Pkotochemisrry and Pkorubiology, 1976, Vol. 24. pp. 507~-513 Pergamon Press. Printed in Great Britain
INDEPENDENCE OF PHOTOPRODUCT FORMATION ON DNA CONFORMATION* MICHAELH. PATRICK and DONALD M. GRAY The University of Texas at Dallas, Box 688, Richardson, TX 75080, U X A (Received 13 April 1976; accepted 30 June 1976)
Abstract-In an ethanolic solution native T7 DNA can undergo conformational transitions from the B conformation (0% ethanol) to the C-like (60% w/w ethanol) and the A (80% wjw ethanol) conformations. We have investigated the formation of three classes of thymine-derived photoproducts in T7 DNA irradiated (280 nm) in the B, C-like, and A conformations, which were monitored by circular dichroism measurements. We find that the predominant class of thymine-derived photoproducts in
any conformational state is cyclobutyl dipyrimidines. While the 'spore product,' 5-thyminyl-5,6-dihydrothymine, which belongs to another class of photoproducts, does form in native DNA in the A conformation, its yield in denatured DNA at 80% ethanol is the same as that in native DNA. The yield of pyrimidine adduct, a third photoproduct class, is a maximum at 5060% ethanol. This effect of ethanol is probably not due to the ethanol-induced C-like conformation, however, since pyrimidine adduct formation is not enhanced when T7 DNA is irradiated in the C conformation in 6 M CsCl or in intact phage. We conclude from these and other data in the literature that the degree of hydration rather than the conformational state is the critical factor in determining which of the photoproducts will form in native DNA.
As these studies show, the photochemistry of DNA differs for the double-stranded, single-stranded, and The photochemical reactivity of deoxyribonucleic dehydrated forms. However, we know of no evidence acid (DNA) depends not only upon the primary base for or against possible differences in the photochemissequences but also upon the secondary structure of try among the double-stranded B, C, and A conforthe molecule and the environment during irradiation mations. The work to be described here was under(For recent reviews see Burr, 1968; Lomant and taken to assess the importance of the conformational Fresco, 1972; Rahn, 1972; Setlow and Setlow, 1972; state of DNA in photoproduct formation. Recent work on conformational polymorphism of Varghese, 1972; Patrick and Rahn, 1976). For example, the overall rate of Thy()Thy formation in polynucleotides in solution has shown that the condenatured DNA is up to two-fold greater than in formation depends on base sequence, and that a native DNA irradiated at the same temperature (e.g. polynucleotide region containing a long cluster of a 25"C, Dellweg and Wacker, 1962; see also Patrick given base sequence may have a conformation different from regions of random sequence (Bram, 1971; and Rahn, 1976), yet pyrimidine adduct (e.g. Thy-Cyt adduct) yield in native DNA is roughly four-fold Brahms et al., 1973; Arnott and Selsing, 1974a,b; greater than in denatured DNA (Patrick, 1976). Arnott et al., 1974). The DNA in T2, T5, and TI phages, and over 50% of the DNA in calf thymus Moreover, the ratio, (Thy()Thy (trans-syn)/(Thy ()Thy (cis-syn)), increases from below 0.02 for native chromatin may be in the C conformation (TunisDNA to 0.14 in denatured DNA (Rahn and Landry, Schneider and Maestre, 1970; Dorman and Maestre, 1971). At temperatures below VC, the rate of forma- 1973; Hamlon et al., 1974); moreover, DNA strands tion of Thy()Thy in native DNA decreases as the may acquire an A conformation during transcription temperature is lowered, but that of Thy(w5)hThy (and in order to pair with RNA strands, which are restricto a lesser extent, Thy-Cyt adduct) increases, reaching ted to A-conformations (Arnott et al., 1968). Circular dichroism (CD) measurements are sensi-100°C (Smith and Yashikawa, a maximum at 1966; Rahn and Hosszu, 1968; Patrick, 1976). In addi- tive to the conformation of DNA in solution. Starting tion, an increase in the yield of both Thy(a-5)hThy from DNA in a B conformation in neutral aqueous and Thy-Cyt adduct and a corresponding decrease solution, the C D spectrum can undergo a continuous in Thy()Thy yield have been observed in irradiated change upon the addition of salts, ethylene glycol, films of E. coli DNA, when the relative humidity was methanol, or up to 60% by weight of ethanol (Ivanov decreased below 65% (Rahn and Hosszu, 1969; et al., 1973; Green and Mahler, 1968; Nelson and Johnson, 1970; Girod et al., 1973) to approach a specPatrick, 1976). trum that is like that usually assigned to DNA in 'Address all correspondence to: Michael H. Patrick, The the C conformation (Tunis-Schneider and Maestre, University of Texas at Dallas, P.O. Box 688, Richardson, 1970). Ethanol appears to be unique among the above solvent components in being able to elicit a further TX 75080, U.S.A. INTRODUCTION
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MICHAEL H. PATRICK and DONALDM. GRAY
cooperative change (between about 70% and 80% ethanol by weight) in the CD spectrum that has been assigned to the A conformation (Ivanov et al., 1974; Gray and Ratliff, 1975). In the experiments t o be described here, we irradiated phage T7 DNA, labeled with 3H(methyl)Thy, in aqueous ethanol solutions, monitored the conformational changes by CD measurements, and determined the distribution of radioactivity in the photoproducts Thy()Thy (cis-syn), Thy( )Thy (trans-syn), Thy( )Ura (the deamination product of Thy( )Cyt originally produced in situ), Thy(6-4)Pyo (the dehydration product of the Thy-Cyt adduct formed in situ), and Thy(cr-5)hThy. W e find that the predominant class of Thy-derived photoproducts in the B, C, or A conformation is T h y O P y r , and conclude from these results that the degree of hydration, rather than a specific conformational state of DNA, is the critical factor in determining which class of photoproducts will form in DNA. MATERIALS AND METHODS
Preparation of DNA samples. Tritiated DNA was prepared from T7 phage grown on E . coli B3 (Thy-), according to the method of Yamamoto et ul. (1970). Cells were grown in 1500 mP of M9-glucose medium containing 2 pg/mt Thy and 1 pCi/mL 3H(methyl)Thy (6 Ci/m mol, Schwartz BioResearch). Following purification of the labeled phage by isopycnic sedimentation in CsCI, the suspension (2 mP, 3 x phage/mrP) was dialyzed at 4°C against 1.0 M NaCI, 0.02 M Tris, pH 7.5, 0.01 M Na2 EDTA, then dialyzed against the same buffer but with a ten-fold lower NaCl concentration. To the phage suspension, a sodium dodecyl sulfate solution was added to give a final concentration of 1% (v/v). Phenol, previously distilled under nitrogen and partial vacuum and saturated in a buffer of the same composition as the final phage suspension dialysate was added to give a final concentration of 50% (v/v), and the protein was extracted by gentle mixing followed by low speed (2 min, 4000 rpm in a Sorvall SS-36 rotor) centrifugation and removal of the phenol and protein interface. This procedure was repeated six times, following which the aqueous phase was dialysed exhaustively at 4°C against 0.01 M Na,HPO,, 0.001 M Na,EDTA, 0.182 M NaCI, pH 7.0. The DNA solution (650 pg/mP, 1.85 x lo5 dpm/pg; A280/A260= 0.52, A230/A2h0= 0.45) was stored at 4°C until used. Unlabeled T7 DNA was also isolated and purified in the same manner, yielding essentially the same concentration and spectral properties. For each experiment, labeled and unlabeled DNA preparations were mixed to give a sample having an of 1 to 2, and 0.5 to 1 x lo5 dpm/pg. These samples were dialyzed into 0.001 M NaZHPO, (0.002 M Na') adjusted to pH 7.0 with H3P04, Samples containing ethanol were prepared by adding reagent quality absolute alcohol (US.Industrial Chemicals Co.) to weighed aliquots of the DNA preparations to give the desired percent ethanol by wt. No additional salts were added. Our reported final ethanol concentrations are accurate to within 2% by wt. Samples irradiated at higher Na' concentrations or in the presence of Cs' were prepared by the addition of solid NaCl or CsCl (optical grade, Schwarz BioResearch) to the dialyzed DNA sample to give the desired salt concentration. Samples of denatured DNA were prepared by heating a sealed spectrophotometer cell containing the DNA in 0.002 M Na+ (phosphate buffer, pH 7.0) to 80°C for 10 min and by then quenching the cell to 20°C. The hyperchromicity that remained after this treatment was 28-29". (% Hyperchromicity = 100 x [ A 2 6 o (denatured)
- A''' ( n a t i ~ e ) ] / A '(native) ~~ at 20°C.) Samples of denatured DNA containing ethanol were prepared by adding ethanol to weighed aliquots of the previously denatured DNA. Circular dichroism measurements. Absorption spectra were obtained with a Cary Model 14 spectrophotometer, and CD spectra were obtained with a Cary Model 61 circular dichrometer. Calibration of the circular dichrometer, instrument operation, on-line computer acquisition of data, and control of sample temperatures have been described (Gray et al., 1973). Circular dichroic data are presented as the CD per mol of monomer, eL - eR = 8/[32.98]1c) in units of P/(mol.cm), where eL and eR are the molar extinction coefficients for left and right circularly polarized light, respectively; 0 is the measured ellipticity in degrees; I is the path length in cm; and c is the molar concentration of monomer subunits. Concentrations of the stock DNA preparations were determined by measuring the optical densities at 260 nm of aliquots diluted with 0.002 M Na' (phosphate buffer, pH 7.0); we used a molar extinction coefficient ( E ) at 260nm of 6570 (P/[mol.cm]) which is equivalent to an E: & of 198 (deciliter/[g.cm]) as reported for the sodium salt of T7 DNA (Crothers and Zimm, 1965). DNA concentrations in diluted samples containing ethanol were calculated from the known DNA stock concentrations, the measured weight percent ethanol concentrations, and known densities of water-ethanol mixtures. Final optical densities of the diluted samples ranged from 0.4 to 1.5 at 260 nm. All spectra were obtained at 20°C. CD and absorption spectra were recorded both before and after irradiation of each sample in a 1 cm path length, glass stoppered, cylindrical quartz cell (2.8 mi). A miniature stirring bar was added to each cell so the samples could be continuously stirred during irradiation. Care was taken to ensure that the stirring bar did not lie in the path of the beam in either the spectrophotometer or circular dichrometer. UV irradiation. The DNA samples, in the same quartz cells described above, were exposed (with constant stirring) to 2.5 kJm-' monochromatic UV light (A = 280 +_ 5 nm) obtained from a Bausch & Lomb grating monochromator (Model 33-86-45-49) illuminated with a 500-W high pressure mercury arc lamp (Philips SP-500). Intensities were measured with a Keithley 150B d.c. breaker amplifier and a thermopile calibrated against a National Bureau of Standards carbon-filament standard lamp; intensities were between l'and 10 Wm-2. Isolation and identification of photoproducts. Irradiated DNA samples were dialyzed exhaustively against distilled water, evaporated to dryness, taken up in trifluoroacetic acid or 98% formic acid, and hydrolyzed in sealed glass tubes for 60min at 105°C. The hydrolysates were spotted on 3 cm-wide Whatman No. I paper strips and developed by descending chromatography at room temperature in n-butanol-acetic acid-water (80: 12:30) for 20 h. Average R , values in this solvent system in the products of interest here are (cf. Patrick and Rahn, 1975): Thy, 0.62; ThyOUra, 0.20; Thy()Thy (cis, syn), 0.30; Thy()Thy (trans, syn), 0.43; and Thy(a-5)h-Thy, 0.36. Each strip was cut into I-cm pieces and eluted 2 h at room temperature with 1 m l water in glass scintillation vials, following which 10 me of a scintillation solution (8 g butylphenylbiphenyloxadiozate-1,3,4; 150 mC Beckman BioSolv 3, loo0 md toluene) was added and the samples were counted in a Nuclear Chicago Mark I1 counter (3H counting efficiency was 48% with a background rate of 2Ocpm). Thy-Cyt adduct formation was monitored by applying the acid hydrolysates directly to a Dowex 50W-X12 column (H", 200 mesh, 0.5 x 5 cm), and eluting with distilled water. 2-me fractions were collected and counted as described above. In a separate series of experiments (M. H. Partrick and J. M. Snow, unpublished data), generalized Pyr adduct
509
DNA conformation and photoproduct formation formation was monitored by its fluorescence properties (Hauswirth and Wang, 1973). The observed relative fluor= 310 nm; Hitaescence emission intensity (400 nm; leXcit chi-Perkin-Elmer spectrofluorimeter using a lP28 phototube) of native T7 DNA irradiated at different ethanol concentrations was in agreement with the corresponding yields of Thy(6-4)Pyo assayed by radioactivity. From the fluorescence quantum yields for different polynucleotides, Hauswirth and Wang (1973) concluded that it’ is predominantly thymidylyl- or cytidylyl-(3’-5’)cytidine sequences in DNA in which adducts form. We shall, therefore, refer to results obtained by determining Thy(6-4)Pyo yield as Pyr adduct formation. RESULTS AND DISCUSSION
CD spectra of nutive and denatured D N A in different concentrations of ethanol. The CD spectra of native T7 DNA at various concentrations of ethanol (% by weight) are shown in Fig. 1. Ethanol concentrations up to 60% cause the CD values above 250 nm to become reduced or more negative. In 6 A4 CsCl (see Fig. 1C) or in dehydrated films (Tunis-Schneider and Maestre, 1970), DNA has a CD spectrum in which the positive bands above 260 nm have almost completely disappeared. Such a spectrum is usually attributed to the C secondary conformation (TunisSchneider and Maestre, 1970). For simplicity, we will refer to DNA in 6 M CsCl as having the C conformation and C-type spectrum, as is usually done. Since the spectrum of DNA in 60% ethanol approaches the C-type spectrum, we will refer to it as being “C-like.” A second conformational change occurs between 60 and 80% ethanol to give a spectrum that is undoubtedly due to the A conformation of DNA (Ivanov et ul., 1973; Ivanov et al., 1974; Gray and Ratliff, 1975). The cooperative nature of the change between 60 and 80% ethanol is shown in the inset of Fig. lA, in which the CD at 270 nm is shown for samples at various ethanol concentrations. The ethanol-dependent CD changes that we find for T7 DNA are similar to those previously reported for other natural DNA’s (Ivanov et al., 1974; Girod et a/., 1973) and for synthetic poly d(AC).d(GT) (Gray and Ratliff, 1975). It is well-known that DNA at low salt concentrations can be denatured by the addition of ethanol. However, the spectral changes we find for denatured DNA in the presence of ethanol differ from those presented in Fig. 1A. Figure 1B shows the 20°C spectrum of previously heat-denatured T7 DNA (solid curve). This spectrum differs from that of native T7 DNA (solid curve in Fig. 1A) in having a larger positive band at short (218-220 nm) wavelengths and a crossover at 260 nm which has been shifted by 2 nm from the crossover at 258 nm in the spectrum of the native DNA. At 50% ethanol, the denatured DNA spectrum (dashed curve in Fig. 1B) is reduced in magnitude, with an apparent reduction in base stacking interactions to give a spectrum similar to that of aqueous solutions of denatured DNA at elevated temperatures (Brahms and Mommaerts, 1964; Gray, unpublished observations). The CD spectrum of dena-
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Wavelength ( n m )
6
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XEtDH 0
Denatured T 7 DNA
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Wavelength ( n m )
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Native T7 DNA
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Wavelength( n m )
Figure I. (A) CD spectra of native T7 DNA at 20°C in oU/, ethanol, 2.0 mM Na’ (-); 60% ethanol, 0.71 mM 70% ethanol, 0.52 mM Na’ (-,-.-); 80% Na’ (----); ethanol, 0.34 mM Na+ (.....). Inset shows the CD at 270 nm for a series of samples at different ethanol concentrations. (Ethanol concentrations are % by wt.) (B) CD spectra of T7 DNA (previously heat-denatured) at 20°C in 0% ethanol, 2.0 mM Na+ (-); 50% ethanol, 0.91 mM Na’ (----); 80% ethanol, 0.34 mM Na’ (....) . (C) CD spectra of native T7 DNA in 0.1 M NaCl (-) and 6 M CsCl plus 0.07 M NaCl (----).
MICHAELH. PATRICK and DONALD M. GRAY
510
tured T7 DNA in 50% ethanol is different from the spectrum of native T7 DNA in 50-60% ethanol in having a smaller magnitude of the negative band at 247-248 nm. (Compare dashed curves in Fig. 1A and 1B.) The spectrum of denatured T7 DNA in 80% ethanol has a crossover which is blue-shifted to 255 nm (dotted curve in Fig. lB), and it may be that the high ethanol concentration has some effect on the conformation of single-stranded DNA. The CD change which occurs for denatured DNA on going from 50 to 80% ethanol is not like that which occurs for native DNA on going from 60 to 80% ethanol and is not likely due to a transition to the A conformation of some renatured portion of the denatured sample. Photoproducts formed at diferent ethanol concentrations. Typical radiochromatographic profiles of the photoproducts formed in T7 DNA irradiated at different ethanol concentrations are shown in Fig. 2 and 3. A more comprehensive and quantitative description of our results is given in Table 1. The major features we wish to draw attention to are: (1) U p to S M O % ethanol, the only Thy-derived photoproducts found in native DNA are Thy()Pyr and Thy-Cyt adduct; (2) The trans-syn isomer of Thy()Thy is produced in denatured DNA at all ethanol concentrations; and (3) Only at high (80%)ethanol concentrations is there a readily discernable peak of radioactivity corresponding to Thy@-5)hThy, and this is present in both native and denatured DNA. We shall discuss first the influence of DNA secondary structure and the presence of ethanol on the formation of Thy()Pyr and Thy(cr-5)hThy. While there is an indication of a slight amount of Thy(a-5)hThy in native DNA at ethanol concentrations of 60 and 70%, these values must be regarded as only suggestive since they are close to the limit of reproducibility of this system in our hands. We prefer to interpret these
106
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results as showing that Thy@-5)hThy is formed in appreciable amounts only at concentrations of ethanol exceeding 70% and thus past the range in which a C-like conformation i s induced in DNA. Furthermore, even though the yield of Thy(cr-5)hThy is greatest at 80% ethanol, where the DNA is in the A conformation as indicated by its CD spectrum, cyclobutyl dipyrimidines are still the major class of photoproducts formed. Even more important, however, is that the yield of Thy(cr-5)hThy is the same in both native and denatured DNA. While an argument could be made for a unique conformation for denatured DNA in high ethanol concentrations, the CD spectra in Fig. 1A and 1B clearly show that this is not A-like. Consequently, the simplest and most reasonable conclusion appears to be that the conformational state of DNA is not critical in determining the formation of Thy()Pyr or Thy@-5)hThy. Rather, we feel that it IS the lowered water activity itself, at high ethanol concentrations, rather than the conformational change such an environment induces in DNA, which is the critical factor in formation of Thy(cr-5)hThy. Thus, while these ethanol concentrations are sufficient to induce a B to A conformational change as well as cause a slight increase in the rate of Thy(w5)hThy formation, the effective water concentration (1 1 M at 80% w/w ethanol concentration) is still large enough to favor Thy()Thy rather than Thy(a-5)hThy (cf. Varghese, 1970). These results, therefore, confirm and extend the work of Rahn and Hosszu (1966) on DNA films, who concluded from their results that the absence of water i s necessary for the formation of Thy(u-5)hThy. The influence of ethanol on the formation of photoproducts in DNA could be due to (a) ethanol-induced conformational changes, the possibitity we are trying to assess, (b) dehydration or removal of water, or (c)
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Figure 2. Chromatographic distribution of radioactivity in formic acid hydrolysates of T7 C3H(methyl)Thy] DNA irradiated (2.5 kJm-*, 280 nm) at different ethanol concentrations. (The same samples used for CD measurements represented in Fig. 1A. See Materials and Methods). Results are shown for native DNA (M and )denatured DNA (o---o).
DNA conformation and photoproduct formation
51 I
and Thy(a-5)hThy photoproducts are, respectively, reduced and increased in solutions at high ethanol concentrations and in films at low humidities, we have interpreted our results in these cases without explicitly distinguishing dehydration from a direct influence of ethanol on the photochemistry. Our main conclusion is that the influence of ethanol on these photoproducts does not seem to be uniquely due to possibility (a). Like Thy()Pyr formation, the yield of Thy(6-4)Pyo (the acid stable product derived from Thy-Cyt adduct formed in situ) is reduced by the presence of ethanol during irradiation of T7 DNA (cf. Table 1 and Fig. 3; note the different fluences used in these experiments for native and denatured DNA, compared to those used for determination of Thy()Pyr and Thy(a-5)hThy formation). For denatured T7 DNA, suppression of Pyr adduct formation is a monotonic function of the ethanol concentration. In contrast to this and to the results obtained for Thy()Pyr formation, Pyr adduct formation in native DNA does not decrease monotonically with increasing ethanol con10' 0 5 10 15 20 25 30 35 centration; rather, superimposed on the inhibition is FA A C T I0 I\] NU hl B [IR a pronounced enhancement in the yield between 50 and 60% ethanol. At this ethanol concentration T7 Figure 3. Elution profile of formic acid hydrolysate of irra- DNA is in a C-like conformation, as indicated by diated (10 kJm-', 280 nm) 3H-Iabeled native T7 DNA from a Dowex column (see Materials and Methods). The the C D spectrum (cf. Fig. 1A). The same qualitative radioactivity in fractions 1 through 13 correspond to thy- behavior for Pyr adduct formation in native T7 DNA mine (T) and all stable Thy-derived photoproducts (T*) as a function of ethanol concentration was observed other than Thy (64) Pyo. 0% ethanol (u 30%) eth-,in an independent series of experiments using fluoresanol (e-+), 50% ethanol (A---A), and 80% ethanol cence emission to assay adduct yield (M. H. Patrick ( A . . . A .) and J. M. Snow, unpublished data; cf. also, Materials and Methods). Since enhanced adduct yield for DNA in a C-like altered photochemistry involving the ethanol itself.* conformation in ethanol is seen even though there Of course, combinations of these effects are also poss- may be a general suppression of this photoproduct ible. There is not sufficient published information by the ethanol, then it may be that adducts are proavailable to provide an independent evaluation of the duced in even higher relative yield in in uiuo situarelative importance of effects (b) and (c) in inter- tions. DNA is thought to be in the C conformation preting our experiments. Since the Thy()Thy(c,s) in phages (Tunis-Schneider and Maestre, 1970; Dorman and Maestre, 1973) and the DNA in chromatin is largely in the C conformation (Hanlon et al., 1974). In addition, one can take advantage of the observation that with increasing salt concentration, the C D *The presence of alcohol has been shown to reduce the efficiency of PyrOPyr formation in irradiated dimethyl spectrum of DNA shows a decrease in the positive thymine (Morrison and Kloepfer, 1968) and TpT (Lamola, band; at sufficiently high salt concentration (e.g. 6 1968; Tramer et al., 1969), as well as to induce photo-addi- M LiCl and CsCl, or 5.5 M NH,Cl) the C D spectrum tion reactions with Pyr (Wang, 1960) and Pur (Connolly is the C-type. Therefore, to further test the possibility and Linschitz. 1968; Evans and Wolfenden, 1960; Steinmus et a/., 1970; Leonov and Elad, 1974) bases in solution. that Pyr adducts form preferentially in DNA having a C conformation, we irradiated T7 DNA in situ (as This may account for the decreasing yield of Thy()Pyr as the ethanol concentration is raised (Table 1). The more part of the intact phage) and in the presence of 6 drastic suppression observed for denatured DNA may re- M CsCl; the C D spectrum and photoproduct yield flect the much increased base-solvent interaction in singlestranded DNA. Ben-Ishai et d. (1973) found, in addition were determined, and the results are shown in Fig. to suppression of ThyOPyr formation in irradiated I C and Table 2), respectively. Consistent with our (2 = 254 nm or > 290 nm) native or denatured E . coli findings for DNA in ethanolic solutions, the results DNA by the presence of 2-propanol or ethanol, evidence obtained for irradiation of T7 phage DNA in situ and for photoalkylation of Gua and Ade moieties by 2-pro- in 6 M CsCl. show that Thy()Pyr and Thy(a-5)hThy panol. They also detected an as yet unidentified Thyderived photoproduct (RJ 0.8 in the solvent system used formation is not affected by whether the DNA is in here) from denatured DNA irradiated in the presence of the B or C conformation. However, it is obvious that 2-propanol. in contrast to the conclusions drawn from the studies
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MICHAEL H. PATRICK and DONALDM. GRAY Table 1. Photoproduct yield* in irradiatedt T7 DNA at different ethanol concentrations ~
Ethanol (%)
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Native (N) or denatured (D) DNA N D N D N D N
0
30
50 60
ThyOUra
Percent of Thy radioactivity as: Thy()Thy(c,s) Thy()Thy(t,s) Thy(a-5)hThy
2.2 4.2 1.9
5.8 11.0 5.7
2.1 2.2 1.9
6.4 6.0 6.3
0.3
2.3
6.0
0.4
1.6 1.4
4.6 4.4
0.8 0.8
Thy(6-4)Pyo 0.59 0.66 0.36 0.58 0.49 0.47 0.42 0.42 0.16 0.28 0.12 0.22
.-
-
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D 70
N D N D
80
*These values represent the average of experiments done at least twice; a dash line represents < O.l?(, of Thy the “/b radioactivity in the region having the R, of the indicated photoproduct. In the case of Thy(-)Pyo, radioactivity in the product is with respect to Thy and all Thy-derived photoproducts eluted in the first 15 fractions from a Dowex column (see Fig. 3). Results obtained by spectrofluorimetric analysis (see Materials and Methods) of generalized Pyr adduct formation were in qualitative agreement with the results shown above for the radioactivity analysis of Thy-Cyt adduct formation. That is, the fluorescence emission at 400 nm (Hauswirth and Wang, 1973) was greater for DNA irradiated at 50 and 60% ethanol, and decreased sharply at higher’ concentrations. We had difficulty, however, in obtaining quantitatively reproducible fluorescence spectra for ethanolic solutions of irradiated DNA. TFluence: 2.5 kJm.-’ at 280 nm for native and denatured DNA assayed for Thy()Pyr and Thy(&-5)hThy; 4 kJm-’ at 280 nm for native DNA assayed for Thy(64)Pyo; 10 kJm-’ 280 nm for denatured DNA assayed for Thy(64)Pyo. Since the yield of Pyr adducts in duplex DNA is significantly less than the PyrOPyr yield, and this difference is even greater in denatured DNA, different fluences were used which depended on the nature of the photoproduct to be studied and the state of the DNA. In general, the ratio, Pyr adduct/Pyr()Pyr, is fluence dependent; but since we are interested here in the photoproduct yield as a function of the ethanol concentration (rather than the fluence dependent photoproduct ratio), the different fluences used for detection of certain photoproducts do not influence the property we wish to measure. Table 2. Photoproduct yield in native T7 DNA irradiated (280 nm) in different aqueous environments
Irradiation condition Intact phage 0.1 M Na+ 6.0 M Cst
Thy()Ura 1.3 1.5 1.9
Percent of Thy radioactivity* as: Thy()Thy(c,s) Thy(6-4)Pyo Thy(a-5)hThy 5.1 5.2 5.3
0.62 0 63 0.64
< 0.1
INDEPENDENCE OF PHOTOPRODUCT FORMATION ON DNA CONFORMATION* MICHAELH. PATRICK and DONALD M. GRAY The University of Texas at Dallas, Box 688, Richardson, TX 75080, U X A (Received 13 April 1976; accepted 30 June 1976)
Abstract-In an ethanolic solution native T7 DNA can undergo conformational transitions from the B conformation (0% ethanol) to the C-like (60% w/w ethanol) and the A (80% wjw ethanol) conformations. We have investigated the formation of three classes of thymine-derived photoproducts in T7 DNA irradiated (280 nm) in the B, C-like, and A conformations, which were monitored by circular dichroism measurements. We find that the predominant class of thymine-derived photoproducts in
any conformational state is cyclobutyl dipyrimidines. While the 'spore product,' 5-thyminyl-5,6-dihydrothymine, which belongs to another class of photoproducts, does form in native DNA in the A conformation, its yield in denatured DNA at 80% ethanol is the same as that in native DNA. The yield of pyrimidine adduct, a third photoproduct class, is a maximum at 5060% ethanol. This effect of ethanol is probably not due to the ethanol-induced C-like conformation, however, since pyrimidine adduct formation is not enhanced when T7 DNA is irradiated in the C conformation in 6 M CsCl or in intact phage. We conclude from these and other data in the literature that the degree of hydration rather than the conformational state is the critical factor in determining which of the photoproducts will form in native DNA.
As these studies show, the photochemistry of DNA differs for the double-stranded, single-stranded, and The photochemical reactivity of deoxyribonucleic dehydrated forms. However, we know of no evidence acid (DNA) depends not only upon the primary base for or against possible differences in the photochemissequences but also upon the secondary structure of try among the double-stranded B, C, and A conforthe molecule and the environment during irradiation mations. The work to be described here was under(For recent reviews see Burr, 1968; Lomant and taken to assess the importance of the conformational Fresco, 1972; Rahn, 1972; Setlow and Setlow, 1972; state of DNA in photoproduct formation. Recent work on conformational polymorphism of Varghese, 1972; Patrick and Rahn, 1976). For example, the overall rate of Thy()Thy formation in polynucleotides in solution has shown that the condenatured DNA is up to two-fold greater than in formation depends on base sequence, and that a native DNA irradiated at the same temperature (e.g. polynucleotide region containing a long cluster of a 25"C, Dellweg and Wacker, 1962; see also Patrick given base sequence may have a conformation different from regions of random sequence (Bram, 1971; and Rahn, 1976), yet pyrimidine adduct (e.g. Thy-Cyt adduct) yield in native DNA is roughly four-fold Brahms et al., 1973; Arnott and Selsing, 1974a,b; greater than in denatured DNA (Patrick, 1976). Arnott et al., 1974). The DNA in T2, T5, and TI phages, and over 50% of the DNA in calf thymus Moreover, the ratio, (Thy()Thy (trans-syn)/(Thy ()Thy (cis-syn)), increases from below 0.02 for native chromatin may be in the C conformation (TunisDNA to 0.14 in denatured DNA (Rahn and Landry, Schneider and Maestre, 1970; Dorman and Maestre, 1971). At temperatures below VC, the rate of forma- 1973; Hamlon et al., 1974); moreover, DNA strands tion of Thy()Thy in native DNA decreases as the may acquire an A conformation during transcription temperature is lowered, but that of Thy(w5)hThy (and in order to pair with RNA strands, which are restricto a lesser extent, Thy-Cyt adduct) increases, reaching ted to A-conformations (Arnott et al., 1968). Circular dichroism (CD) measurements are sensi-100°C (Smith and Yashikawa, a maximum at 1966; Rahn and Hosszu, 1968; Patrick, 1976). In addi- tive to the conformation of DNA in solution. Starting tion, an increase in the yield of both Thy(a-5)hThy from DNA in a B conformation in neutral aqueous and Thy-Cyt adduct and a corresponding decrease solution, the C D spectrum can undergo a continuous in Thy()Thy yield have been observed in irradiated change upon the addition of salts, ethylene glycol, films of E. coli DNA, when the relative humidity was methanol, or up to 60% by weight of ethanol (Ivanov decreased below 65% (Rahn and Hosszu, 1969; et al., 1973; Green and Mahler, 1968; Nelson and Johnson, 1970; Girod et al., 1973) to approach a specPatrick, 1976). trum that is like that usually assigned to DNA in 'Address all correspondence to: Michael H. Patrick, The the C conformation (Tunis-Schneider and Maestre, University of Texas at Dallas, P.O. Box 688, Richardson, 1970). Ethanol appears to be unique among the above solvent components in being able to elicit a further TX 75080, U.S.A. INTRODUCTION
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MICHAEL H. PATRICK and DONALDM. GRAY
cooperative change (between about 70% and 80% ethanol by weight) in the CD spectrum that has been assigned to the A conformation (Ivanov et al., 1974; Gray and Ratliff, 1975). In the experiments t o be described here, we irradiated phage T7 DNA, labeled with 3H(methyl)Thy, in aqueous ethanol solutions, monitored the conformational changes by CD measurements, and determined the distribution of radioactivity in the photoproducts Thy()Thy (cis-syn), Thy( )Thy (trans-syn), Thy( )Ura (the deamination product of Thy( )Cyt originally produced in situ), Thy(6-4)Pyo (the dehydration product of the Thy-Cyt adduct formed in situ), and Thy(cr-5)hThy. W e find that the predominant class of Thy-derived photoproducts in the B, C, or A conformation is T h y O P y r , and conclude from these results that the degree of hydration, rather than a specific conformational state of DNA, is the critical factor in determining which class of photoproducts will form in DNA. MATERIALS AND METHODS
Preparation of DNA samples. Tritiated DNA was prepared from T7 phage grown on E . coli B3 (Thy-), according to the method of Yamamoto et ul. (1970). Cells were grown in 1500 mP of M9-glucose medium containing 2 pg/mt Thy and 1 pCi/mL 3H(methyl)Thy (6 Ci/m mol, Schwartz BioResearch). Following purification of the labeled phage by isopycnic sedimentation in CsCI, the suspension (2 mP, 3 x phage/mrP) was dialyzed at 4°C against 1.0 M NaCI, 0.02 M Tris, pH 7.5, 0.01 M Na2 EDTA, then dialyzed against the same buffer but with a ten-fold lower NaCl concentration. To the phage suspension, a sodium dodecyl sulfate solution was added to give a final concentration of 1% (v/v). Phenol, previously distilled under nitrogen and partial vacuum and saturated in a buffer of the same composition as the final phage suspension dialysate was added to give a final concentration of 50% (v/v), and the protein was extracted by gentle mixing followed by low speed (2 min, 4000 rpm in a Sorvall SS-36 rotor) centrifugation and removal of the phenol and protein interface. This procedure was repeated six times, following which the aqueous phase was dialysed exhaustively at 4°C against 0.01 M Na,HPO,, 0.001 M Na,EDTA, 0.182 M NaCI, pH 7.0. The DNA solution (650 pg/mP, 1.85 x lo5 dpm/pg; A280/A260= 0.52, A230/A2h0= 0.45) was stored at 4°C until used. Unlabeled T7 DNA was also isolated and purified in the same manner, yielding essentially the same concentration and spectral properties. For each experiment, labeled and unlabeled DNA preparations were mixed to give a sample having an of 1 to 2, and 0.5 to 1 x lo5 dpm/pg. These samples were dialyzed into 0.001 M NaZHPO, (0.002 M Na') adjusted to pH 7.0 with H3P04, Samples containing ethanol were prepared by adding reagent quality absolute alcohol (US.Industrial Chemicals Co.) to weighed aliquots of the DNA preparations to give the desired percent ethanol by wt. No additional salts were added. Our reported final ethanol concentrations are accurate to within 2% by wt. Samples irradiated at higher Na' concentrations or in the presence of Cs' were prepared by the addition of solid NaCl or CsCl (optical grade, Schwarz BioResearch) to the dialyzed DNA sample to give the desired salt concentration. Samples of denatured DNA were prepared by heating a sealed spectrophotometer cell containing the DNA in 0.002 M Na+ (phosphate buffer, pH 7.0) to 80°C for 10 min and by then quenching the cell to 20°C. The hyperchromicity that remained after this treatment was 28-29". (% Hyperchromicity = 100 x [ A 2 6 o (denatured)
- A''' ( n a t i ~ e ) ] / A '(native) ~~ at 20°C.) Samples of denatured DNA containing ethanol were prepared by adding ethanol to weighed aliquots of the previously denatured DNA. Circular dichroism measurements. Absorption spectra were obtained with a Cary Model 14 spectrophotometer, and CD spectra were obtained with a Cary Model 61 circular dichrometer. Calibration of the circular dichrometer, instrument operation, on-line computer acquisition of data, and control of sample temperatures have been described (Gray et al., 1973). Circular dichroic data are presented as the CD per mol of monomer, eL - eR = 8/[32.98]1c) in units of P/(mol.cm), where eL and eR are the molar extinction coefficients for left and right circularly polarized light, respectively; 0 is the measured ellipticity in degrees; I is the path length in cm; and c is the molar concentration of monomer subunits. Concentrations of the stock DNA preparations were determined by measuring the optical densities at 260 nm of aliquots diluted with 0.002 M Na' (phosphate buffer, pH 7.0); we used a molar extinction coefficient ( E ) at 260nm of 6570 (P/[mol.cm]) which is equivalent to an E: & of 198 (deciliter/[g.cm]) as reported for the sodium salt of T7 DNA (Crothers and Zimm, 1965). DNA concentrations in diluted samples containing ethanol were calculated from the known DNA stock concentrations, the measured weight percent ethanol concentrations, and known densities of water-ethanol mixtures. Final optical densities of the diluted samples ranged from 0.4 to 1.5 at 260 nm. All spectra were obtained at 20°C. CD and absorption spectra were recorded both before and after irradiation of each sample in a 1 cm path length, glass stoppered, cylindrical quartz cell (2.8 mi). A miniature stirring bar was added to each cell so the samples could be continuously stirred during irradiation. Care was taken to ensure that the stirring bar did not lie in the path of the beam in either the spectrophotometer or circular dichrometer. UV irradiation. The DNA samples, in the same quartz cells described above, were exposed (with constant stirring) to 2.5 kJm-' monochromatic UV light (A = 280 +_ 5 nm) obtained from a Bausch & Lomb grating monochromator (Model 33-86-45-49) illuminated with a 500-W high pressure mercury arc lamp (Philips SP-500). Intensities were measured with a Keithley 150B d.c. breaker amplifier and a thermopile calibrated against a National Bureau of Standards carbon-filament standard lamp; intensities were between l'and 10 Wm-2. Isolation and identification of photoproducts. Irradiated DNA samples were dialyzed exhaustively against distilled water, evaporated to dryness, taken up in trifluoroacetic acid or 98% formic acid, and hydrolyzed in sealed glass tubes for 60min at 105°C. The hydrolysates were spotted on 3 cm-wide Whatman No. I paper strips and developed by descending chromatography at room temperature in n-butanol-acetic acid-water (80: 12:30) for 20 h. Average R , values in this solvent system in the products of interest here are (cf. Patrick and Rahn, 1975): Thy, 0.62; ThyOUra, 0.20; Thy()Thy (cis, syn), 0.30; Thy()Thy (trans, syn), 0.43; and Thy(a-5)h-Thy, 0.36. Each strip was cut into I-cm pieces and eluted 2 h at room temperature with 1 m l water in glass scintillation vials, following which 10 me of a scintillation solution (8 g butylphenylbiphenyloxadiozate-1,3,4; 150 mC Beckman BioSolv 3, loo0 md toluene) was added and the samples were counted in a Nuclear Chicago Mark I1 counter (3H counting efficiency was 48% with a background rate of 2Ocpm). Thy-Cyt adduct formation was monitored by applying the acid hydrolysates directly to a Dowex 50W-X12 column (H", 200 mesh, 0.5 x 5 cm), and eluting with distilled water. 2-me fractions were collected and counted as described above. In a separate series of experiments (M. H. Partrick and J. M. Snow, unpublished data), generalized Pyr adduct
509
DNA conformation and photoproduct formation formation was monitored by its fluorescence properties (Hauswirth and Wang, 1973). The observed relative fluor= 310 nm; Hitaescence emission intensity (400 nm; leXcit chi-Perkin-Elmer spectrofluorimeter using a lP28 phototube) of native T7 DNA irradiated at different ethanol concentrations was in agreement with the corresponding yields of Thy(6-4)Pyo assayed by radioactivity. From the fluorescence quantum yields for different polynucleotides, Hauswirth and Wang (1973) concluded that it’ is predominantly thymidylyl- or cytidylyl-(3’-5’)cytidine sequences in DNA in which adducts form. We shall, therefore, refer to results obtained by determining Thy(6-4)Pyo yield as Pyr adduct formation. RESULTS AND DISCUSSION
CD spectra of nutive and denatured D N A in different concentrations of ethanol. The CD spectra of native T7 DNA at various concentrations of ethanol (% by weight) are shown in Fig. 1. Ethanol concentrations up to 60% cause the CD values above 250 nm to become reduced or more negative. In 6 A4 CsCl (see Fig. 1C) or in dehydrated films (Tunis-Schneider and Maestre, 1970), DNA has a CD spectrum in which the positive bands above 260 nm have almost completely disappeared. Such a spectrum is usually attributed to the C secondary conformation (TunisSchneider and Maestre, 1970). For simplicity, we will refer to DNA in 6 M CsCl as having the C conformation and C-type spectrum, as is usually done. Since the spectrum of DNA in 60% ethanol approaches the C-type spectrum, we will refer to it as being “C-like.” A second conformational change occurs between 60 and 80% ethanol to give a spectrum that is undoubtedly due to the A conformation of DNA (Ivanov et ul., 1973; Ivanov et al., 1974; Gray and Ratliff, 1975). The cooperative nature of the change between 60 and 80% ethanol is shown in the inset of Fig. lA, in which the CD at 270 nm is shown for samples at various ethanol concentrations. The ethanol-dependent CD changes that we find for T7 DNA are similar to those previously reported for other natural DNA’s (Ivanov et al., 1974; Girod et a/., 1973) and for synthetic poly d(AC).d(GT) (Gray and Ratliff, 1975). It is well-known that DNA at low salt concentrations can be denatured by the addition of ethanol. However, the spectral changes we find for denatured DNA in the presence of ethanol differ from those presented in Fig. 1A. Figure 1B shows the 20°C spectrum of previously heat-denatured T7 DNA (solid curve). This spectrum differs from that of native T7 DNA (solid curve in Fig. 1A) in having a larger positive band at short (218-220 nm) wavelengths and a crossover at 260 nm which has been shifted by 2 nm from the crossover at 258 nm in the spectrum of the native DNA. At 50% ethanol, the denatured DNA spectrum (dashed curve in Fig. 1B) is reduced in magnitude, with an apparent reduction in base stacking interactions to give a spectrum similar to that of aqueous solutions of denatured DNA at elevated temperatures (Brahms and Mommaerts, 1964; Gray, unpublished observations). The CD spectrum of dena-
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Wavelength ( n m )
6
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-(El’
--- so
4-
-4
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XEtDH 0
Denatured T 7 DNA
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200
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Wavelength ( n m )
- (C) 4-
200
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Native T7 DNA
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Wavelength( n m )
Figure I. (A) CD spectra of native T7 DNA at 20°C in oU/, ethanol, 2.0 mM Na’ (-); 60% ethanol, 0.71 mM 70% ethanol, 0.52 mM Na’ (-,-.-); 80% Na’ (----); ethanol, 0.34 mM Na+ (.....). Inset shows the CD at 270 nm for a series of samples at different ethanol concentrations. (Ethanol concentrations are % by wt.) (B) CD spectra of T7 DNA (previously heat-denatured) at 20°C in 0% ethanol, 2.0 mM Na+ (-); 50% ethanol, 0.91 mM Na’ (----); 80% ethanol, 0.34 mM Na’ (....) . (C) CD spectra of native T7 DNA in 0.1 M NaCl (-) and 6 M CsCl plus 0.07 M NaCl (----).
MICHAELH. PATRICK and DONALD M. GRAY
510
tured T7 DNA in 50% ethanol is different from the spectrum of native T7 DNA in 50-60% ethanol in having a smaller magnitude of the negative band at 247-248 nm. (Compare dashed curves in Fig. 1A and 1B.) The spectrum of denatured T7 DNA in 80% ethanol has a crossover which is blue-shifted to 255 nm (dotted curve in Fig. lB), and it may be that the high ethanol concentration has some effect on the conformation of single-stranded DNA. The CD change which occurs for denatured DNA on going from 50 to 80% ethanol is not like that which occurs for native DNA on going from 60 to 80% ethanol and is not likely due to a transition to the A conformation of some renatured portion of the denatured sample. Photoproducts formed at diferent ethanol concentrations. Typical radiochromatographic profiles of the photoproducts formed in T7 DNA irradiated at different ethanol concentrations are shown in Fig. 2 and 3. A more comprehensive and quantitative description of our results is given in Table 1. The major features we wish to draw attention to are: (1) U p to S M O % ethanol, the only Thy-derived photoproducts found in native DNA are Thy()Pyr and Thy-Cyt adduct; (2) The trans-syn isomer of Thy()Thy is produced in denatured DNA at all ethanol concentrations; and (3) Only at high (80%)ethanol concentrations is there a readily discernable peak of radioactivity corresponding to Thy@-5)hThy, and this is present in both native and denatured DNA. We shall discuss first the influence of DNA secondary structure and the presence of ethanol on the formation of Thy()Pyr and Thy(cr-5)hThy. While there is an indication of a slight amount of Thy(a-5)hThy in native DNA at ethanol concentrations of 60 and 70%, these values must be regarded as only suggestive since they are close to the limit of reproducibility of this system in our hands. We prefer to interpret these
106
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,
results as showing that Thy@-5)hThy is formed in appreciable amounts only at concentrations of ethanol exceeding 70% and thus past the range in which a C-like conformation i s induced in DNA. Furthermore, even though the yield of Thy(cr-5)hThy is greatest at 80% ethanol, where the DNA is in the A conformation as indicated by its CD spectrum, cyclobutyl dipyrimidines are still the major class of photoproducts formed. Even more important, however, is that the yield of Thy(cr-5)hThy is the same in both native and denatured DNA. While an argument could be made for a unique conformation for denatured DNA in high ethanol concentrations, the CD spectra in Fig. 1A and 1B clearly show that this is not A-like. Consequently, the simplest and most reasonable conclusion appears to be that the conformational state of DNA is not critical in determining the formation of Thy()Pyr or Thy@-5)hThy. Rather, we feel that it IS the lowered water activity itself, at high ethanol concentrations, rather than the conformational change such an environment induces in DNA, which is the critical factor in formation of Thy(cr-5)hThy. Thus, while these ethanol concentrations are sufficient to induce a B to A conformational change as well as cause a slight increase in the rate of Thy(w5)hThy formation, the effective water concentration (1 1 M at 80% w/w ethanol concentration) is still large enough to favor Thy()Thy rather than Thy(a-5)hThy (cf. Varghese, 1970). These results, therefore, confirm and extend the work of Rahn and Hosszu (1966) on DNA films, who concluded from their results that the absence of water i s necessary for the formation of Thy(u-5)hThy. The influence of ethanol on the formation of photoproducts in DNA could be due to (a) ethanol-induced conformational changes, the possibitity we are trying to assess, (b) dehydration or removal of water, or (c)
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Figure 2. Chromatographic distribution of radioactivity in formic acid hydrolysates of T7 C3H(methyl)Thy] DNA irradiated (2.5 kJm-*, 280 nm) at different ethanol concentrations. (The same samples used for CD measurements represented in Fig. 1A. See Materials and Methods). Results are shown for native DNA (M and )denatured DNA (o---o).
DNA conformation and photoproduct formation
51 I
and Thy(a-5)hThy photoproducts are, respectively, reduced and increased in solutions at high ethanol concentrations and in films at low humidities, we have interpreted our results in these cases without explicitly distinguishing dehydration from a direct influence of ethanol on the photochemistry. Our main conclusion is that the influence of ethanol on these photoproducts does not seem to be uniquely due to possibility (a). Like Thy()Pyr formation, the yield of Thy(6-4)Pyo (the acid stable product derived from Thy-Cyt adduct formed in situ) is reduced by the presence of ethanol during irradiation of T7 DNA (cf. Table 1 and Fig. 3; note the different fluences used in these experiments for native and denatured DNA, compared to those used for determination of Thy()Pyr and Thy(a-5)hThy formation). For denatured T7 DNA, suppression of Pyr adduct formation is a monotonic function of the ethanol concentration. In contrast to this and to the results obtained for Thy()Pyr formation, Pyr adduct formation in native DNA does not decrease monotonically with increasing ethanol con10' 0 5 10 15 20 25 30 35 centration; rather, superimposed on the inhibition is FA A C T I0 I\] NU hl B [IR a pronounced enhancement in the yield between 50 and 60% ethanol. At this ethanol concentration T7 Figure 3. Elution profile of formic acid hydrolysate of irra- DNA is in a C-like conformation, as indicated by diated (10 kJm-', 280 nm) 3H-Iabeled native T7 DNA from a Dowex column (see Materials and Methods). The the C D spectrum (cf. Fig. 1A). The same qualitative radioactivity in fractions 1 through 13 correspond to thy- behavior for Pyr adduct formation in native T7 DNA mine (T) and all stable Thy-derived photoproducts (T*) as a function of ethanol concentration was observed other than Thy (64) Pyo. 0% ethanol (u 30%) eth-,in an independent series of experiments using fluoresanol (e-+), 50% ethanol (A---A), and 80% ethanol cence emission to assay adduct yield (M. H. Patrick ( A . . . A .) and J. M. Snow, unpublished data; cf. also, Materials and Methods). Since enhanced adduct yield for DNA in a C-like altered photochemistry involving the ethanol itself.* conformation in ethanol is seen even though there Of course, combinations of these effects are also poss- may be a general suppression of this photoproduct ible. There is not sufficient published information by the ethanol, then it may be that adducts are proavailable to provide an independent evaluation of the duced in even higher relative yield in in uiuo situarelative importance of effects (b) and (c) in inter- tions. DNA is thought to be in the C conformation preting our experiments. Since the Thy()Thy(c,s) in phages (Tunis-Schneider and Maestre, 1970; Dorman and Maestre, 1973) and the DNA in chromatin is largely in the C conformation (Hanlon et al., 1974). In addition, one can take advantage of the observation that with increasing salt concentration, the C D *The presence of alcohol has been shown to reduce the efficiency of PyrOPyr formation in irradiated dimethyl spectrum of DNA shows a decrease in the positive thymine (Morrison and Kloepfer, 1968) and TpT (Lamola, band; at sufficiently high salt concentration (e.g. 6 1968; Tramer et al., 1969), as well as to induce photo-addi- M LiCl and CsCl, or 5.5 M NH,Cl) the C D spectrum tion reactions with Pyr (Wang, 1960) and Pur (Connolly is the C-type. Therefore, to further test the possibility and Linschitz. 1968; Evans and Wolfenden, 1960; Steinmus et a/., 1970; Leonov and Elad, 1974) bases in solution. that Pyr adducts form preferentially in DNA having a C conformation, we irradiated T7 DNA in situ (as This may account for the decreasing yield of Thy()Pyr as the ethanol concentration is raised (Table 1). The more part of the intact phage) and in the presence of 6 drastic suppression observed for denatured DNA may re- M CsCl; the C D spectrum and photoproduct yield flect the much increased base-solvent interaction in singlestranded DNA. Ben-Ishai et d. (1973) found, in addition were determined, and the results are shown in Fig. to suppression of ThyOPyr formation in irradiated I C and Table 2), respectively. Consistent with our (2 = 254 nm or > 290 nm) native or denatured E . coli findings for DNA in ethanolic solutions, the results DNA by the presence of 2-propanol or ethanol, evidence obtained for irradiation of T7 phage DNA in situ and for photoalkylation of Gua and Ade moieties by 2-pro- in 6 M CsCl. show that Thy()Pyr and Thy(a-5)hThy panol. They also detected an as yet unidentified Thyderived photoproduct (RJ 0.8 in the solvent system used formation is not affected by whether the DNA is in here) from denatured DNA irradiated in the presence of the B or C conformation. However, it is obvious that 2-propanol. in contrast to the conclusions drawn from the studies
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MICHAEL H. PATRICK and DONALDM. GRAY Table 1. Photoproduct yield* in irradiatedt T7 DNA at different ethanol concentrations ~
Ethanol (%)
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Native (N) or denatured (D) DNA N D N D N D N
0
30
50 60
ThyOUra
Percent of Thy radioactivity as: Thy()Thy(c,s) Thy()Thy(t,s) Thy(a-5)hThy
2.2 4.2 1.9
5.8 11.0 5.7
2.1 2.2 1.9
6.4 6.0 6.3
0.3
2.3
6.0
0.4
1.6 1.4
4.6 4.4
0.8 0.8
Thy(6-4)Pyo 0.59 0.66 0.36 0.58 0.49 0.47 0.42 0.42 0.16 0.28 0.12 0.22
.-
-
-
D 70
N D N D
80
*These values represent the average of experiments done at least twice; a dash line represents < O.l?(, of Thy the “/b radioactivity in the region having the R, of the indicated photoproduct. In the case of Thy(-)Pyo, radioactivity in the product is with respect to Thy and all Thy-derived photoproducts eluted in the first 15 fractions from a Dowex column (see Fig. 3). Results obtained by spectrofluorimetric analysis (see Materials and Methods) of generalized Pyr adduct formation were in qualitative agreement with the results shown above for the radioactivity analysis of Thy-Cyt adduct formation. That is, the fluorescence emission at 400 nm (Hauswirth and Wang, 1973) was greater for DNA irradiated at 50 and 60% ethanol, and decreased sharply at higher’ concentrations. We had difficulty, however, in obtaining quantitatively reproducible fluorescence spectra for ethanolic solutions of irradiated DNA. TFluence: 2.5 kJm.-’ at 280 nm for native and denatured DNA assayed for Thy()Pyr and Thy(&-5)hThy; 4 kJm-’ at 280 nm for native DNA assayed for Thy(64)Pyo; 10 kJm-’ 280 nm for denatured DNA assayed for Thy(64)Pyo. Since the yield of Pyr adducts in duplex DNA is significantly less than the PyrOPyr yield, and this difference is even greater in denatured DNA, different fluences were used which depended on the nature of the photoproduct to be studied and the state of the DNA. In general, the ratio, Pyr adduct/Pyr()Pyr, is fluence dependent; but since we are interested here in the photoproduct yield as a function of the ethanol concentration (rather than the fluence dependent photoproduct ratio), the different fluences used for detection of certain photoproducts do not influence the property we wish to measure. Table 2. Photoproduct yield in native T7 DNA irradiated (280 nm) in different aqueous environments
Irradiation condition Intact phage 0.1 M Na+ 6.0 M Cst
Thy()Ura 1.3 1.5 1.9
Percent of Thy radioactivity* as: Thy()Thy(c,s) Thy(6-4)Pyo Thy(a-5)hThy 5.1 5.2 5.3
0.62 0 63 0.64
< 0.1
