulose consisted of 107 pg of dsDNA (323 nmol nucleotide) attached to 14.3 mg of cellulose (Fig. 1A). Binding of Cu(I1) to the same amount of blank cellulose is also displayed. There are 236 nmol copper binding sites in the dsDNA-cellulose. The saturation curve for cellulose indicates that there are 5.6 nmol of copper binding sites per milligram of cellulose. The specific binding of Cu(I1) to dsDNA is obtained by subtracting the copper bound to cellulose from the copper bound to dsDNA-cellulose. The presence of 156 nmol of Cu(I1) binding sites in the 323 nmol nucleotide present in the incubation mixture can be estimated by considering the copper specifically bound to dsDNA. This corresponds to 1 Cu(I1) binding site per 2.1 bases (or 1.46 nmol/pg of dsDNA). The binding of Cu(I1) to ssDNA was studied in a similar fashion and is displayed in Fig. 1B. The total binding of copper to 100 pg of ssDNA (302 nmol nucleotide) attached to 17.9 mg of cellulose was 19 1 nmol. The specific binding to ssDNA was 97 nmol of copper binding sites per 302 nmol of nucleotide. This corresponds to 1 site per 3.1 nucleotide (or 0.97 nmol sites/pg of ssDNA). Saturation of 156 nmol of sites in dsDNA was achieved with 300 nmol of Cu(I1); 500 nmol of Cu(I1) was necessary to saturate 97 nmol of ssDNA. This suggests a higher affinity of Cu(I1) for dsDNA sites than for SSDNA sites.
SAGRIPANTI,
480
Double-Stranded
I-
GGERING,
AND LAMANNA
The saturation curve for Cu(I1) binding sites in dsDNA in solution is shown in Fig. 2. The nonsaturable component of copper binding was considered to be nonspecific. The specific DNA bound copper was calculated by subtracting the nonspecific component from the total binding. A maximal specific binding capacity value corresponding to 80 PM Cu(I1) for a base concentration of 170 I.LM in dsDNA was obtained. This corresponds to approximately one copper binding site per two nucleotides. The ethanol precipitation method showed that the 56 pg of dsDNA present in the in-
DNA
/
120
100
Single-Stranded
DNA 60
0
200
400
600
600
1'
Total Cu &M)
FIG. 1. Analysis of copper bound to double- and singlestranded DNA-cellulose and DNA-free cellulose. Concentrations of total DNA-cellulose-bound Cu(I1) (closed circles), specific DNA-bound Cu(I1) (open circles), and nonspecific cellulose-bound Cu(I1) (triangles) as a function of the total copper concentration in the incubation mixture are shown. Various concentrations of Cu(I1) (50 to 700 FM) were incubated with either double- or single-stranded DNA-cellulose in a 1.0 ml total volume. Cu(I1) was incubated with an aliquot of dsDNA-cellulose consisting in 107 pg of dsDNA (323 nmol nucleotide) attached to 14.3 mg of cellulose. The ssDNA was aliquoted in 100 pg of dsDNA (302 nmol nucleotide) attached to 17.9 mg of cellulose. Each point represents the mean from four independent determinations and standard deviations were < 10% of the means.
The binding of copper to free cellulose was a deterrent to further binding studies, particularly in the range of low Cu(I1) concentrations. Binding of Cu(I1) to DNA in solution with subsequent separation of copper bound to DNA from free copper by ethanol precipitation was more successful and was used throughout the remainder of the studies.
5
z aa
60
40
I
20
0 L 0
r/ 100
200
Non-specific
300
400
Total Cu @M)
FIG. 2. Analysis of copper bound to double-stranded DNA in solution. Concentrations of total DNA-bound Cu(I1) (closed circles), specific DNA-bound Cu(I1) (open circles), and nonspecific Cu(I1) binding (triangles) as a function of the total copper concentration in the incubation mixture are shown. Various concentrations of Cu(I1) (10 to 400 pM) were incubated with 56 fig of dsDNA (170 nmol nucleotide) in 1.O ml total volume. Free and DNAbound copper were separated by an ethanol precipitation procedure described under Methods and copper was analyzed by flame atomic absorption spectrophotometry. Each point represents the mean from seven independent determinations and standard deviations were < 10% of the means.
COPPER-DNA
cubation mixture was saturated with 80 PM Cu(I1). This agrees closely with the results obtained with the method employing dsDNAcellulose (Fig. 1A), where 107 pg of dsDNA was saturated in the presence of 156 PM Cu(I1). Therefore, both methods indicate that saturation of copper binding sites is achieved with approximately 1.4 PM Cu(I1) per microgram of dsDNA. Scatchard analysis (Scatchard, 1949) of copper-DNA interactions over a broad range of Cu(I1) concentrations from seven independent experiments exhibited several interesting features which are depicted in Fig. 3. At a low proportion of occupied sites corresponding up to one copper molecule bound per four bases (=G40 PM Cu(I1) in the figure), a positive slope was observed, indicative of positive cooperativity of Cu(I1) binding to DNA. When the proportion of bound copper molecules per base fell in the range between 1:4 and 1:2, (40 to 80 PM in the figure), an approximate linear relationship with negative slope was obtained. Analysis of the regions of the Scatchard plot showing a negative slope revealed that the concentration of copper binding sites was 86 I.LM for 170 PM of nucleotides in dsDNA, corresponding to one site per two bases. The best fit slope for the descending portion of the binding curve revealed an affinity constant (&) of 2.4 x lo4 M-‘. Although ethanol separation of DNAbound and free Cu(I1) was useful for Scatchard analysis and the studies with metal antagonists that will be discussed later, the method appeared suboptimal for kinetic studies. No binding differences could be detected between binding at equilibrium and the shortest incubation time that could be achieved by this technique (1 min). No differences in binding between Cu(I1) and dsDNA could be detected for incubations carried out in a range of temperature between 4 and 37°C. The capacity of nine metals to antagonize Cu(I1) binding to DNA was determined and is shown in Fig. 4. None of the metals interfered with the FAAS detection method for
481
INTERACTIONS
IO
Bound
Cu (JLM)
FIG. 3. The binding of copper to DNA as analyzed by the method of Scatchard ( 1949). Analysis was conducted using values for specific DNA-bound Cu(II) shown in Fig. 2. Each point represents the mean from sevenindependent determinations.
copper. In all cases, the background absorbance was unchanged by the antagonist metals even at the highest concentration analyzed (10,000 PM). The displacement of Cu(I1) was approximately a linear function of the log antagonist concentration in the range tested. All metal antagonists demonstrated a negative displacement trend except for Cr(VI), which showed an apparent enhancement of copper binding to DNA. The concentration of metal which displaces 50% of bound Cu(I1) (IC50) can be compared in Table 1. The ability to displace Cu(I1) from DNA was Ni = Cd = Mg b Zn = Hg > Ca > Pb p Mn. In the presence of 150 FM Cu(II), 50% displacement of copper was achieved by addition of 3- to 5-fold excess of Ni(II), Cd(II), and Mg(I1). A 20- to 25-fold excess of Zn(I1) and Hg(II), and an approximately 40-fold excess of Ca(I1) and Pb(I1) were required to achieve a 50% copper displacement. In the presence of 150 PM Cu(II), 456 PM Ni(I1) was able to displace 50% bound copper. Assuming
482
SAGRIPANTI,
GOERING,
Yit......
=
loo
-
Q E 8 8o B E 6o z i Li 4o a
.
k
. . . WI .
’ Mn CA,’ ’
20 t
FIG. 4. Concentration-dependent effect of various metals on copper binding to DNA. The Cu(I1) concentration in alI incubates was 150 pM and 100% represents DNA-bound Cu(II) in the absence of competing ions. Each point represents the mean of five to nine independent determinations.
that both metals compete for the same chemical structures, this result indicates that Ni(I1) ions have about half the affinity of Cu(I1) for the copper binding sites. Mn(I1) essentially did not displace Cu(I1) from DNA; the IC50 was 14,55 1 PM. Cr(V1) apparently enhanced copper binding to DNA. DISCUSSION Copper has been shown to be deleterious to chromosomes and increases the frequency of
AND LAMANNA
noncomplementary nucleotides in DNA (Aaseth and Norseth, 1986). Copper in the presence of peroxide produces DNA lesions located specifically in polyguanosine sequences. This work suggested that the key event in producing DNA damage is the binding of copper to specific sites within DNA (Sagripanti and Kraemer, 1989). The present study attempted to extend these findings by characterizing the copper-DNA interaction and the extent of the specificity of the binding sites for the metal. Our saturation studies indicate that there is an average of one Cu(I1) molecule bound for every two nucleotides equivalent to 1.50 pmol copper per milligram of dsDNA. Bryan et al. (198 1) found 0.4 nmol of copper tightly bound per milligram of DNA in chromatin. Taken together, these results indicate that only 1 in 3700 copper sites are actually occupied in DNA in vivo. Unsaturated binding sites may allow for a regulatory role of copper at the DNA level. In ssDNA there is an average copper binding site every three nucleotides as compared with one average Cu(I1) site every two bases in dsDNA (Fig. 1). This indicates that appropriate DNA base-pairing accounts for some of the Cu(I1) binding. We found the interaction of Cu(I1) with ssDNA to be of lower affinity TABLE 1 ANTAGONISM
OF COPPER BINDING VARIOUS METALS
TO DNA
Antagonist
1C50 (PM)”
Ni(I1) Cd(I1) M&II) Zn(I1) WIU Ca(I1) WI) Mn(I1) ctiw
456 670 709 3,407 3,557 5.361 7,008 14,551 Enhanced binding
BY
u Concentrations of various metals that displace 50% of Cu(I1) bound to dsDNA under the conditions described in the legend to Fig. 4.
COPPER-DNA
than with dsDNA (Fig. 1); however, the implications of copper binding to ssDNA as found in open transcribing chromatin could be significant for gene regulation. Scatchard plots with marked deviation from linearity and with positive slopes at low occupancy of DNA sites have been shown for carcinogenic divalent metals such as Hg(II), Cd(II), and Ni(I1) (Simpson, 1964; Waalkes and Poirier, 1984; Kasprzak et al., 1986). The steep positive slope we observed for copper could indicate a very high affinity between this metal and the low abundance sites in DNA (Fig. 3). The presence of one site per four nucleotides in dsDNA agrees with a previous report about the formation of a DNA base-copper complex with a stoichiometry of one complex per four bases (Minchenkova and Ivanov, 1967). One copper binding site per four nucleotides can be explained in one of two ways. Either Cu(I1) binds to four bases in two adjacent base pairs, or Cu(I1) shows preferential binding for one of the four bases. A previous report (Sagripanti and Kraemer, 1989) describing a copper-mediated lesion specific for guanosine suggests that the low abundance binding site could be base-specific. For the Cu(I1) binding sites in high abundance we obtained an apparent stability constant (&) of 2.4 X lo4 M-’ (Fig. 3). This value is comparable to those obtained previously by spectroscopic analysis under different ionic strengths (1.1 X 10m4 M-’ in 0.25 M NaCl to 1.8 X lo4 M-’ in 0.001 M NaCl; Sayenko et al., 1986). Stacking of complementary bases in DNA and electrostatic binding of Na(1) to phosphates as ionic strength increases most likely makes the penetration of partially hydrated Cu(I1) into the double helix more difficult. Our slightly higher value of K, can be explained by the fact that we carry out our binding studies in water to maximize the access of copper to all potential DNA binding sites. The relatively more abundant copper binding site is present on average once every two
INTERACTIONS
483
nucleotides. The frequency of appearance of the high abundance site is similar to that observed upon the release of Cu(I1) during DNA renaturation (Richard et al., 1973). However, we do not consider this copper binding site simply as counterion neutralization of phosphate charges. The stability constant reported for the interaction of Cu(I1) and the phosphate in cytidine-monophosphate was 7 X 10’ Me1 (Siegel, 1989). Binding to phosphate alone could not explain the higher affinity we found for Cu(II)-DNA. The low competition we have observed for other divalent cations also indicates a more specific interaction between copper and DNA. We interpret the high abundance site in dsDNA as including groups from two nucleotides in complementary strands. It is well known that various metals will antagonize the effects of other metals both in vitro and in vivo. For example, cadmium binding to DNA in vitro was antagonized by zinc, magnesium, and calcium (Waalkes and Poitier, 1984). Magnesium, manganese, and calcium reduced nickel binding to DNA in vitro (Kasprzak et al., 1986). The relative capacities of these metals to antagonize binding of cadmium and nickel to DNA correlates remarkably with their abilities to prevent cadmium and nickel carcinogenesis (Waalkes and Poitier, 1984; Kasprzak et al., 1986). Other studies have shown that zinc administration attenuates copper toxicity in Wilson’s disease (Shilsky et al., 1989; Brewer et al., 1989). In the present study, several physiologically essential and nonessential cations were found to compete with binding of Cu(I1) to DNA. The relative efficacy of these metals in antagonizing Cu(I1) binding was: Ni(I1) = Cd(I1) = Mg(I1) >> Zn(I1) = Hg(I1) > Ca(I1) > Pb(I1) B Mn(I1). The low antagonism of Zn(I1) observed in this study suggests that the mitigating effect of zinc in anticopper therapy for Wilson’s disease (Schilsky et al., 1989; Brewer et al., 1989) is not mediated by displacement of Cu(I1) from DNA binding sites.
484
SAGRIPANTI,
GOERING,
The value for the apparent stability constant of 2.4 X lo4 M-t we obtained for Cu(I1) binding sites in high abundance is lower than the reported values for cadmium (4 X lo4 M-l; Waalkes and Poirier, 1984) and for nickel ( 19 X lo4 M-t; Kasprzak et al., 1986). The fact that metals with higher affinity constants for DNA, such as cadmium and nickel, displaced Cu(I1) minimally, leads us to speculate that the primary binding site for copper is different than the binding site for the other metals. The distorted octahedral coordination sphere proposed for Cu(I1) and the strong tendency to coordinate donor atoms equatorially (Siegel and Martin, 1982) may be responsible for the specific interaction with DNA. The biological mechanisms underlying copper toxicity could be rooted in the binding of copper to specific sites within DNA. In spite of the body of data accumulated regarding metal-DNA interactions, the characteristics and biological consequences of the binding of copper to DNA remains an exciting field to be explored. ACKNOWLEDGMENT The authors recognize Edward Gordon for performing the log regression analyses in this report.
REFERENCES ALBERTS, B., AND HERRICK, G. (1971). DNA-cellulose chromatography. In Methods in Enzymology (L. Grossman and K. Moldave, Eds.), Vol. 21, pp. 198-217. Academic Press, San Diego. AASETH, J., AND NORSETH, T. (1986). Copper. In Handbook on the Toxicology of Metals (L. Friberg, G. F. Nordberg, and V. B. Vouk, Eds.), Vol. 2, pp. 233-254. Elsevier, New York. BREMNER, I. (1987). Nutritional and physiological significance of metallothionein. Experientia Suppl. 52, 8 l107. BREWER, G. J., YUZBASIYAN-GURKAN, V., LEE, D.-Y., AND APPELMAN, H. (1989). Treatment of Wilson’s disease with zinc. VI. Initial treatment studies. J. Lab. Clin. Med. 114,633-638. BRYAN, S. E., VIZARD, D. L., BEARY, D. A., LABICHE, R. A., AND HARDY, K. J. (198 1). Partitioning of zinc
AND LAMANNA
and copper within subnuclear nucleoprotein particles. Nucleic Acids Res. 9, 581 l-5823. EICHORN, L. E., AND SHIN, Y. A. (1968). Interaction of metal ions with polynucleotides and related compounds. XII. The relative effect of various metal ions on DNA helicity. J. Am. Chem. Sot. 90, 7323-7328. FURST,P., AND HAMER, D. (1989). Cooperative activation of a eukariotic transcription factor: Interaction between Cu(I) and yeast ACE1 protein. Proc. Natl. Acad. Sci. USA 86,5267-527 1. IZATT, R. M., CHRISTENSEN,J. J., AND RYTTING, J. H. (197 1). Sites and thermodynamic quantities associated with proton and metal ion interaction with RNA, DNA and their constituent bases,nucleosides, and nucleotides. Chem. Rev. 71,439-48 1. JENDRYCZKO,A., DROZDZ, M., TOMALA, J., AND MAGNER, K. (1986). Copper and zinc concentrations, and superoxide dismutase activities in malignant and nonmalignant tissue of female reproductive organs. Neoplasma 33,239-244. KASPRZAK, K. S., WAALKES, M. P., AND POIRIER, L. A. (1986). Antagonism by essential divalent metals and amino acids of nickel(II)-DNA binding in vitro. Toxicol. Appl. Pharmacol. 82,336-343. KUWAHARA, J., SUZUKI, T., FUNAKOSHI, K., AND SUGIURA, Y ( 1986). Photosensitive DNA cleavageand phage inactivation by copper(camptothecin. Biochemistry 25, 1216-1221. MANIATIS, T., FRITSCH,E. F., AND SAMBROOK. J. (1982). Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. MICHENKOVA, L. E., AND IVANOV, V. I. (1967). Influence of reductants upon optical characteristics of the DNACu(I1) complex. Biopolymers 5, 6 15-625. RICHARD, H., SCHREIBER,J. P., AND DUANE, M. (1973). Interactions of metallic ions with DNA. V. DNA renaturation mechanism in the presence of Cu(I1). Biopolymers 12, 1- 10. SAGRIPANT~, J.-L., AND KRAEMER, K. H. (1989). Sitespecific oxidative DNA damage at polyguanosines produced by copper plus hydrogen peroxide. J. Biol. Chem. 264, 1729-1734.
SAGRIPANTI, J. L., SWICORD. M. L., AND DAVIS, C. C. (1987). Microwave effects on plasmid DNA. Radiat. Res. 110, 219-231. SAGRIPANTI, J. L.. SANTACOLOMA, T. A., AND CALVO, J. C. (1984). A simple computer program for Scatchard plot analysis of hormone receptors including statistical analysis on a low cost desktop calculator. Acta Fisiol. Latinoam. 34,45-53. SAMUNI, A., CHEVION, M., AND CZPASKI,G. (1984). Roles of copper and O(2) in the radiation-induced inactivation of T7 bacteriophage. Radiat. Res. 99, 562-572. SAYENKO, G. N., BABII, A. P., AND BAGAVEYEV. I. A. (1986). Change in the ultraviolet spectra of copper(R)
COPPER-DNA complexes with native DNA with the ionic strength of solution. Biophysics 31,45 l-456. SCATCHARD, G. (1949). The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51,660612.
SCHILSKY, M. L., BLANK, R. R., CZAJA, M. .I., ZERN, M. A., SCHEINBERG, I. H., STOCKERT, R. J., AND STERNLIEB,I. (1989). Hepatocellular copper toxicity and its attenuation by zinc. J. Clin. Invest. 84, 1562-l 568. SIEGEL, H. (1989). Metal nucleotide interactions. In Metal DNA Chemistry. pp. 158-203. American Chemical Society, Washington, DC. SIEGEL, H., AND MARTIN, B. R. (1982). Coordinating properties of the amine bond. Stability and structure of metal ion complexes of peptides and related ligands. Chem. Rev. 82,385-426. SIMPSON, R. B. ( 1964). Association constants of methylmercuric and mercuric ions with nucleosides. J. Amer. Chem. Sot. 86,2059-2065.
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STERNLIEB, I. (1980). Copper and the liver. Gastroenterology78, 1615-1628. SUGIURA, Y., TAKITA, T., AND UMEZAWA, H. (1985). Bleomycin antibiotics: Metal complexes and their biological action. Metal Ions Biol. Syst. 19, 8 l-108. S~G~JRA, Y., Kuwm, J., AND Suzrrru, T. (1984) DNA interaction and nucleotide sequence cleavage of copperstreptonigrin. Biochem. Biophys. Acta 782,254-26 1. TAJMIR, H. A., LANGLAIS, M., AND SAVOIE, R. (1988). A laser Raman spectroscopic study of the interaction of calf thymus DNA with Cu(II) and Pb(II) ions: Metal binding and DNA conformational changes. Nucl. Acid Res. 16, 15 I-762. WAALKES, M. P., AND POIRIEQ L. A. (1984). In vitro cadmium-DNA interactions: Cooperativity of cadmium binding and competitive antagonism by calcium, magnesium, and zinc. Toxicol. Appl. Pharmacol. 75,539-546. WACKER, W. E., AND VALLEE, B. L. (1959). Nucleic acids and metals. J. Biol. Chem. 234,3257-3262.
