Pholo~lr~ , r i r isrr .? uridPkorohrolop~. 1973, Vol. 18. pp. 437-439. Pergdmon Press. Printed in Great Britain

RESEARCH NOTE EFFECT OF ULTRAVIOLET IRRADIATION OF DNA

ON T H E DISSOCIATION TRANSITION OF THE STRONG DNA-ACRIFLAVINE COMPLEX

DANIEL ROTH Department of Pathology. New York University Medical School, 550 First Ave.. New York, N.Y.

10016. U.S.A.

(Received 19 December 1972; accepted 11 April 1973)

The strong binding of aminoacridines to DNA, when ionic strength and dye: DNA mole ratios are low, is determined by electrostatic interaction between the dye cation and polymeric phosphates, in cooperation with an intimate association be- tween the acridine and nucleotide aromatic rings. According to Lerman’s ( 1 96 1, 1964) conception of this association, an acridine molecule inter- calates between two adjacent base pairs and ex- tends completely across the DNA duplex. I n the modifications proposed by Mason and McCaffery (1964). Drummond et al. (1965), Pritchard et al. (1966), and Neville and Davies (1966), the dye molecules are conceived to be disposed toward one of the strands, yet to retain their interactions with the base rings. In both situations the overlapping ring electron orbitals can induce similar changes in the absorption and fluorescence spectra of the acri- dine. With respect to acriflavine, these consist of hypochromicity, fluorescence quenching, and a red shift in its absorption spectrum. All of these have been employed analytically as specific expressions of the strong, or ‘intercalative’, binding mode, in studies such as those of Morthland et al. (19541, Heilweil and Van Winkle (1959, and Tubbs et al. (1964). In aggregate, the reports of Peacocke and Skerrett ( 1 956), Weill ( 1 969 , KleinwPchter et al. (1969), Thomes et al. (1969), Lober and Achtert (1969), Chan and Van Winkle (1969), MacInnes and McClintock (1970), and Bidet et al. (1971) indicate that A-T pairs may play a major role in the fluorescence quenching of intercalated aminoacri- dines, both through the relatively large dye-binding constant at A-T rich regions, and through their participation in transferring the excitation energy of an A-T-bound dye to a quenching site at an ad- jacent guanine residue within the ordered structure of the DNA molecule.

This suggested that intercalative dye binding might be applied advantageously to certain studies

of the photochemistry of DNA. The present study employs fluorescence quenching as an expression of intercalative dye binding, in an examination of the effects that prior UV irradiation of DNA has upon the ‘dissociation transition’ of its complex with acriflavine. The term ‘dissociation transition’ was originally applied by Chambron et af. (1966) to the biphasic thermal dissociation kinetics exhibited by complexes of DNA with intercalating cations.

MATERIALS AND METHODS Calf thymus DNA (Sigma Chemical Co.) was

dissolved at a concentration of 50 pg/ml into phos- phate-buffered saline solution, pH 6.2, ionic strength 0.006, and was irradiated by a UVS-11 mercury lamp. Dosage was measured on a Yellow Springs Instrument Co. (Yellow Springs, Ohio) model 65 radiometer linked to a Farrand Optical Co., Inc. (New York) UV monochromator. To aliquots of 0.1 ml were added 4 x mmole of acriflavine hydrochloride in 2 ml of the buffered saline solution. After a 15 min staining period in the dark, dye fluorescence was measured in a Photovolt model 540 filter fluorometer. Exciting light was supplied by a 100 W mercury lamp through a 438 nm interference filter. Emitted light was passed through a Corning glass filter, C.S. No. 4-105. Samples were brought up to desired temperatures in a heating block, held there for 1 min, and analyzed immediately. Samples and con- trols were treated in duplicate and were analyzed in a sequence which averaged out temperature vari- ations at each reading point.

The DNA was denatured by holding at 95°C for I5 min, cooling rapidly to O’C, and then equilib- rating with room temperature.

RESULTS AND DISCUSSION At room temperatures the dye exhibited inter-

calative binding to both denatured and native

437

438 D. ROTH

DNA, as indicated by similar quenching of its fluorescence (Fig. 1). When bound to single- stranded DNA in the incompletely intercalated complex, its thermal dissociation slope was linear over the 25-60°C range; the free dye levels of fluorescence were restored at 60°C. In contrast, the dissociation curve of the double-stranded com- plex was biphasic, exhibiting a low rate of increase in fluorescence yield at low temperatures, a transi- tion region at about 6WC, and a rapid increase thereafter. This is attributed to enhanced thermal stability of the complex conferred by complete intercalation of the "Lerman" type, in accordance with studies such as those of Gersch and Jordan (1969, Walker (1963, and Finkelstein and Weinstein (1967). At less than saturating concen- trations (one dye molecule per 200 base pairs), it is thought that the major portion of the bound dye is intercalated, that the quenching action is de- pendent upon the integrity of A-T hydrogen bonds, and that, at temperatures which rupture those bonds, quenching conditions are abolished.

0 10 20 30 40 50 60 70 80 90 100 Tern pe ra t u r e

Figure 1. Thermal dissociation curves ("C) as reflected by fluorescence of a DNA-acriflavine complex. (0) nal tive calf thymus DNA; (0) calf thymus DNA denatured at 95°C. DNA: 2.5 pg/ml; acriflavine hydrochloride: 2 X lo-* M in 2 ml of phosphate-buffered saline solution, pH 6.2. ionic strength 0.006. Broken line: fluorescence

intensity of free dye standard.

Additional support for this interpretation is shown in Fig. 2. Native DNA was heated at 72°C for 1 h, rapidly cooled to O"C, then reacted with acriflavine at room temperature as before. The thermal dissociation slope is now seen to be linear and without a transition region. However, when a sample of similarly heated DNA was allowed to cool slowly to promote reassociation of thermally ruptured hydrogen bonds, the curve regained its

0 1 0 10 20 30 40 50 60 70 80 90100

Temperature

Figure 2. Effects of pre-heating DNA at 72°C on the dis- sociation transition of the DNA-acriflavine complex. (0) DNA heated at 72°C cooled rapidly to 0°C. then complexed with acriflavine; (0) DNA heated at 72°C. cooled slowly, then complexed with acriflavine; (0) native DNA control complexed with acriflavine. Com-

plexing done as in Fig. 1.

original transition pattern, approaching that of native DNA. Thus a relationship between fluores- cence quenching and DNA secondary structure at A-T pairs was demonstrable for our experi- mental conditions.

These results are taken to indicate that dye fluor- escence values at 60°C may be employed to dis- criminate between single-stranded and double- stranded DNA-acriflavine complexes in the region of A-T pairs.

Figure 3 shows dissociation curves for acriflavine complexed to DNA which had initially been ir- radiated at 254nm with doses differing by one order of magnitude. The curves exhibit a progres- sive obliteration of the transition knuckle and an approach toward linearity with increasing ultra- violet (UV) dose. This is ascribed to regional de- naturation of DNA around UV photoproducts of thymine, alluded to by Shugar (1961) and by Lazur- kin ef al. (1970). To the extent that hydration photoproducts can occur in double-stranded DNA, and to the extent, if any, that they affect dye-bind- ing sites, hydration photoproducts might contri- bute to these altered dissociation curves. However, when native DNA was irradiated at 44°C to avoid hydration of 5-6 double bonds in the pyrimidine rings, no changes from the original curves were obtained. Therefore it is considered unlikely that hydration products contribute significantly to the phenomenon.

The 27 and 88 per cent increases in fluorescence yield at the 60°C mark, which developed with the

Effect of ultraviolet irradiation of DNA 439

21 1 I 0 10 20 30 40 50 60 70 80 90100

Temperature

Figure 3. Thermal dissociation curves of DNA-acrifla- vine complex following U V irradiation. (0) Native DNA control; (0) DNA irradiated at 254 nm, incident dose of 6X 103J/m2; (0) irradiated at incident dose of 6 x lo4 J/m2. DNA concentration during irradiation was 50 pglml. Complexing and dissociation studies done under

same conditions as in Figs. 1 and 2.

two experimental UV doses, may be crude expres- sions of the number of dimers formed between adjacent thymine residues. Since it is not known whether or not the extent of strand separation was the same for each dimer, nor how many sequential thymines were involved (which would alter the net length of the denatred regions), an exact propor- tional relationship cannot presently be derived. Available data place a sensitivity limit for the reported technique at about 1/20 th of the smaller of the two experimental doses of radiation, or 300J/m2 incident at 254nm. With this dosage approximately 0.8 per cent of the thymine re- sidues are dimerized (data obtained in collabora- tion with Dr. G. Teebor, New York University Medical School).

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