Photochembhy and Photobiology Vol. 56, No. 2, pp. 185-193, 1992 Rinted in Great Britain. All rights reserved

0031-8655/92 S05.00+0.00 Copyright 0 1992 Pergamon Press Ltd

PSORALENS CLEAVE pBR322 DNA UNDER ULTRAVIOLET RADIATION JACQUES KAGAN*',XINSHENG CHEN', TIANP. WANG'and P. FORLO? 'Department of Chemistry, m/c 111, University of Illinois at Chicago, P.O. Box 4348, Chicago, IL 60680, USA and 2LaboratoiresBergaderm, 46 rue de la Crosse Pierre, 94562 Rungis Cedex, France (Received 25 July 1991; accepted 18 December 1991)

Abstract-supercoiled (SC) pBR322 was used to probe the recent claim that 5-geranoxylpsoralen (5GOP) did not photoreact with DNA. Contrary to expectations, 5-GOP was found to damage DNA in the presence of UV-A through two competing pathways: (a) single strand breaks, identified by the conversion of supercoiled into open circular and linear DNA, and (b) cross-linking, revealed by the fluence-dependent decrease in the extent of denaturation of the double stranded supercoiled DNA to single stranded circular DNA. In addition, a fluence-dependent modification reduced the ability of the restriction enzyme EcoR I to linearize the photosensitized DNA, and alkali-labile lesions were generated. Psoralen, 5-methoxypsoraler1,and 8-methoxypsoralen, which are well-known to undergo cycloaddition to DNA, had a more pronounced effect on supercoiled DNA. Single strand breaks occurred more readily than with 5-COP, and the surviving SC form remaining had reduced electrophoreticmobility in agarose gels. In all cases, the DNA damage was more prominent when oxygen was absent. INTRODUCTION

Psoralens have been found to interact with many biological targets in the presence of ultraviolet light. However, it is their interactions with nucleic acids, particularly DNA, which have dominated photobiochemical studies. The most predictable reaction is a [2 + 21-cycloaddition between a pyrimidine base and one double bond of the psoralen, either in the furan or in the pyrone moiety. When a monoadduct is first formed on the furan side, further irradiation of the remaining coumarin chromophore may also produce a cycloadduct on the pyrone side, generating a cross-linked DNA. The book Psoralen-DNA Photobiology (Gasparro, 1988) and recent reviews by Averbeck (1989), Potapenko (1991), and Dall'Acqua and Martelli (1991) admirably summarize the recent work concerning the phototoxic properties of psoralens, and provide leading references to the extensive earlier literature. One mode of photoreactivity in psoralens which does not lead to the formation of photoadducts with DNA is the sensitized generation of reactive oxygen species, which may produce lesions such that DNA cleavage occurs upon subsequent treatment with alkali or hot piperidine. Many other photosensitizers are known to produce similar oxidative

*To whom correspondence should be addressed. tAbbreviationr: DNA, deoxyribonucleic acid; EDTA, cthylenediaminetetraacetic acid; %MOP, 8-methoxypsoralen; 5-COP, 5-geranyloxypsoralen; 5-MOP, 5methoxypsoralen; HPLC, high performance liquid chromatography; L, linear; OC, open circular; P, psoralen; SC, supercoiled; SEM, standard error on the mean; SSC, single-stranded circular; Tris, 2-amino-2(hydroxymethyl)-l,3-propanediol.

damage of DNA (Kochevar and Dunn, 1990). Plasmid circular DNA, normally double-stranded and supercoiled (SC)t, is sensitive to damage by a variety of physical and chemical agents. Cleavage (nicking) of one strand produces a relaxed, but still double-stranded, open circular (OC) DNA. Further cleavage of the other strand within a short distance of the first site of cleavage (less than ca 10 base pairs away) generates a linear (L) DNA. Although the SC, OC, and L double-stranded forms of a plasmid DNA have negligible differences in molecular weight, their physical properties are sharply different. In particular, these DNA species can easily be distinguished by the difference in their migration rates during agarose gel electrophoresis. Denaturation of the double-stranded SC and L forms of DNA produces two different kinds of species, namely circular and linear single-stranded DNA (the latter as a mixture of molecules which may have different molecular weights), while O C DNA is denatured to a mixture of circular and linear single-stranded DNA. These single-stranded species can also be revealed electrophoretically in a more alkaline agarose gel. In the course of studies on photosensitized damage to DNA (Tuveson er al., 1990; Kagan et al., 1990; Wang er al., 1991), it was desirable to compare experimental results with those produced by wellknown sensitizers. 5-Geranoxypsoralen (5-GOP, bergamottin) was chosen because of its unusual properties: it was recently shown to be highly phototoxic and yet not to cross-link DNA (Morliere et al., 1991). 8-Methoxypsoralen (8-MOP), psoralen (P) and 5-methoxypsoralen (5-MOP, bergapten) were included in the study for comparison because they are powerful phototoxic compounds which are

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renowned for forming adducts with DNA. In this report we provide preliminary evidence proving for the first time that all the psoralens tested were capable of photosensitizing direct DNA strand breaking in vitro in competition with adduct formation, and that the strand breaking was more prominent in the absence than in the presence of oxygen. Quantitative data on the location of the breaks and the quantum yields of the various processes are sought in our ongoing research and will be reported later. MATERIALS AND METHODS

Chemicals and equipment. Psoralen was obtained from Sigma Chemical Co. (St Louis, MO), 5-MOP from Extrasynthese (B.P. 62, 69730 Genay, France), 5-GOP from Laboratories Bergaderm (46 rue de la Grosse Pierre, 94563 Rungis, France), and 8-MOP from Aldrich Chemical Co. (Milwaukee, WI) (Fig. 1). All gave single peaks on HPLC. The supercoiled pBR322 DNA was prepared and purified in the laboratory (Wang et al., 1991). Its concentration in a Tris (10 mM)-EDTA (1 mM) buffer (pH 7.4) was 50 pg mL-'. The enzyme EcoR I (20 units pL-') was purchased from Sigma (St. Louis, MO). Irradiation experiments. A Rayonet apparatus (Southern New England Co., Hamden, CT) equipped with either 4 or 9 tubes (RPR-3500, maximum emission at 350 nm) was used in this work. Measured with a YSI-Kettering Model 65A radiometer, the fluence was either 5.5 or 12.5 Wm-2. A stock solution (0.5 mg mL-I) of each sensitizer was prepared in ethanol. The experiments were performed in Pyrex tubes (10 cm in length, 1.5 cm in diameter) which contained 5 pL of a mixture prepared by adding 1 pL of sensitizer solution to 9 pL of DNA stock solution. The experiments under argon were performed with gas which had been allowed to bubble through distilled water before Rowing through the tube for 2 h. Some evaporation, less than 20%, took place despite this precaution. The tube irradiated without argon was closed with plastic foil. Electrophoretic analyses. The analyses were performed in 1% agarose gels prepared and run horizontally in a buffer containing 50 mM Tris, 50 mM boric acid, and 1 mM EDTA. Each sample (5 to 8 pL) was mixed with 2 pL of 40% sucrose solution containing 0.2% bromophenol. After electrophoresis for 5 h at 40 V, the gels (14 x 18 cm) were stained with a solution of ethidium bromide (0.5 mg L-I) and photographed under long-wavelength UV light using Polaroid P/N 55 film. The negatives were reproduced with an office duplicating machine and were scanned in absorbance mode with a Hoefer GS300 densitometer linked to a Hewlett-Packard integrator. The electrophoreses under denaturing conditions were performed in 1% agarose gels at 20 V for 10 h using 30 mM NaOH and 2.0 mM EDTA. Treatment with EcoR I . The enzyme solution was prepared by diluting 1 pL of the commercial enzyme with 9 pL of 1.0 M Tris-HCI buffer (pH 7.5) containing MgCI, (50 mM), NaCl (0.5 M),BSA (1.0 g L-I), and triton X100 (1.5%). A mixture of 10 p L (0.5 ng) of irradiated pBR322 and 1.1 pL of freshly prepared enzyme solution was incubated for 1 h at 37°C. Denaturation of DNA. Supercoiled DNA (5 pL) was mixed with 0.55 pL of a solution containing NaOH (2 M ) and EDTA (0.1 M) and incubated for 15 min at 37°C prior to analysis. Linearized DNA was treated identically with a solution of NaOH (2 M) and EDTA (0.2 M ) . Detection of alkali-labile damage. A 4-pL sample of photosensitized pBR322, mixed with 1 pL of 0.25 M NaOH, was incubated at 37°C for 20 min before electrophoretic analysis.

P R,=H,%=H %MOP R,=OCH3,%=H 5-GOP R1= OC~C(CH,)=CHCH2CH*CHIC(CH3)2, Rz t H &MOP R, = H.% OCH3 Figure 1. Structure of the psoralens investigated. RESULTS

Effect of the concentration

The effect of the sensitizer was dependent upon concentration when the duration of the photolysis was kept constant, as shown on agarose gel electrophoretic separation of pBR322 D N A samples after a 2 h irradiation with 5-MOP and 8-MOP (Fig. 2) and with P and 5-GOP (Fig. 3). In every case, greater sensitizer concentrations led to increasingly severe modifications. D N A irradiated without sensitizer for 2 h served as control. Two processes were clearly at play, affecting the starting SC DNA as well as its conversion into OC and eventually even into L DNA: Supercoiled DNA modifications. The mobility of SC D N A was reduced and the band broadened when pBR322 was irradiated in the presence of 5MOP, 8-MOP, and P. These effects were observed whether the irradiations were performed in air or under argon. As controls, unirradiated mixtures of DNA and sensitizer (5-MOP, 8-MOP, or P) were also analyzed electrophoretically. The SC D N A in these mixtures and in a pure sample migrated identically, proving that no electrophoretically identifiable modifications of the D N A took place when DNA and the sensitizers were kept in the dark (Fig. 4). The change in mobility for SC DNA after photosensitization was not observed with 5-GOP. Open circular DNA formation. Competing with the photosensitized modification of the SC DNA, strand breaks occurred and produced O C species. The extent of strand breaking reflected in the intensity of the electrophoretic bands clearly depended upon concentration; photosensitized damage was most pronounced in the presence of argon. At the highest concentration used (50 pg mL-'), very little or no SC DNA remained after irradiation with 8MOP (Fig. 2) and with P (Fig. 3), but most of it was still present after irradiation with 5-GOP (Fig. 3). The difference between the migration of the OC band of the DNA control and that of the photosensitized samples was very small, much smaller than those observed for the SC bands with P, 5MOP, and 8-MOP. Linear DNA. Both 5-MOP and P, but particularly the latter, sensitized the formation of L DNA under argon at the lower fluence (Figs. 2 and 3).

Psoralens cleave DNA

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sc Figure 2. Irradiation of pBR322 (45 pg mL-') for 2 h with 5-MOP (lanes 2-7) and with 8-MOP (lanes 8-13) in air (lanes 5-7 and 11-13) and under Ar (lanes 2-4 and 8-10). Irradiated DNA without sensitizer under Ar (lane 1) and in air (lane 14) are the controls. The concentrations of sensitizer are 6.6 (lanes 2, 5, 8, l l ) , 13.3 (lanes 3, 6, 9, 13) and 50 pg mL-l (lanes 4, 7, 10, 12). The position of the origin (0),OC, L, and SC bands is indicated. The fluence was 5.5 Wm-*.

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sc Figure 3. Irradiation of pBR322 (45 pg mL-l) for 2 h with P (lanes 2-7) and with 5-GOP (lanes 8-13) in air (lanes 5-7 and 11-13) and under Ar (lanes 2-4 and 8-10). DNA (lane l), and irradiated DNA without sensitizer under Ar (lane 14) and in air (lane 15) are the controls. The concentrations of semitizers are 6.6 (lanes 2, 5, 10, l l ) , 13.3 (lanes 4 , 6 , 9 , 12) and 50 pg mL-' (lanes 3, 7, 8, 13). The position of the origin (0),OC, L, and SC bands is indicated. The fluence was 5.5 Wm-*.

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Figure 4. Electrophoresis of mixtures of pBR322 kept in the dark with P (lane 3), 8-MOP (lane 5 ) , and 5-MOP(lane 7), compared to samples irradiated for 2 h (lanes 4 , 6 and 8, respectively), and with unirradiated (lane 1) and irradiated DNA (lane 2). The concentration of sensitizers was 50 pg mL-1 in all cases. The position of the origin (0),OC, and SC bands is indicated. The tluence was 5.5

Wm-*.

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1 2 3 4 5 6 7 8 9 1 0

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Effect of the fruence

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Figure 5 . Cleavage of pBR322 photosensitized by 5-GOP. DNA controls are in lanes 1 (no light) and 10 (irradiated in air 80 min). Lanes 2-5 show the results of irradiations with 5-GOP in air for 10, 20, 40 and 80 min respectively. Lanes 6-9 refer to corresponding experiments under argon. The h e n c e was 12.5 Wm-*.

At the same fluence in the presence of oxygen, some SC was converted into OC, but no L DNA was formed. The other psoralens usually did not induce the formation of L DNA when irradiated in the same conditions, whether or not oxygen was present. At high fluences, a small amount of L DNA was also observed upon sensitization with 5GOP under argon (Fig. 5).

100

DNA samples (45 pg mL-’) were irradiated with 5-GOP (50 pg mL-’) in the presence of air and under argon. The presence of air significantly decreased the extent of photosensitized cleavage (Fig. 5). The results of two separate experiments showed that the phenomenon was very reproducible. Because the nicking produced by 5-GOP was much less efficient than that observed with P, 5MOP, and %MOP, the cleavage of DNA sensitized by these three psoralens (Fig. 6) was compared at the same concentration but under reduced fluence (5.5 compared with 12.5 Wm-2). The negatives of the photographs of the electrophoretic gels were analyzed by densitornetry, but the data were not corrected for any differential fluorescence of the SC and O C bands after staining with ethidium bromide. The actual percentage of cleavage of SC into OC DNA is thus probably overestimated, perhaps by as much as ca 60% (Ciulla et al., 1989). Detection of alkali-labile lesions

DNA (45.5 p,g mL-’) irradiated for 2 h with P, %MOP, and 5-MOP (each 50 pg mL-’) was treated with 50 mM NaOH for 20 min prior to electrophor-

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Figure 6. Time dependence of pBR322 cleavage for 5-GOP compared to P, 5-MOP, and 8-MOP. The panel for 5-GOP shows the means of two determinations (with SEM) obtained with DNA alone and with DNA plus sensitizer irradiated with lamps providing 12.5 Wm-2. The comparison data with the other sensitizers were obtained at lower fluences (5.5 Wm-*)where the unsensitized DNA photodamage was negligable. The concentration of pBR322 was 45 pg mL-’, that of the sensitizers 50 +g mL-l.

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Psoralens cleave DNA

etic analysis. The relative intensity of the OC band greatly increased compared with the original irradiated samples (Fig. 7). As control, SC DNA irradiated for 2 h was also treated with NaOH, but no significant formation of OC was detected. The sensitizer concentration affected the formation of alkali-labile lesions in DNA photosensitized with 5-GOP when the fluence was kept constant, and the same observation was made upon photosensitization with P (Fig. 8).

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Linearization with EcoR I of photosensitized p BR322

Samples of pBR322 photosensitized with 5-GOP for increasing durations were treated with EcoR I and compared with untreated DNA. Agarose electrophoresis of both the reference sample and the sample which had been treated with 5-GOP for 20 min and 40 min proved that the DNA had been linearized completely (Fig. 9). The sample photosensitized for 80 min showed that much DNA had resisted linearization with the restriction enzyme. As mentioned above, the enhancement of the intensity of the OC band produced upon treatment with NaOH demonstrates that alkali-labile lesions were created during the photosensitized treatment (Fig. 9).

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Denaturation of pBR322 photosensitized with 5GOP

Unirradiated pBR322 DNA was treated with 0.2 M NaOH and 10 mM EDTA prior to agarose electrophoresis. Complete d-aturation to singlestranded circular DNA (SSC) was revealed by the appearance of a new band of greater mobility. This band was also present as a minor component with the mixture of OC and SC DNA analyzed after 20 min and, to a smaller extent, after 40 min of photosensitized treatment with 5-GOP. However, it was no longer present in the sample which had been irradiated for 80 min (Fig. 9). DISCUSSION

Figure 7. Comparison of pBR322 irradiated alone (lane 1). with P (lane 2), and with 8-MOP (lane 3), and identical samples analyzed after treatment with 50 mM NaOH for 20 min at 37°C (P,lane 4; DNA irradiated alone, lane'5, 8-MOP, lane 6). The position of the origin (0),OC, and SC bands is indicated. The fluence was 5.5 Wm-Z.

pBR322 is an Escherichia coli plasmid cloning vehicle which has been used extensively; it contains 4361 base pairs, and a multitude of restriction sites are known. Photosensitization of plasmid DNA by psoralens has been investigated on many occasions, but without specific analysis for strand breaks in the SC species. For example, Popa et al. (1986) performed alkali denaturation, Wei et al. (1986) linearized pBR322 DNA prior to its analysis for adduct formation, Gasparrro et al. (1982), Yoon (1982), Koo el al. (1986), and Sinden and Hagerman (1984) used pBR322 DNA digests, and Saffran and Cantor (1984a,b) used pBR322 which incorporated a psoralen, but which was nicked and denatured before analysis. The sample of pBR322 prepared and purified by HPLC in our laboratory (Wang et al., 1991) had undergone only minor degradation upon storage (Fig. 2), and it was therefore used without further purification. Because of the low solubility of the psoralens in aqueous media, stock solutions in ethanol were used, from which small amounts were added to the DNA solution. In this work, the resulting mixtures were irradiated without estab-

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Figure 9. Photosensitization (12.5 Wm-*) with 5-GOPin the presence of air. Three experiments are recorded: (a) irradiated DNA (lanes 2-4); (b) irradiated DNA treated with 0.2 M NaOH and 10 mM EDTA (lanes 6-8); (c) irradiated DNA treated with EcoR I (lanes 10-12). Lanes 1, 5, and 9 are the unirradiated DNA controls treated as indicated in the following group. In each group, the lanes correspond to irradiation times of 20, 40 and 80 min respectively. The position of the single-stranded circular DNA is marked as SSC.

lishing whether they were truly homogeneous. Irradiation of our pBR322 did not lead to significant differences in electrophoretic properties, although biological properties may have been appreciably modified (Paramio et al., 1991). Photosensitization with 5-GOP

Irradiation of pBR322 in the presence of 5-GOP produced no changes in the appearance of the SC DNA band (Figs. 3 and 5). This result appeared to agree with the conclusions of Morliere et al. (1991), who found no interactions between 5-GOP and calf thymus DNA. Unexpectedly, however, SC D N A was converted to OC DNA, only slightly in the presence of air, but more extensively under argon (Figs. 5 and 6). The extent of reaction was dependent on the duration of the irradiations at constant 5-GOP concentration and on the concentration of 5-GOP at constant irradiation time. Covalently bound psoralens affected the electrophoretic mobility of OC D N A much less than SC DNA (Figs. 2 and 3), but the OC species also appeared to be slightly retarded in the analysis of the pBR322 irradiated with P, 5-MOP, and 8-MOP. While we could be confident that the OC DNA species generated upon photolysis in the presence of 5-MOP, 8-MOP, and P contained adducts, since they were formed more slowly than the modification of the SC forms, we have not yet determined whether adduct formation occurred with 5-GOP after the OC had been generated. The behavior of 5-GOP with respect to photoaddition reactions with SC D N A was found to be very similar to that of the other psoralens with which it was compared, but different techniques had to be used because the extent of reaction was definitely

smaller under our experimental conditions. First, serious modifications of pBR322 occurring upon photosensitization with 5-GOP were revealed by the fact that the restriction enzyme EcoR I, which readily linearizes the D N A which had not been photosensitized, became increasingly less effective in producing L D N A from pBR322 as the irradiation time increased. This qualitative observation, unfortunately, does not reveal the nature of the photosensitized damage inflicted upon the plasmid DNA, whether covalent attachment of the psoralen at or near the restriction site of the enzyme, more distant attachment which modifies the shape of the molecule in a manner interfering with the enzyme binding andlor reaction, or other types of damage. The clue that pBR322 modification beyond simple nicking could take place with 5-GOP sensitization was further investigated by denaturing the photosensitized pBR322. The extent of denaturation of the mixture of SC and OC D N A clearly diminished as the duration of the irradiation increased (Fig. 5 ) , and this observation was interpreted as proof that increasing cross-linking of DNA strands prevented their separation under denaturing conditions. However, we have not yet assessed whether the crosslinking involved one molecule of sensitizer bridging two strands of DNA, as other psoralens are known to do, or simple [2 + 2]-cycloaddition reactions between pyrimidines on adjacent strands. In any event, this observation confirmed the finding that photosensitized pBR322 with which linearization with EcoR I had been attempted was only partially linearized upon treatment with 0.2 M NaOH. Finally, it is interesting to note that the strand breaking in SC D N A observed in this work takes place to a much greater degree in the absence of oxygen. This agrees with observations first made by Oginsky et al. (1959), who found greater phototoxicity of 8-MOP to bacteria in the absence of oxygen, and who explained it by suggesting enhanced photosensitized cycloaddition reactions. Since then, a variety of photosensitized effects of psoralens have been found to occur both in the presence and in the absence of oxygen (Midden, 1988). The earlier conclusions by Morliere et a!. (1991), concerning the lack of photoreactions of 5-GOP with calf thymus DNA, were derived from denaturation-renaturation experiments. Probably, those experiments were not sensitive enough to detect small amounts of D N A modification, and supercoiled plasmid D N A is much more prone to undergo andor reveal strand breaks than linear

DNA. Photosensitization by 5 - G 0P photoproducts

Because 5-GOP had been reported to photodecompose (Morliere et al., 1991), it was interesting to compare the photosensitizing ability of 5-GOP

Psoralens cleave DNA itself and its photoproduct(s). Experiments performed in the presence of air showed that the sensitizer had completely lost its ability to sensitize the conversion of SC into OC DNA when it was irradiated alone for 2 h (results not shown). It is clear, therefore, that the photoproduct(s) from 5GOP are not more potent photosensitizers than 5GOP itself. Rather, they appear to be less effective. Ashwood-Smith et al. (1992) observed that four degradation products were formed before the photobiological activity was completely lost upon irradiation. Photosensitization with P , 5-MOP, and 8-MOP

191

iments had been cleaved in several places in both strands. It is also probable that unless an extremely selective interaction of psoralens with pBR322 allowed successive cleavage on adjacent strands, the linearization of DNA observed with P, 5-MOP, and 5-GOP resulted from random breaking on both strands, where one break happened to be within about 10 base pairs of a break already created on the other strand. However, we do not yet know whether cleavage was photosensitized by a covalently bound molecule (which would have to be on the furan side) or by a non-covalently bound psoralen. A more detailed analysis of the DNA damage by these sensitizers is under way and the results will be reported in due course.

The wealth of prior information on the photosensitized interaction between psoralens and DNA Detection of alkali-labile sites supports the notion that covalent bonds are formed It is well known that photosensitization of DNA in the presence of UV light, depending both on the length of irradiation at constant concentration and under oxidative conditions may generate alkali-laon the sensitizer concentration at constant fluence. bile lesions. Further treatment with alkali produces It is therefore reasonable to attribute the modifi- strand breaks (Kochevar and Dunn, 1990). The sencation in electrophoretic mobility and the broaden- sitizers tested in this work produced alkali-labile ing of the band corresponding to SC DNA (Figs. 2 lesions in addition to the direct damage demonand 3) to the increasing covalent binding of the strated electrophoretically (Figs. 5-9). The effect psoralens to the DNA as the sensitizer concen- depended on the concentration of sensitizer (Fig. tration was increased. Since the individual contri- 8) * bution by monoadducts and crosslinks were unknown, we were unable to dissect their effects on Has psoralen-photosensitized strand breaking in the observed electrophoretic mobilities. Although plasmid DNA already been described? P and 5-MOP did lead to the eventual formation of Our search through the literature failed to L DNA, this process was not observed under the same experimental conditions with 8-MOP. How- uncover any reports where plasmid DNA was ever, the irradiation of pBR322 with 8-MOP pro- proved to be cleaved (rather than being cross-linked duced OC DNA that was extensively modified, to the sensitizer) upon photolysis in the presence of since it was no longer linearized by the enzyme psoralens. However, this reaction may have been EcoR I, which normally cleaves pBR322 at position encountered earlier by Nakamura (1989), who did 4361. Complete linearization was observed on a not appreciate the significance of his results. He control irradiated without sensitizer and analyzed designed a bleomycin mimic possessing a psoralen moiety (in order to link photochemically to DNA), on the same gel (data not shown). The strand breaking which converted SC into OC which was attached to a metal-ion complexing site DNA appeared to take place somewhat more slowly (to cleave the DNA through oxygen-dependent and to depend less on concentration than did the redox chemistry with Fez'). As control experadduct formation in the SC DNA. This suggests iments, this 8-substituted psoralen and 8-MOP were that the strands which underwent cleavage most each irradiated under argon in the presence of Col probably already had formed adducts with the sensi- El DNA, but without Fez+. Although a significant tizer. Identical mixtures of pBR322 and sensitizer decrease in SC (from 81% to 46% and 43% kept without irradiation for the same duration were respectively) and corresponding increase in OC used as controls. Their analysis proved that what- (from 19% to 46% and 43% respectively) and L ever interactions had occurred in the dark did not DNA (from 0 to 5%) were reported after a 1 h affect the electrophoretic properties of SC DNA irradiation, these changes were ascribed not to the effect of the irradiation, but to the ultrafiltration (Fig. 4). It is important to note that observing the electro- step with a Centricon cartridge (it is not clear phoretic band for OC DNA provides no information whether the plasmid used as reference had been at all on the number of sites where cleavage has submitted either to the Centricon filtration or to occurred. It takes only the first break on one strand irradiation). to relax the SC DNA molecule and to produce Usually, ultrafiltration with Centricon devices the OC form. Subsequent events do not produce does not lead to plasmid cleavage, and certainly not electrophoretically distinguishable species. It is of the magnitude reported by Nakamura. In any quite likely that the OC DNA obtained in our exper- event, the plasmid used in our work was never

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centrifuged in Centricon filters, and the changes described here are unquestionably photoinduced. In accord with our observations, Nakamura reported that photo-crosslinked SC DNA migrated more slowly and showed tailing in the electrophoretic separations. However, he did not compare the photodamage produced in the presence and in the absence of oxygen. CONCLUSlON

DNA was cleaved photochemically in the presence of all the psoralens tested. With P, 5-MOP, and 8-MOP, there were competing modifications of SC DNA, most probably through crosslinking, which slowed the electrophoretic migration of that band and were therefore very conspicuous. With 5GOP, in contrast, SC DNA modifications were not directly detected electrophoretically, but strand cleavage was observed, particularly under argon. Denaturation experiments and treatment with a restriction enzyme and with alkali, however, disclosed that sensitization with 5-GOP had produced important modifications of SC DNA. Cross-linking was certainly induced upon photosensitization of pBR322 with 5-GOP, although it had not been found with calf thymus DNA (Morliere etal., 1991). The difference in migration patterns found between 5-GOP and the other three psoralens perhaps partially reflected differences in populations of topoisomers induced photochemically, as well as differences in the efficiency of the cross-linking reactions. These possibilities will be evaluated in our forthcoming studies. With linear DNA such as calf thymus DNA, the structural integrity of cross-linked DNA may not be greatly affected by strand breaks, since they do not produce separate fragments (unless two breaks happen to occur within a short distance on opposite strands at two different locations). It is the strain energy uniquely embodied in the structure of SC DNA which allows a large topological change whenever one strand is broken, and which creates the diagnostic tool for the chemical damage. Supercoiled DNA could therefore become a useful adjunct to calf thymus DNA and other linear DNA for studies on modifications of DNA sensitized by psoralens. In addition to the cleavage of Col El (Nakamura, 1989), there is a report on the laser-photosensitized destruction of A phage DNA at low temperature in the presence of 8-MOP (Zhizhina et al., 1990). Psoralen may therefore photosensitize DNA strand breaks more generally than presently recognized. The demonstration with pBR322 that strands of supercoiled and open circular DNA do break in vitro suggests that a careful analysis for psoralenphotosensitized DNA strand breaks in vivo is desirable. Of course, even if demonstrated, these breaks could be without deleterious biological conse-

quences if efficient repair mechanisms existed. On the other hand, it is conceivable that the correlation between the observed photobiological activity of psoralens and their photophysical and photochemical parameters could be greatly improved by adding the sensitizers' contribution to DNA strand scission to all the previously known effects on cellular components. Acknowledgements-This work was initiated when support

of our research was received from the Public Health Service (ES04397). Support by a Bristol-Myers Squibb Company Award of Research Corporation is also gratefully acknowledged. REFERENCES

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Psoralens cleave pBR322 DNA under ultraviolet radiation.

Supercoiled (SC) pBR322 was used to probe the recent claim that 5-geranoxylpsoralen (5-GOP) did not photoreact with DNA. Contrary to expectations, 5-G...
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