In the Laboratory
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Photoinduced Oxidative DNA Damage Revealed by an Agarose Gel Nicking Assay A Biophysical Chemistry Laboratory Experiment Vladimir Shafirovich,* Carolyn Singh, and Nicholas E. Geacintov Chemistry Department and Radiation and Solid State Laboratory, New York University, New York, NY 10003-5180; *
[email protected] This article describes a set of biophysical chemistry experiments that are designed to explore several important concepts relevant to oxidative DNA damage photosensitized by the acridine derivative, proflavine (PF). This compound binds to DNA by a noncovalent intercalation mechanism and undergoes an electron-transfer reaction with DNA when it is photoexcited with visible light. The end product of this photoinduced electron-transfer reaction is the cleavage of one of the DNA strands (nicking), which can be quantitatively evaluated by a sensitive agarose gel electrophoresis assay using supercoiled DNA as a model system (1–3). Different aspects of these phenomena or experiments can be emphasized depending on the interests of the students and the instructor. Electron-Transfer Reactions in DNA Electron-transfer reactions in DNA are an active and rapidly developing area of research that has been reviewed in this Journal by Netzel (4). Oxidative modifications of DNA can arise from electron-transfer reactions initiated by the attack of strong chemical oxidants, radicals, or ionizing radiation. The use of DNA photocleavage agents has been studied extensively and the basic mechanisms have been reviewed (5, 6). In many cases, electron-transfer reactions in DNA can be initiated by synthetic DNA cleavage agents activated photochemically. Among the four natural nucleobases in DNA, guanine has the lowest redox potential (7), and is therefore the most easily oxidized base in photoinduced electron-transfer reactions (8–10). Once the guanine in DNA is oxidized by an oxidative electron-transfer reaction, a series of complex, oxygen-dependent chemical reactions ensue that can lead to the cleavage of the phosphodiester backbone (11, 12). Proflavine as a Photosensitizer of DNA Cleavage There are a number of basically planar, aromatic acridine derivatives such as proflavine (Figure 1) that bind to DNA by a reversible noncovalent intercalation mechanism (5). Such PF–DNA complexes are characterized by red-shifted absorption spectra exhibiting a maximum at 460 nm, while
Figure 1. Proflavine (PF).
H2N
N H
NH2
in the absence of DNA the absorption maximum occurs at 444 nm. Upon photoexcitation of the intercalated PF residues, electron transfer occurs (8),
[PF–DNA] + h ν
[PF*–DNA]
[PF · −–DNA· +]
(1)
oxidative DNA cleavage
where PF* is a photoexcited PF molecule. Instead of resulting in oxidative DNA cleavage, the intermediate radical ions can also decay by a recombination mechanism that regenerates the original ground state molecules,
[PF · −–DNA· +]
(2)
[PF–DNA]
thus diminishing the yield of the DNA cleavage reaction (1). The cationic compound methylviologen (MV2+; Figure 2), a well-known electron scavenger or acceptor, substantially enhances the yield of DNA cleavage (13) by the following reaction:
[PF · −–DNA· +] + MV2+
[PF–DNA·+] + MV ·+
(3) enhanced DNA cleavage yield
Overview of Experiments The sensitive supercoiled DNA–agarose gel nicking assay is utilized to detect direct DNA strand breaks arising from the oxidative cleavage of the sugar–phosphate backbone (1). A single cleavage event gives rise to the complete relaxation of a supercoiled DNA molecule leading to a nicked, covalently closed circular DNA molecule (1–3). The nicked circular forms exhibit slower electrophoretic mobilities than the supercoiled DNA molecules and, therefore, can be easily distinguished by gel-electrophoresis techniques involving ethidium bromide staining techniques. The experiments can be done in three parts, or independently, depending on the time available.
Figure 2. Methylviologen (MV2+).
H3C
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Materials and Methods
Materials Commercially available supercoiled pBR322 plasmid DNA (Promega) is suitable for the experiments described here. Proflavine hemisulfate (Aldrich), methylviologen chloride (Aldrich), other chemicals and buffers are listed in the Supplemental Material.W Light Sources for DNA Photocleavage Any source of visible light, for instance, a slide projector, is suitable for these experiments. Experiment III requires a monochromator and a light power meter. Acquiring and Digitizing EB Fluorescence Images of the Gels An inexpensive gel scanning system was developed for analyzing the relative proportions of nicked and supercoiled DNA molecules. Ethidium bromide (Sigma) is a widely used fluorescent stain of DNA that exhibits weak fluorescence in aqueous solution, but its fluorescence efficiency increases by a factor of ∼20 on binding to DNA by an intercalation mechanism. The fluorescence images of these gels were acquired utilizing an inexpensive digital camera with a resolution of equal or greater than 2 mega pixels CCD fitted with RG-610 and KG-5 Schott glass filters and interfaced with a personal computer. A program used to analyze the data, 0.5 Mbytes in size, can be downloaded from one of the author’s Web site (14) or from JCE Online.W Hazards Ethidium bromide and proflavine are potential mutagens, typically sold as powders. Students should wear gloves while handling solutions of these compounds (prepared by the instructor), and special care should be taken to clean all spills. The transilluminator produces UV light, another hazard, and should be installed in a closed cabinet equipped with a digital camera. Those students who wish to inspect the fluorescence bands visually must wear full face shields that are completely opaque to ultraviolet light below 400 nm. Of course, the skin should be fully covered and not exposed to the UV light. Results and Discussion
Experiment I. Photoinduced Cleavage of pBR 322 DNA Molecules Sensitized by Proflavine A typical fluorescence image is shown in Figure 3. The fastest migrating (lowest) bands in the control experiments (lanes 1 and 7) are due to form I supercoiled DNA molecules. The form II nicked circular DNA molecules are characterized by the slowest electrophoretic mobility (e.g., the top bands in lanes 1 and 7 in Figure 3; it should be noted that some nicked DNA is always present even in the best commercial samples of supercoiled plasmid DNA). In the absence of PF molecules, the 460 nm light does not generate any measurable quantities of nicked DNA (Figure 3, compare lanes 1 and 7). In the absence of any irradiation and the small concentrations of PF (~3 µM) utilized in these experiments, there are no observable effects on the shapes and positions 1298
Figure 3. Image of an ethidium-stained agarose slab gel showing photodamage of pBR322 supercoiled DNA (83 µM) induced by irradiation of intercalated PF molecules (3 µM) by the 460 nm output of a 100 W Xe arc lamp (fluence rate ≈ 5 mW/cm2) during fixed periods of irradiation time (∆t ) in the absence and the presence of methylviologen (10 µM) electron acceptor molecules.
of the supercoiled and nicked DNA bands (lanes 1 and 2 in Figure 3). This suggests that, at these low PF concentrations, the degree of unwinding of supercoiled DNA molecules (3) induced by the intercalation of PF is negligible. However, at greater PF concentrations, unwinding is observed that manifests itself in terms of an appearance of new bands with lower electrophoretic mobilities than that of the supercoiled DNA molecules (3, 15). Excitation of the PF–DNA complexes in solution results in a significant increase in the quantity of form II nicked DNA and a decrease in the quantity of form I supercoiled DNA (Figure 3, lanes 3–6).
Experiment II. Enhancement of Photodamage Induced by the MV2+ Electron Scavenger The extent of DNA photodamage is significantly enhanced in the presence of electron acceptors that, by scavenging an electron from the reduced PF• ᎑ radical anions, prevent radical recombination (eq 3), thus enhancing the yield of DNA radicals and strand cleavage (Figure 3). The positively charged MV2+ molecules bind to the surface of the double helical, negatively-charged DNA molecules and can effectively interact with the negatively charged or photoexcited intercalated PF molecules. Excitation of the aqueous samples containing supercoiled pBR 322 DNA, PF, and MV2+ by visible light (460 nm) generates the same DNA cleavage patterns (Figure 3, lanes 10– 13) as in the absence of MV 2+ (Figures 1, lanes 3–6). However, in the presence of MV2+ the DNA cleavage rates are markedly enhanced and shorter irradiation times are sufficient to generate greater quantities of cleaved form II DNA in the presence of MV2+ (0.5–3 min in lanes 10–13) than in the absence of MV2+ (1–5 min in lanes 3–6). The rise in the net quantity of nicked DNA induced by the irradiation, [form II]net (the observed quantity of nicked DNA minus the quantity initially present), occurs significantly faster in the presence than in the absence of 10 µM MV2+ in the samples irradiated for the same time intervals (Figure 4).
Journal of Chemical Education • Vol. 80 No. 11 November 2003 • JChemEd.chem.wisc.edu
In the Laboratory
spreadsheets utilizing personal computers. The experiments described here can be used as a flexible platform for developing more advanced, small individual student projects, and to introduce undergraduate students to exciting contemporary research in biophysical chemistry, biophysics and biochemistry. We have successfully used this approach to train a number of College of Arts undergraduate students at New York University.
[MV2ⴙ] = 10 µM
[form II]net
1.0
0.5
[MV2ⴙ] = 0
0.0
Acknowledgments 0
1
2
3
Time / min Figure 4. Kinetics of pBR 322 supercoiled DNA damage induced by irradiation of intercalated PF molecules in the absence and presence of MV2+ electron-acceptor molecules. The fractions of the net amounts of nicked DNA induced by the irradiation, [form II]net were calculated by integration of the histograms obtained by scanning the original image of an ethidium-stained agarose slab gel.
Experiment III. Action Spectrum of DNA Photocleavage It is not clear, a priori, whether intercalated PF or free PF molecules generate the observed photocleavage of DNA. To distinguish between these two possibilities, the extent of DNA cleavage is measured as a function of the excitation wavelength. The result of a typical experiment using light passing through a monochromator, shows that the excitation maximum for the photoinduced DNA cleavage sensitized by PF is located at 460 nm (see Supplemental MaterialW for details). Thus, the DNA photodamage is induced by intercalated PF (λmax = 460 nm) rather than by free PF (λmax = 444 nm) molecules.
This work has been supported by the National Science Foundation, Grant CHE-9700429, and by a grant from the Kresge Foundation. Carolyn Singh was the recipient of a Research Experiences for Undergraduates Program supplement to this NSF grant. The development of this laboratory experiment was in part supported by the National Science Foundation, Grant DUE-0126958. W
Supplemental Material
Detailed experimental procedures, student handouts, instructor notes, and a program used to analyze the data are available in this issue of JCE Online. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.
Summary
9.
This set of biophysical chemistry experiments provide opportunities for exposing the students to the principles of photoinduced electron transfer reactions (5, 6) in biological macromolecules such as DNA (4), the oxidative chemistry of nucleobases in DNA that are initiated by electron transfer reactions (12), and the properties of supercoiled DNA (1– 3) that allow for the sensitive detection of strand cleavage reactions associated with oxidative DNA chemistry. Students are exposed to UV–vis absorbance spectrophotometric techniques, gel electrophoresis, the binding of an aromatic acridine chromophore to supercoiled DNA, the basic principles of acquiring and processing imaging data, interfacing a digital camera to a computer, and data analysis using standard
10. 11. 12.
13. 14. 15.
Scovell, W. M. J. Chem. Educ. 1986, 63, 562–565. Sinden, R. R. J. Chem. Educ. 1987, 64, 294–301. Keck, M. V. J. Chem. Educ. 2000, 77, 1471–1473. Netzel, T. L. J. Chem. Educ. 1997, 74, 646–651. Kochevar, I. E.; Dunn, D. A. In Bioorganic Photochemistry; Morrison, H., Ed.; Wiley: New York, 1990; pp 273–315. Armitage, B. Chem. Revs. 1998, 98, 1171–1200. Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617–618. Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992, 114, 3656–3660. Shafirovich, V. Y.; Courtney, S. H.; Ya, N.; Geacintov, N. E. J. Am. Chem. Soc. 1995, 117, 4920–4929. Seidel, C. A. M.; Schulz, A.; Sauer, M. H. M. J. Phys. Chem. 1996, 100, 5541–5553. Douki, T.; Cadet, J. Int. J. Radiat. Biol. 1999, 75, 571–581. Cadet, J.; Bellon, S.; Berger, M.; Bourdat, A. G.; Douki, T.; Duarte, V.; Frelon, S.; Gasparutto, D.; Muller, E.; Ravanat, J. L.; Sauvaigo, S. Biol. Chem. 2002, 383, 933–943. Dunn, D. A.; Lin, V. H.; Kochevar, I. E. Biochemistry 1992, 31, 11620–11625. N. E. Geacintov Home Page. http://www.nyu.edu/projects/ geacintov/Gel_page.htm (accessed Aug 2003). Swenberg, C. E.; Carberry, S. E.; Geacintov, N. E. Biopolymers 1990, 29, 1735–1744.
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