Demonstration of DNA Strand Breakage Induced by Ultraviolet Light An Experiment To Show Molecular Events in Carcinogenesis Ruth N. Russo and James E. Russo Whitman College, Walla Walla, WA99362 The initial event in cellular carcinogenesis likely involves changes in the DNA at both the gene and chmmosome levels, resulting from damage to the cell by outside agents ( I ) . We have developed a visually striking experiment that demonstrates the damage to DNAcaused by the common carcinogen, ultraviolet radiation. The protocol, suitable for undergraduate biochemistry and molecular biology courses, can be executed in one 3 4 h r laboratory period. Students become familiar with several common biochemical techniques, including electrophoretic separation of DNA molecules on horizontal agarose gels, measurement of microliter volumes, and the use of ultraviolet light sources. The experiment can be used to introduce diverse topics, including DNA biochemistry, reactive oxygen species, and cancer prevention.
breaks in the sugar-phosphate backbone, convening the DKA first to thc open circular form, and finally to the linear form. Materials and Methods The DNA sample used here was the plasmid pUC7. Other suitable plasmids (e.g., QX174, pUC8) are available &om suppliers (Sigma, Gibco-BRL) for $2-$4/pg. Milligram quantities of plasmid can be obtained inexpensively via propagation of the plasmid in a n appropriate bacterial host (8). The restriction enzyme EcoRI is available from several suppliers (Sigma, Gibco-BRL). Electrophoresis was performed in a Mini-Sub horizontal gel apparatus (Biorad Laboratories). Inexpensive electrophoresis units can also be constructed in-house (9).Adjustable Pipetman
Background Ultraviolet (UV)radiation induces dimerization of thymine bases and breakage of the sugar-phosphate DNA backbone as a result of the formation of hydroxyl radicals (2)(Fig. 1).In the normal individual, cellular enzymes usually repair DNA lesions. However, individuals with inherited, recessive defects in the DNA repair enzymes express the condition xeroderma pigmentosum. Since these individuals are highly cancer prone, especially to W radiation (i.e., sunlight) induced skin cancer, they exhibit roughly 1000-fold greater re valence of skin cancer than the general population (3,4). Likewise, sunlight exposure probably contributes to the approximately 400,000 skin cancers developed in the United States each year (51. This experiment demonstrates that W radiation pmduces breaks in the sugar-phosphate backbone of DNAand that the extent of the DNA strand scission is dose-dependent.'We take advantage of the fact that double-stranded plasmid DNA molecules generally exhibit three conformations that reflect the integrity of the DNA: open circular (a single break on one of the DNA strands); linear (one or more breaks on both strands): and su~ercoiled(no breaks) (Fig. 2). During nondenaturing agarose gel electrophoresis, the migration of a DNAmolecule is a function of both the size and the wnformation of the molecule. All plasmid molecules areof ldenticill size in this cxperiment:thcrefore, differences in elcctro~horeticmoblllty result only from differences in plasmid ionformation. 1; 'Iris-ace& buffer, the supercoiled form migrates farthest from the origin, the open-circular form migrates the least, and the linear form migrates between the superwiled and open-circular forms (7).With no UV treatment, the predominant plasmid species is the supercoiled form. However, with increasing doses of UV radiation, the plasmid DNA accumulates 'Another consequence of radianon damage, the formationof thym ne dlmers n DhA, can be aemonstratea by thermal aenaturatlon as described in reference 6.
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Figure 1. (A) Thymine dimers formed by UV irradiation of DNA. Dashed lines represent covalent bonds between adjacent thymine bases on the same DNA strand. Sugar = deoxyribose. (6) Sites of DNAstrand breakage induced by UV irradiation. The arrows indicate carban-oxygenbonds labile to attack by hydroxyl radicals.
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Figure 2. Schematic diagram of the three conformations of plasmid DNA. The heavy and light lines refer to two complementary strands of double helical DNA. micropipettors (Gilson Co.) were used to measure and transfer DNA samples. C a u t i o n : Safety gla9sesarr necessary to block IN illurninn-
tion. Gloves must bc worn when handling ethldlurn bromide; methods for dtsposol of this mutagen arc descrhad elirwhere (8)
Methods for electrophoresis and the preparation of TrisEDTA (TE) buffer, Tris-acetate-EDTA (TAE) buffer, gelloading dye, and ethidium bromide are described elsewhere (8, 10, 11). Plasmid solutions were diluted in TE buffer. EcoRI digestion of plasmid DNA was performed a s described (10). Agarose gels (1% wlv) were prepared in TAE buffer. Gels were run for approximately 50 min a t 6.5 Vlcm in TAE buffer. The gels were stained in a n aqueous solution of t h e fluorescent dye ethidium bromide (0.5 pg/mL) for 20 min, and destained for 20 minutes in 1mM MgS04. DNAbands were visualized by illumination of the gel by a UV source a t 254 nm. Procedure The ulasmid solution was diluted to 0.03 wgIpL and 100 pI. wai transferred t o u small plastic weigh boat sitting on crushed ice, which prevented evaporation of the sample. A 10-KL aliquot (cbntrol) w a s - r e m o v e d t o a 1.5-pL microcentrifuge tube. The weigh boat was placed 4 cm below a UV source (we used a hand-held, shortwave UV lamp a t 254 nm).After 1 4 0 - m i n exposure intervals, 10-pL aliquots were transferred from t h e weigh boat to a microcentrifuge tube. Each 1 0 p L DNA sample was resolved on a n agarose gel after the addition of 2 pL gel-loading dye. The time course for DNA damage must be empirically determined since the extent of damage depends on the in-
F~gdre3 Ge e enrophores s of plasmlo samp es afterLV lrrao atlon Lanes 1-4 DNA rrad~atedfor0 5, 10,and 2J m n. respect vely -ane 5 mear piasmod DNA resLlt ng from d gest on of !he p asm 0 w t n a reslrlcllon enoonuc ease rEcoRI, tnar cleaves bolh DNA stranos at a single site. The positions of DNAmarkers are indicated to the left, and the bold line marks the qel . origin. . DNA molecular sizes are given in kilobase pairs. tensitv of each W source. Variations on the method that reduci DNA damage can be tested by different students. These include increasing the distance between source and sample, shielding the ;ample with a borosilicate glass slide, and testing the effect of 0.01 mM p-uninobenzoic acid (PABA), a UV blocking agent i n commercial sunscreen~.~ Results and Discussion Results from a representative experiment are shown in Figure 3. With no exposure of the DNA to UV radiation (Lane 11, the most intensely staining band migrates farthest from the origin. This corresponds to the supercoiled plasmid molecules that contain no breaks in the sugarphosphate backbone. The upper band in Lane 1 corresponds to the open circular plasmid DNA form? After 5 min of W irradiation (Lane 21, the sharp band of supercoiled plasmid disappears and a diffuse band is visible, which migrates more slowly than the lower band in Lane 1. After 10 min of irradiation (Lane 3), the diffuse band appears to be less intense, indicating that most of the plasmid migrates with the upper band. Lane 4 shows that by 20 min of UV irradiation, virtually all of the plasmid migrates a s one band. This band co-mimates with uUC7 digested with the restriction enzyme
2For the PABA test. 2 CL of PABA (0 5 M n 50°. v v erhano ) were aadeo to tne we gn ooat pror to exposure lo LV lghr A welgn boat contanma DhA an0 2 9- of 50°. etnanol twtno~tPABA) was then included Tn the experiment as a control. 3To confirm the identity of the upper band in Lane 1, we treated oUC7 DNAwith toooisomerase I. which converts suoercoiled to ooen c rcular p asmld. ine dpper bano of tne dnlreatei plasm o co:m grateo w lh me topos omerase I lrealea plasm d d ~ng r e eclropnores s; thds. 11sconformal~onis prooab y open c!rcJtar 7 -
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EcoRI (Lane 51, confirming that the plasmid sample in Lane 4 has been converted to the linear form by W-induced strand scission. Observant students will recognize that the upper bands in Lanes 2 and 3 exhibit intermediate mobilities between that of the open-circular plasmid (upper band, Lane 1) and the linear plasmid (Lane 5). If strand breakage were the only damage to DNA, then discrete conversions from supercoiled, to open circular, to linear plasmid forms should result from increasing UV irradiation. Perhaps other W-induced DNA damage, such as thymine dimer formation, alters the mobilities of the plasmid molecules by creating new conformations. Summary
This experiment demonstrates the damage to DNA caused bv W radiation. Careful lahoratorv techniaue and obsemance of safety precautions are required, making these experiments beneficial learning- ex~eriences for undergraduates. The simple, visual demonstration of DNA damage caused by W radiation fosters an understanding of the molecular mechanisms involved in the initial stages
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ofcarcinogenesis and provides an awareness of exposure to carcinugens outdoors and in the laboratory Acknowledgement
This project was developed during a course for secondary school teachers titled "The Initiation of Cancer", supported by an institutional grant from the Howard Hughes Medical Institute. We gratefully acknowledge John Hilton and Earl Fleck for helpful discussions, and Tracey Setoda for technical assistance. Literature Cited 1. Weinatein. 1. B. Cancer Rea 1888.48.4135-4143. 2. Coggie, J. E.BiologicolEffctt ofRndhfion.2ndd.; lntemationalPublieatimsService: New Sork, 1983:Chapter 2. 3. Levitan, M . Teztbook ofHumon Dpnrtior, 3rd 4.;Oxf~xf~xfdUnivelaity Press:New York. 1988:Chanter 17. 4. s~~~&,R.B.N& 197S,272,713-717. 5. ConcorFocts end Figwe*;American Cancer society: Newsork, 1990. 6. LOW&,C. M.; Ntzgibh.' N.;Chang, R.J Cham. Educ. 1989,66,526528. 7. Scwell, W M. J Cham. Edue 1988.63 ,562665. 8. Sambmok. J.; Frirseh, E.F.; Maniatia, T , Eda.; Molecular Cloning, 2nd d.;Cold Spring Herbar Laboratoly Reas: Cold Spring Harbor, m, 198% 9. Hopiuns, T.R.; Sreekrishna, K J Cham. Edue 1987.64,279. 10. Kaneko, K J.;Burke,J. M.; Kaplan, L. J. J C k m . Edue. 1887,64,274-278. 11. FarreU, S.O.;Famell,L.E.;Dircks,L.KJ Ck~mEdue.lMl,68,707-709.
